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Elements  essential  to  life  and  health  
• Bulk  elements  –  Structural  components  of  cells  required  in  daily  gram  
amounts  (H,C,N,O,Na,Mg,K,Ca)  
• Trace  elements  –  Required  in  daily  mg  amounts  
• Cells  comprise  very  few  types  of  atoms  
o H,C,N,O  comprise  99%  of  an  organism  
o Contrasts  the  non-­‐living,  inorganic  environment  
 
A  cell  is  made  of  carbon  compounds  
• Most  molecules  within  cells  are  comprised  of  carbon  due  to  its  ability  to  
form  large  molecules  
• C  is  small,  has  4  electrons  and  4  vacancies  in  outer  shell  à  Forms  4  
covalent  bonds  
• Highly  stable  C-­‐C  covalent  bonds  form  linear/branched  chains  and  rings  
• C  skeletons  can  bind  functional  groups  (methyl,  hydroxyl,  carbonyl  etc)  
 
Small  organic  molecules  are  used  to  form  macromolecules  
• Sugars  à  Polysaccharides  
• FA  à  Fats,  lipids,  membranes  
• AA  à  Proteins    
• Nucleotides  à  Nucleic  acids  (DNA,  RNA)  
 
Water  is  the  most  abundant  substance  in  cells  
• Most  cellular  interactions  occur  in  an  aqueous  environment  
• Water  is  a  polar  molecule  à  Unequal  sharing  of  electrons  results  in  
partial  positive  charge  on  H  and  partial  negative  charge  on  O  à  Polarity  
and  cohesiveness  due  to  H  bonding  
• Water  is  a  good  solvent  for  charged  and  polar  substances  
o AA  and  peptides  
o Small  alcohols  
o CHO  
• Water  is  a  poor  solvent  for  nonpolar  substances  
o Nonpolar  gases  
o Aromatic  moieties  
o Aliphatic  chains  
 
Water  can  participate  directly  in  chemical  reactions  (act  as  a  reactant)  
• Condensation  reaction  à  Formation  of  a  bond  between  OH  and  OH  
expelling  water    
o New  bond  formed  between  OH  groups  on  2  different  molecules    
o Water  generated  
• Hydrolysis  reaction  à  Cleavage  of  this  bond  accompanied  by  the  addition  
of  water  
o 2  molecules  cleaved  to  form  OH  groups  on  the  2  monomers  
• Cage  formation  of  water  around  hydrophobic  regions  
 
Revision  
• H,  O,  C,  N  are  the  4  main  elements  in  the  human  body  

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Carbon  is  well  suited  to  life  because  it  is  small  and  can  form  4  covalent  
bonds  and  C  skeletons  can  bind  functional  groups  
Hydrophilic  –  Polar  or  charged  molecules  that  dissolve  readily  in  water  
Hydrophobic  –  Non-­‐polar  molecules  or  groups  that  do  not  dissolve  
readily  in  water  
Hydrolysis  reaction  cleaves  bond  in  a  molecule  and  adds  water  molecules  
Condensation  reaction  forms  a  bond  between  hydroxyl  groups  of  2  
molecules  and  expels  water  

 
Enzymes  promote  reactions  
• Cellular  chemical  reactions  require  higher  temperatures  than  that  found  
in  cells;  overcome  by  use  of  specialised  proteins  (enzymes)  
• Enzymes  function  as  catalysts  to  speed  up  the  reaction  rate  of  a  specific  
substrate  by  lowering  activation  energies  
• Do  not  affect  equilibrium  of  a  reaction  and  thus  can  be  bidirectional  
(substrate  à  Product  and  product  à  substrate)  
• Often  conjugated  to  a  cofactor  (inorganic  ion)  or  coenzyme  (complex  
molecule)  to  function  
• Lowers  amount  of  total  energy  required  for  a  forward  reaction  
 
Factors  affecting  the  activity  of  enzymes  and  metabolic  processes  (enzyme  
regulation)  
• Amount  of  enzyme  à  Influenced  by  rate  of  transcription  of  enzyme  genes  
o Extracellular  signals  (down  or  up  regulation)  
o Protein  degradation  (ubiquitin)  
o mRNA  degradation  
• Catalytic  activities  of  enzymes  à  Allosteric  control,  feedback  inhibition,  
covalent  modification  (phosphorylation)  or  proteolytic  cleavage  
• Accessibility  of  substrates  à  Compartmentalisation/compartmentation  
o Reactions  occur  in  certain/specific  parts  of  the  cell/body  
 
Enzyme  regulation  
• Factors  affecting  amount  of  enzyme  
o Extracellular  signals  à  down  or  up-­‐regulation  
o Transcription  of  specific  enzyme  gene  
o mRNA  degradation    
o mRNA  translation  on  ribosome  
o Protein  degradation  (ubiquitin,  proteasome)  
o Enzyme  sequestered  in  subcellular  organelle  
(compartmentalisation)  
• Factors  affecting  enzyme  activity  
o Enzyme  binds  substrate  
o Enzyme  binds  ligand  (allosteric  effector)  
o Enzyme  undergoes  phosphorylation  or  de-­‐phosphorylation  
o Enzyme  combines  with  regulatory  protein  
 
Allosteric  control  of  enzymes  

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Allosteric  control  is  the  regulation  of  an  enzyme  by  a  regulatory  molecule  
that  interacts  at  a  site  other  than  the  active  site  (allosteric  site)  
Allosteric  modulators  can  be  either  stimulatory  (positive)  or  inhibitory  
(negative)  

 
Feedback  inhibition  of  enzymes  
• Inhibition  of  the  first  irreversible  reaction  (committed  step)  in  linear  
pathways  
• Branched  pathways    
o Inhibition  of  common  initial  step  by  own  product  and  activation  by  
product  of  another  pathway  
o Enzyme  multiplicity  à  Inhibition  of  the  committed  step  catalysed  
by  isoenzymes    
o Cumulative  feed  inhibition  à  Partial  inhibition  of  a  common  step  
by  each  of  the  final  products  
 
Covalent  modification  of  enzymes  à  Phosphorylation  
• Tyr,  Ser,  Thr,  His  
• Protein  kinase  (ATP  à  ADP)  phosphorylates  
• Protein  phosphatase  dephosphorylates  
 
Some  enzymes  (zymogens)  are  regulated  by  proteolytic  cleavage  
• Zymogens  à  Inactive  precursors  that  become  active  following  cleavage  
• Trypsinogen  +  enteropeptidase  à  Trypsin  (active)  
• Chymotrypsinogen  +  trypsin  à  Pi-­‐Chymotrypsin  (active)  
• Pi-­‐chymotrypsin  à  Alpha-­‐chymotrypsin  
 
Living  organisms  require  energy  from  the  environment  
• Energy  comes  in  the  form  of  chemical  fuels  (food)  or  light  from  
environmental  sources  
• Energy  is  required  for  3  main  purposes  for  living  organisms  
o Mechanical  work  (muscle  contraction  and  cellular  movement)  
o Active  transport    
o Synthesis  of  macromolecules  from  simple  precursors  
 
Basic  principles  underlying  energy  flow  
• Fuels  are  degraded  and  large  molecules  are  constructed  in  a  series  of  
reactions  (metabolic  pathways)  
• ATP  links  energy  releasing  pathways  with  energy  requiring  pathways  
(crucial  carrier  used  for  formation  of  complex  molecules)  
• Oxidation  of  carbon  fuels  powers  ATP  formation  
o Carbon  fuels  are  highly  reduced  elements  that  when  broken  down  
yield  electrons  for  ATP  formation  
• A  limited  number  of  reaction  types  and  intermediates  are  common  to  
many  pathways  
• Metabolic  pathways  are  highly  regulated  and  compartmentalised  
o Compartmentalisation  and  allosteric  modulation  regulate  
metabolic  pathways  

  
Metabolism  
• Highly  coordinated  cellular  activity  in  which  multi-­‐enzyme  systems  
cooperate  to    
o Obtain  energy  by  degrading  energy  rich  nutrients  or  by  capturing  
solar  energy  
o Convert  nutrient  molecules  to  cell’s  own  molecules  
o Polymerise  monomers  to  polymers  (polysaccharides,  lipids,  
proteins,  membranes,  nucleic  acids)  
o Synthesise  and  degrade  biomolecules  
o Maintain  distinctive  composition  of  different  cell  compartments  
 
Catabolic  and  anabolic  metabolism  
• Catabolism  and  anabolism  are  the  major  metabolic  pathways  
• Catabolism  is  the  breakdown  food  molecules  into  useful  forms  of  energy  +  
heat  lost  
• Anabolism  uses  the  energy  produced  from  catabolism  to  produce  new  
molecules  from  small  molecules  
• Enzymes  catalyse  these  two  pathways  which  constitute  the  metabolism  of  
a  cell  
 
Metabolism  
• Where  the  processes  occur  can  be  cell  specific  
• Centres  around  generation  of  pyruvic  acid  and  acetyl  CoA  
• CHO  stored  as  glycogen  which  provides  energy  between  meals  (glucose  
à  Glycolysis  à  Pyruvic  acid  à  Acetyl  CoA  à  CA  cycle  à  Electron  energy  
carriers  à  Oxidative  phosphorylation  à  ATP  generation)  
• Pyruvic  acid  à  Glucose  can  generate  glycogen  during  gluconeogenesis  
• Protein  not  stored,  only  broken  down  during  starvation  state  
• Proteins  à  AA  à  CA  cycle  à  Pyruvic  or  acetyl  CoA  and  some  may  enter  
TCA  cycle  directly  
• Lipids  stored  as  TAG  à  FA  +  glycerol  à  Glycerol  can  enter  glycolysis  but  
the  bulk  of  the  energy  comes  from  the  FA  chains  which  break  down  to  
acetyl  CoA  à  CA  cycle  
• Production  of  energy  from  the  oxidation  of  food  molecules  (either  
digested  or  stored)  which  are  highly  reduced  molecules  (rich  in  
electrons)  
• Energy  carriers  carry  electrons  to  the  pathways  which  produce  ATP  
 
Three  forms  of  energy  stores  in  biology  
• Energy  carriers  à  Contain  1  or  more  energy  rich  covalent  bonds  
• Macromolecules  
o Highly  reduced  molecules  
o Large  branched  polysaccharides  (glycogen/starch)  
o Fatty  acids  (Animal  cells  derive  energy  from  between  meals)  
o Sugars  and  fats  both  degraded  to  acetyl  CoA  in  the  mitochondria  to  
drive  ATP  production  
• Electrochemical  gradients  also  a  source  of  energy  

o Electrical  force  (drive  by  differences  in  membrane  potential)  
o Chemical  force  (ion  concentration)  

 
Energy  carriers  are  essential  for  biosynthesis  
• Food  molecules  (digested  or  stored)  are  highly  reduced  and  rich  in  energy  
à  Through  catabolism  they  are  oxidised  to  yield  their  electrons  à  
Electrons  carried  by  activated  carrier  molecules  and  used  to  drive  
metabolic  pathways  in  the  production  of  new  molecules  required  by  the  
cell  for  storage  or  use  
• About  the  flow  of  electrons  
 
Different  forms  of  energy  carriers  
• ATP  à  Energy  release  following  transfer  of  groups  
o Used  to  drive  metabolic  processes  
• NADPH,  NADH  and  FADH2  release  high  energy  electrons  and  H+  
o Important  in  driving  the  processes  through  the  oxidation  of  food  
sources  
• Coenzyme  (CoA)  à  Energy  release  from  high  energy  bond  of  the  acyl  
(acetyl)  group  
 
ATP  is  the  most  abundant  and  widely  used  energy  carrier  
• Formed  by  oxidation  of  carbon  fuels  
• Breakdown  is  exergonic  (thermodynamically  favourable)  
• Cells  maintain  ATP  concentrations  far  above  concentration  equilibrium  in  
order  to  drive  chemical  reactions  
• Participates  in  the  enzyme  catalysed  reaction  and  supplies  it  with  free  
energy  
• Provides  energy  by  transfer  of  groups    
o Transfer  or  addition  of  one  or  more  phosphate  or  AMP  provides  
energy  
• ATP  hydrolysis  is  not  the  source  of  energy  
• ATP  structure  
o Ribose  group  
o Adenine  group  
o 3  phosphate  groups  which  yield  the  energy  
 
NADH  and  NADPH  are  activated  carriers  of  electrons  
• Act  as  cofactors  for  many  enzymes  
• Shuttle  electrons  and  H+  between  anabolic  and  catabolic  processes  
o Reduced  metabolites  (food)  become  oxidised  through  catabolism  
o During  this  reaction  pathway,  NAD  becomes  reduced  (becomes  
electron  rich)  and  gives  up  the  electrons  to  drive  pathways  to  build  
new  molecules  (anabolism)  
• Extra  phosphate  group  on  NADPH  (reduced  form)  
o No  effect  on  the  electron  transfer  properties  (no  extra  energy  
value)  
o Allows  NADPH  to  bind  different  substrates  compared  to  NADH  

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o NADPH  mainly  used  in  synthesis  of  molecules  and  NADH  used  
mainly  in  ATP  generation  
NADH  and  NADPH  derived  from  Vitamin  B3  (niacin)  

 
Coenzyme  A  is  an  activated  carrier  of  acyl  groups  
• Ribose  and  adenine  group,  2  phosphates,  chain  of  various  groups  
• Acetyl  group  is  transferable  and  held  by  a  high  energy  bond  
• Majority  of  coenzyme  A  structure  facilitates  recognition  by  specific  
enzymes  
 
Oxidation  of  organic  molecules  is  an  important  fuel  source  
• Oxidation  and  reduction  both  apply  when  there  is  a  shift  in  electrons  
between  atoms  linked  by  a  covalent  bond  
• Oxidation  is  the  removal  of  electrons  from  a  molecule  
• Reduction  is  the  addition  of  electrons  to  a  molecule  
 
Carbon  oxidation  is  paired  with  reduction  
• Oxidation  of  organic  molecules  à  Loss  of  electron  and  proton  (H+)  
• In  organic  molecules  
o Oxidation  occurs  if  the  number  of  C-­‐H  bonds  decreases  
o Reduction  occurs  if  the  number  of  C-­‐H  bonds  increases  
 
Flow  of  electrons  in  metabolism  
• Responsible  for  all  work  done  in  living  organisms  
• Source  of  electrons  is  food  or  stored  molecules  (reduced  compounds)  in  
non-­‐photosynthetic  organisms  à  Catabolism  à  Electron  carriers  and  
electrons  à  ATP  
• Electrons  move  from  a  range  of  metabolic  intermediates  to  specialised  
electron  carriers  
• Glucose  à  Glycolysis  à  Pyruvate  à  Acetyl  CoA  
• FA  à  Acetyl  CoA  
• AA  à  Acetyl  CoA  
 
Compartmentation  
• Affects  accessibility  of  substrates  
• Particular  metabolic  pathways  need  to  occur  in  different  parts  of  the  cell  
• Breakdown  of  FA  à  ATP  in  mitochondrion;  Lipids  made  in  ER  
• Glycolysis,  CA  cycle  à  Brain  
• Beta  oxidation,  CA  cycle  à  Heart  
• Beta  oxidation,  TAG  synthesis  à  Adipose  tissue  
• Beta  oxidation,  glycolysis,  proteolysis  à  Muscle  
 
Revision  
• Anabolic  metabolic  pathways  are  biosynthetic    
• Energy  carriers,  macromolecules  and  electrochemical  gradients  are  the  
three  forms  of  energy  stores  in  biology  
• ATP  is  the  most  abundant  energy  carrier  

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NADH,  NADPH  and  FADH2  are  energy  carriers  which  release  high  energy  
electrons  and  H+  
Oxidation  of  organic  molecules  is  important  for  energy  production  
A  loss  of  a  C-­‐H  bond  from  an  organic  molecule  is  indicative  of  oxidation  
Compartmentation  is  the  concept  used  to  describe  metabolic  reactions  
that  occur  in  separate  compartments  of  the  cell  or  organism  

 
Glucose  
• Important  energy  carrying  molecule  
• The  oxidation  of  glucose  via  glycolysis  produces  pyruvate  
• Can  be  stored  as  glycogen  and  starch  
 
Cellular  respiration  
• Primarily  responsible  for  ATP  synthesis  which  is  used  for  everything  
• Process  in  which  cells  consume  O2  and  produce  CO2  
• Provides  energy  in  the  form  of  ATP  
• Occurs  in  three  major  stages  
o Acetyl  CoA  production  à  Cytoplasm  
o Acetyl  CoA  oxidation  à  Mitochondrial  matrix  
o Electron  transfer  and  oxidative  phosphorylation  à  Inner  
mitochondrial  membrane  
• Pyruvate  is  the  precursor  to  acetyl  CoA  
• Sources  of  acetyl  CoA  include  pyruvate  generated  from  glycolysis,  amino  
acids  and  fatty  acids  
• Glucose  à  Oxidation  via  glycolysis  à  Pyruvate  
 
Glycolysis  
• Chemical  pathway  common  to  almost  every  cell  
• Occurs  in  the  cytosol  (complex  eukaryotic  cells  are  compartmentalised)*  
• Energy  is  released  and  captured  as  ATP  
• One  glucose  molecule  à  2  pyruvate  molecules  
• Stereoisomers  (D  and  L)  of  sugars  à  D  sugars  are  most  common  
• 6C  in  glucose,  3C  in  pyruvate  
• Reduce  NAD+  à  NADH  
• ADP  +  PO43-­‐  à  2ATP  
• Glycolysis  produces  2  ATP  
 
NAD  and  NADP  (common  redox  cofactors)  
• NAD  is  electron  carrier  molecules  (source  of  reduction  potential)  
• Vitamin  B3  (Niacin)  is  needed  to  form  the  nicotinamide  group  
• Chronic  niacin  deficiency  leads  to  a  disease  called  pellagra  
 
Significance  of  glycolysis  
• For  some  tissues  (such  as  brain,  kidney  medulla  and  contracting  skeletal  
muscle)  and  some  cells  (erythrocytes,  sperm  cells)  glucose  is  the  only  
source  of  metabolic  energy  (critically  dependent  on  oxidation  of  glucose)  
• The  product  of  glycolysis  (pyruvate)  is  a  versatile  metabolite  used  in  
many  ways  and  can  have  multiple  fates  

o Glycolysis  provides  precursors  
o In  most  tissues  when  oxygen  is  plentiful  (aerobic  conditions),  
pyruvate  is  oxidised  (loses  carboxyl  group  as  CO2)  and  the  
remaining  2  carbon  unit  becomes  the  acetyl  group  of  acetyl  CoA
...
 When  animal  tissues  cannot  be  supplied  with  
sufficient  oxygen  to  support  aerobic  oxidation  of  pyruvate  and  NADH  
produced  in  glycolysis,  NAD+  is  regenerated  by  reduction  of  pyruvate  to  
lactate  
• Some  tissues  and  cell  types  that  have  no  mitochondria  cannot  oxidise  
pyruvate  to  CO2  and  produce  lactate  from  glucose  even  under  aerobic  
conditions  
 
• Reaction  1  –  Phosphorylation  of  glucose  à  G6P  
o Biochemical  pathways  often  regulated  at  the  first  step  
o Hexokinase  attaches  a  P  from  the  ATP  to  the  6th  carbon  of  glucose  
to  make  G6P  
o The  activity  of  hexokinase  is  regulated  

o This  chemical  reaction  must  be  shielded  from  water  to  keep  the  
phosphate  from  being  cleaved  off  ATP  by  a  water  molecule  
o Hexokinase  performs  an  induced  fit,  closing  around  ATP  and  
glucose  once  they  are  bound  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
•

•

 
 
 
 
 
 
 
 

Reaction  2  –  Conversion  of  glucose-­‐6-­‐phosphate  à  fructose-­‐6-­‐phosphate  
o Isomerization  –  A  reaction  that  changes  the  shape  of  a  single  
molecule  but  doesn’t  permanently  add  or  remove  any  atoms  
o Isomer  –  Same  number  of  atoms,  different  arrangement  
o Fructose  has  a  5  atom  ring  structure,  glucose  has  6  atom  ring  
§ No  atoms  gained/lost  
Reaction  3  –  Phosphorylation  of  fructose-­‐6-­‐phosphate  à  fructose-­‐1,6-­‐
bisphosphate  (F-­‐1,6-­‐BP)***  EXAM  QUESTION  
o Pi  from  ATP  transferred  to  sugar  à  F-­‐1,6-­‐BP  
§ 2  phosphate  molecules  attached  to  two  different  points  (1st  
and  6th  carbons)  
o Phosphofructokinase-­‐1  (PFK1)  is  the  gatekeeper  of  glycolysis  à  
Catalyses  the  committed  step  of  the  glycolytic  pathway  
o PFK1  proposed  to  have  important  roles  in  metabolic  
reprogramming  in  cancer  
o Somatic  cancer  mutations  of  PFK  alter  enzymatic  activity  and  
allosteric  regulation    
o Regulated  allosterically  
o PFK1  is  a  bacterial  enzyme  with  4  identical  subunits
...
 The  enzyme  also  has  regulatory  
binding  sites  at  the  top  and  bottom  

•

•

•

•

•

•

•

Reaction  4  –  Cleavage  of  fructose-­‐1,6-­‐biphosphate  (F-­‐1,6-­‐BP)  
o Aldolase  splits  sugar  into  two  3  carbon  molecules    
§ Dihydroxyacetone  phosphate  
§ Glyceraldehyde  3-­‐phosphate  
Reaction  5  –  Interconversion  of  the  triose  phosphates  
o Triose  phosphate  isomerase  (enzyme)  rearranges  atoms  of  
dihydroxyacetone  phosphate  into  glyceraldehyde  3-­‐phosphate  
(end  up  with  2  equivalents  of  glyceraldehyde  3-­‐phosphate)  
o The  TIM  barrel  (structure  that  occurs  widely  in  nature)  is  a  
conserved  protein  fold  of  triose  phosphate  isomerase
...
 This  is  the  pasteur  effect
...
 Therefore,  cancer  cells  
that  are  more  than  100-­‐200um  from  capillaries  depend  on  anaerobic  
glycolysis  for  their  ATP  production  
• F-­‐deoxyglucose  is  taken  up  by  brain  cells  in  direct  proportion  to  the  
amount  of  glycolysis  occurring
...
 
• Locations  in  the  body  where  a  lot  of  glycolysis  is  occurring  à  Solid  
tumours  
 
Summary  
• Glycolysis  
o Occurs  in  the  cytosol  
o 1  glucose  à  2  pyruvate  molecules  
o 2NADH  +  2H+  +  2ATP  generated  
o Does  not  require  O2  
o Regulated  to  maintain  constant  cellular  ATP  concentrations  
o Is  the  only  source  of  ATP  for  some  cell  types  
o Solid  tumours  have  very  high  rates  of  glycolysis  
• Pyruvate  
o Can  be  converted  to  lactate  under  anaerobic  conditions  
o Can  be  converted  to  ethanol  by  yeast  
o Can  be  converted  to  acetyl  CoA  to  feed  into  TCA  cycle  
 
Only  a  small  amount  of  energy  available  in  glucose  is  captured  in  glycolysis  
• Glycolysis  =  Glucose  à  2x  pyruvate  +  146kJ/mol  energy  
• Full  oxidation  (+6O2)  à  6CO2  +  6H2O  +  2840kJ/mol  energy  
 
Cellular  respiration  
• Process  in  which  cells  consume  O2  and  produce  CO2  
• Provides  more  energy  (ATP)  from  glucose  than  glycolysis  
• Also  captures  energy  stored  in  lipids  and  AA  
• Occurs  in  three  major  stages  
o Acetyl  CoA  production  à  Cytoplasm  (pyruvate  à  Acetyl  CoA)  
§ Glucose  à  Pyruvate  (glycolysis)  

Pyruvate  à  Acetyl  CoA  via  pyruvate  dehydrogenase  
complex  
o Acetyl  CoA  oxidation  via  TCA  cycle  à  Mitochondrial  matrix  
o Electron  transfer  and  oxidative  phosphorylation  à  Inner  
mitochondrial  membrane  
§

 
Cellular  respiration  occurs  in  3  major  stages  
• Glucose,  FA  and  some  AA  are  oxidised  to  yield  two  carbon  fragments  in  
the  form  of  the  acetyl  group  of  acetyl  CoA  (CH3CO-­‐)  
o Coenzyme  A  carries  functional  groups  around  the  body  (such  as  
acetyl  groups)  
o Fatty  acid  metabolism  also  produces  acetyl  groups  
• The  acetyl  groups  are  fed  into  TCA  cycle  which  enzymatically  oxidises  
them  to  CO2;  the  energy  released  is  conserved  in  the  electron  carriers  
NADH  and  FADH2  
o TCA  cycle  produces  reducing  factors  
• These  reduced  coenzymes  are  themselves  oxidised,  giving  up  protons  
(H+)  and  electrons
...
 In  the  course  of  electron  transfer,  the  large  amount  of  
energy  released  is  conserved  in  the  form  of  ATP,  by  a  process  called  
oxidative  phosphorylation  
 
Coenzyme  A  
• Coenzymes  are  not  a  permanent  part  of  the  enzymes’  structure,  they  
associate,  fulfil  a  function  and  dissociate  (not  part  of  the  chemistry)  
• ADP  attached  to  pantothenic  acid  (B5)  and  SH  group  which  is  important  
to  the  chemistry  of  acetyl  CoA  
• Chemical  intermediates  attach  to  SH  group  
• The  function  of  CoA  is  to  accept  and  carry  acetyl  groups  
• Coenzyme  A  contains  vitamin  B5  (pantothenic  acid)  for  which  deficiency  
is  exceptionally  rare  
 
Conversion  of  pyruvate  to  acetyl  CoA    
• Occurs  in  mitochondrial  matrix  of  eukaryotes  
• Reducing  factors  produced  in  mitochondria  and  used  to  produce  ATP  in  
oxidative  phosphorylation  
• Required  to  enter  into  TCA  cycle  
• Net  reaction  
o Oxidative  decarboxylation  of  pyruvate  (lose  CO2)  
o Acetyl  group  attaches  to  coenzyme  A  
o First  carbons  of  glucose  to  be  fully  oxidised  
• Catalysed  by  large  pyruvate  dehydrogenase  complex  
o Requires  5  coenzymes  
o Thiamine  pyrophosphate  (TPP),  lipoyllysine  and  FAD  are  
prosthetic  groups  
o NAD+  and  CoA-­‐SH  are  co-­‐substrates  

  
 
 
 
 
 
 
 
 
 
 
Pyruvate  dehydrogenase  
• The  complex  is  organised  in  cubic  symmetry  in  Gram-­‐negative  bacteria,  
having  60  subunits  in  three  functional  proteins  
• E2  subunit  carries  acetyl  groups  
• Multiple  processes  happen  
• In  eukaryotes  and  gram-­‐positive  bacteria  it  is  organised  in  dodecahedral  
symmetry  and  consists  of  96  subunits,  organised  into  three  functional  
proteins  in  the  human  enzyme  
• Lipoic  acid  is  covalently  linked  to  a  lysine  residue  to  give  lipoyllysine  
• The  pyruvate  dehydrogenase  complex  uses  substrate  channelling  so  that  
o The  intermediates  of  the  multistep  reaction  sequence  never  leave  
the  surface  
o The  local  concentration  of  substrates  is  kept  high  
o It  prevents  the  loss  (theft)  of  the  activated  acetyl  group  by  other  
enzymes  that  use  this  group  as  a  substrate  
• The  PDC  is  also  an  example  of  an  enzyme  that  uses  a  vitamin  as  a  cofactor  
o TPP  is  thiamine  pyrophosphate  (a  vitamin  B1  derivative)  
o Thiamin  deficient  animals  are  unable  to  oxidize  pyruvate  normally  
which  leads  to  Beri  beri  (characterised  by  a  loss  of  neural  function)  
o An  elevated  blood  pyruvate  level  is  often  an  indicator  of  defects  in  
pyruvate  oxidation  
 
Pyruvate  dehydrogenase  reaction  
• Loss  of  CO2  from  pyruvate  à  Acetyl  
• Acetyl  attached  to  E1  subunit  and  carried  along  to  lipoyllysine  
• Coenzyme  A  attaches  to  acetyl  group  (formation  of  acetyl  CoA)  
• Reoxidation  of  lipoamide  group  (recycled)  à  Produce  reducing  
equivalent  FADH2  à  Passes  electrons  to  NADH  
 
Overall  reaction  of  PDC  
• Enzyme  1    
o Decarboxylation  of  pyruvate  to  an  aldehyde  
o Oxidation  of  aldehyde  to  a  carboxylic  acid  
• Enzyme  2  
o Formation  of  acetyl  CoA  
• Enzyme  3  
o Reoxidation  of  the  lipoamide  cofactor  

o Regeneration  of  the  oxidised  FAD  cofactor,  generation  of  NADH  

 
The  TCA  cycle  summary    
• Glycolysis  is  not  the  only  source  of  acetyl  CoA  (Break  down  of  AA  and  
fatty  acids)  
• Acetyl  CoA  à  Citrate    
• Citrate  is  progressively  oxidised  to  form  the  end  product,  oxaloacetate    
• Oxaloacetate  is  then  recycled  to  form  citrate    
• CO2  also  produced  in  the  oxidation  of  citrate    
• Pyruvate  is  3C  à  Lose  one  CO2  in  the  formation  of  acetyl  CoA  and  2  CO2  
molecules  in  the  oxidation  of  citrate  
• The  oxidation  of  various  intermediates  throughout  the  cycle  leads  to  
electrons  being  stripped  at  key  points;  these  electrons  are  then  used  to  
produce  NADH  and  FADH2  
• Loss  of  CO2  and  production  of  reducing  factors  (reduce  electron  carriers)  
• Generates**  
o 3  NADH  
o 1  FADH2  
o 1  GTP  
 
FAD  –  A  redox  cofactor  
• Vitamin  B2  (riboflavin)  is  the  precursor  of  FMN  and  FAD  
• Riboflavin  deficiency  symptoms  à  Sore  throat,  lesions  of  the  lips  and  
mucosa  of  the  mouth,  glossitis,  conjunctivitis,  dermatitis  
 
Mitochondrion  
• TCA  cycle  and  pyruvate  dehydrogenase  complex  found  in  mitochondrial  
matrix  (surrounded  by  inner  mitochondrial  membrane)  
• Glycolysis  occurs  in  the  cytoplasm  
• TCA  cycle  occurs  in  mitochondrial  matrix  (except  succinate  
dehydrogenase  which  is  located  in  the  inner  membrane)  
• Oxidative  phosphorylation  occurs  in  inner  membrane  
 
TCA    
• Acetyl  CoA  from  pyruvate  (via  pyruvate  dehydrogenase  complex)  
• Acetyl  CoA  feeds  into  the  cycle    
o Acetyl  CoA  +  oxaloacetate  à  Citrate  
o Reaction  catalysed  by  citrate  synthase  
• Citrate  rearranged  into  iso-­‐citrate    
• CO2  lost  at  step  3  and  step  4  (produce  2  CO2  molecules)  
• Produce  reducing  equivalent  (NAD  à  NADH2  at  step  3,4  and  8)  
• FADH2  produced  from  FAD  at  step  6  
• GTP  produced  at  step  5  (energy  from  the  molecule  transferred  to  GDP  à  
GTP)  
• Oxaloacetate  is  the  end  product  and  is  recycled  back  to  citrate  
 
In  eukaryotes,  CA  cycle  occurs  in  mitochondria  
• Reduced  substrate  (fuel)  donates  electrons  

•
•
•

Electron  carriers  pump  H+  out  as  electrons  flow  to  O2  
Energy  of  electron  flow  stored  as  electrochemical  potential  
ATP  synthase  uses  electrochemical  potential  to  synthesise  ATP  

 
Sequence  of  events  in  the  CA  cycle  
1
...
 Isomerization  to  isocitrate  via  cis-­‐aconitate  
3  &  4
...
 Substrate  level  phosphorylation  to  give  GTP  
6
...
 Hydration  (addition  of  H2O)  
8
...
5  ATP  are  
produced  
• For  each  FADH2  oxidised  in  oxidative  phosphorylation,  1
...
 The  enzyme  selects  glucose  over  water  as  a  
result  of  the  conformational  change  that  occurs  when  the  correct  
substrate  (glucose)  binds  
 
Hexokinase  I,  II  and  III  
• Tissue  specific  differences  in  hexokinase  
• Different  tissues  express  different  hexokinases  (different  genes)  
• There  are  four  different  isozymes  of  hexokinase  
o Hexokinase  I,  II  and  III  are  all  found  in  muscle  cells  
o Hexokinase  II  is  the  predominant  isozyme  
§ High  affinity  for  glucose  (half  saturated  at  0
...
1  (faster  initial  rate  of  activity;  more  active)  
o Relative  activity  varies  very  little  with  fluctuations  of  glucose  
o Main  function  is  to  catabolise  glucose  and  provide  pyruvate  for  
TCA  cycle  
o Glucose  taken  up  by  muscle  cells  is  rapidly  phosphorylated  to  trap  
glucose  in  the  cell  
• Hexokinase  IV  (liver)  
o Km  10mM  
o Main  function  to  regulate  blood  glucose  levels  
o Activity  is  regulated  by  glucose  concentration  (a  small  change  in  
concentration  can  result  in  a  large  change  in  activity)  
o Rate  of  hexokinase  activity  increases  with  glucose  concentration  
(regulatory  control)  à  More  subtle  regulation  of  activity  
 
Hormonal  regulation  
• Important  hormones  that  control  metabolism  include  the  peptide  
hormones  glucagon  and  insulin  
• Peptide  hormones  act  via  cell  surface  receptor  (eg  insulin)  
o Mediate  effects  inside  the  cell  
o Cascade  of  reactions  which  may  involve  phosphorylation  of  
proteins  (form  of  regulation)    
o Production  of  secondary  messengers  (cAMP)  which  send  signals  
à  Changes  in  gene  expression  via  regulation  of  transcription  
factors  

•

•

•

Hormones  do  not  act  directly  with  enzymes  in  a  metabolic  pathway,  but  
bind  to  cell  membranes  and  cause  a  series  of  reactions  that  eventually  
lead  to  modification  of  pathway  enzyme  activity  
o Cell  surface  receptors  (peptide/amine  hormones)  
o Nuclear  receptors  (Steroid/thyroid  hormones  enter  the  cell  and  
the  hormone-­‐receptor  complex  acts  in  the  nucleus)  
A  major  problem  in  glucose  metabolism  is  diabetes  
o Type  I  à  No  insulin  production  
o Type  II  à  Insulin  resistance  
§ Cells  become  insensitive  to  insulin  
When  insulin  combines  with  its  receptor  on  the  cell  surface,  a  series  of  
biochemical  events  (downstream  events)  stimulate  uptake  of  glucose  into  
cells  by  the  GLUT4  transporter  
o GLUT4  is  muscle  specific  glucose  transporter    
o Number  of  transporters  on  cell  surface  increases  (regulation  of  
rate  that  glucose  is  imported)  

 
The  second  and  most  important  control  point  in  glycolysis  is  reaction  3,  
catalysed  by  phosphofructokinase  I  (PFK1)  
• Fructose-­‐6-­‐phosphate  à  Fructose-­‐1,6-­‐bisphosphate  is  the  commitment  
step  in  glycolysis  
• While  ATP  is  a  substrate,  ATP  is  also  a  negative  effector  
o Glycolysis  is  down  regulated  if  there  is  plenty  of  ATP  
• Regulation  of  PFK-­‐1  
o Allosteric  inhibitors  
§ ATP  is  both  a  substrate  and  eventual  product  of  glycolysis  
§ Citrate  (TCA  cycle  intermediate/product)  is  an  indicator  of  
if  the  cell  is  meeting  its  energy  needs  à  Increases  inhibition  
by  ATP  
o Allosteric  activators  
§ ADP  and  AMP  increase  in  concentration  when  ATP  
utilisation  outpaces  production  
§ Fructose-­‐2,6-­‐bisphosphate  increases  in  concentration  when  
blood  glucose  concentrations  decrease  
 
Allosteric  regulation  of  PFK1  by  ATP    
• PFK-­‐1  has  allosteric  sites  that  control  rate  of  reaction  
• ATP  binds  to  an  allosteric  site,  lowering  the  affinity  of  the  active  site  of  
fructose-­‐6-­‐phosphate  
• AMP/ADP  act  allosterically  to  relieve  this  inhibition  
• Increased  ATP  à  Lower  PFK1  activity  
• Increased  ADP/AMP  (low  ATP)  à  Higher  PFK1  activity  
 
Allosteric  regulation  of  PFK1  by  fructose-­‐2,6-­‐bisphosphate  
• Not  a  glycolytic  intermediate  
• A  regulator  specifically  produced  to  regulate  glycolysis  and  
gluconeogenesis  (creation  of  new  glucose)  
• F26BP  is  synthesised  from  F6P  by  PFK-­‐2  

•

•

•

•

o Takes  fructose-­‐6-­‐phosphate  and  makes  fructose-­‐2,6-­‐bisphosphate  
in  a  phosphorylation  reaction  
o F26BP  is  not  a  glycolytic  intermediate  
o PFK2  à  F26BP  à  Stimulates  PFK1  
Broken  down  by  fructose-­‐2,6-­‐bisphosphatase  (FBPase-­‐2)  
o Removes  the  phosphate  group  à  Fructose-­‐6-­‐phosphate  
o This  cycle  synthesises  and  removes  F26BP  
o Fine  regulatory  control  of  PFK1  via  F26BP  à  Important  for  
regulating  glycolysis  
o During  gluconeogenesis  we  must  shutdown  glycolysis  via  this  
system  
Regulation  of  F26BP  levels  
o Process  regulated  by  hormones  (eg  insulin  à  Tells  cells  to  take  up  
glucose  and  glycolysis  begins)  
o Both  PFK2  and  FBPase-­‐2  activities  are  on  the  same  protein    
o Process  regulated  by  phosphorylation  à  Regulates  levels  of  F26BP  
which  is  required  for  the  activity  of  PFK1  
o If  the  protein  is  phosphorylated  then  FBPase-­‐2  becomes  active  and  
glycolysis  shuts  down  
When  F26BP  binds  to  the  allosteric  site  on  PFK1  
o Increases  the  enzymes  affinity  for  F6P  
o Decreases  the  enzymes  affinity  for  ATP  
PFK-­‐1  is  almost  inactive  in  the  absence  of  F26BP  

 
Pyruvate  kinase  is  the  third  control  point  of  glycolysis  (at  reaction  10)  
• Pyruvate  kinase  controls  outflow  from  glycolysis  
• Phosphoenolpyruvate  transfers  a  phosphate  group  to  ADP  à  ATP  and  we  
also  generate  a  molecule  of  pyruvate  (which  then  has  multiple  fates)  
• Tissue  specific  isozymes    
• Allosterically  activated  by  fructose-­‐1,6-­‐bisphosphate  
o High  flow  through  from  glycolysis  
• Allosterically  inhibited  by  
o ATP  
o Acetyl-­‐CoA  and  long  chain  fatty  acids  
o Alanine  (enough  amino  acids)  
o Signs  of  abundant  energy  supply  
 
Hormonal  regulation  of  pyruvate  kinase  
• The  L  (liver)  isozyme,  but  not  the  M  (muscle)  isozyme,  is  subject  to  
covalent  modification  (phosphorylation)  
o Liver  isozyme  phosphorylated  by  PKA    
o Muscle  isozyme  does  not  get  phosphorylated  
• Low  BG  à  Glucagon  release  à  Activates  cAMP  dependent  protein  kinase  
(PKA)  à  Phosphorylates  pyruvate  kinase  (liver  isozyme)  à  Glucose  
metabolism  in  the  liver  is  slowed;  glucose  is  conserved  
• MUST  BE  ABLE  TO  WRITE  REACTION  CATALYSED  BY  PYRUVATE  KINASE  
AND  KNOW  STRUCTURES  OF  PHOSPHOENOLPYRUVATE  AND  PYRUVATE  
 
Conversion  of  pyruvate  to  acetyl-­‐CoA  

Pyruvate  dehydrogenase  has  3  subunits  (3  different  enzymes)  
Pyruvate  gets  attached  to  TPP  à  Hydroxyethyl  group  gets  passed  to  
lipoyllysine  à  Acetyl  CoA  (which  feeds  into  TCA)  
• Production  of  1  NADH  
 
Regulation  of  the  pyruvate  dehydrogenase  complex  
• Bridges  glycolysis  and  TCA  cycle  so  important  regulatory  enzyme  (takes  
pyruvate  à  Acetyl  CoA)  
• Regulated  by  phosphorylation/dephosphorylation  
• PDH  incorporates  pyruvate  into  CoA-­‐SH  to  give  acetyl  CoA  for  entry  into  
the  TCA  cycle  
• Regulated  allosterically  
o ATP,  acetyl  CoA  and  NADH  are  inhibitors  (presence  of  the  
metabolites  indicate  sufficient  amounts)  
o AMP,  CoA,  NAD+  and  Ca2+  are  activators  
§ Ca2+  is  a  second  messenger  inside  the  cell  
• Also  regulated  by  covalent  modification  
o Reversible  phosphorylation  of  a  serine  residue  in  one  of  the  two  
subunits  of  E1  
o PDH  kinase  and  PDH  phosphatase  add  and  remove  phosphate  
§ Kinase  is  regulated  by  ATP  
• High  [ATP]  à  Kinase  more  active  à  Phosphorylated  
PDH  à  Less  acetyl  CoA  
• Low  [ATP]  à  Kinase  less  active/phosphatase  
removes  phosphate  à  More  acetyl  CoA  
§ Regulation  of  PDH  complex  via  
phosphorylation/dephosphorylation  (covalent  
modifications)  
§ E1  has  Ser-­‐OH  group  which  is  where  phosphorylation  takes  
place  on  the  subunit  
§ When  the  PDH  complex  is  phosphorylated  it  is  down  
regulated  (less  acetyl  CoA;  less  active)  
 
Regulation  of  the  TCA  cycle  
• Tightly  regulated  at  several  points  
• 3  steps  where  we  produce  NADH,  1  equivalent  of  FADH2  
• Rates  of  glycolysis  and  the  TCA  cycle  are  integrated  so  that  wasteful  
consumption  of  glucose  does  not  occur  
o Regulate  entry  of  pyruvate  (limit  amount  available  for  pyruvate  
dehydrogenase  complex)  
o Supplies  of  pyruvate  match  demand  for  acetyl  CoA  
• Indicators  of  plentiful  energy  supply  (acetyl  CoA,  ATP,  NADH)  inhibit  key  
reactions    
• Indicators  of  energy  depletion  (AMP,  CoA,  NAD+)  stimulate  key  reactions  
• Coenzyme  A  is  also  an  allosteric  regulator  (More  CoA  =  Depletion  à  
Stimulatory)  
• Citrate  synthase  (1)  is  also  inhibited  by  succinyl  CoA  
o Citrate  synthase  is  where  acetyl  CoA  enters  the  TCA  cycle  
•
•

•

•

o Succinyl-­‐CoA  communicates  flow  at  this  point  back  to  the  start  of  
the  cycle  
§ Presence  of  the  intermediate  communicates  back  to  the  
entry  point  of  the  cycle  
o Alpha-­‐ketoglutarate  is  an  important  branch  for  amino  acid  
metabolism  
Regulated  at  highly  thermodynamically  favourable  and  irreversible  steps  
o Irreversible  steps  regulated  (same  as  in  glycolysis)  
o PDH  complex,  citrate  synthase,  isocitrate  dehydrogenase  and  
alpha-­‐ketoglutarate  dehydrogenase  are  the  enzymes  regulated  
General  regulatory  mechanisms  
o Substrate  availability  
o Inhibited  by  product  accumulation  
o Overall  products  (NADH  and  ATP)  affect  all  regulated  enzymes  in  
the  cycle  
§ NADH  and  ATP  are  inhibitors  
§ NAD+  and  AMP/ADP  are  activators  

 
Respiratory  control    
• Tightly  controlled  
• An  adult  woman  requires  approximately  6000kJ  of  metabolic  energy  per  
day  
• ATP  à  ADP  +  Pi  (-­‐30
...
 (which  carries  an  unpaired  electron  and  is  therefore  a  radical)  
o Production  of  O2  is  related  to  the  concentration  of  potential  
electron  donors,  the  local  concentration  of  O2  
• Regulation  via  hypoxia  inducible  transcription  factor  1  (HIF1)  
o Regulation  of  PDH  via  PDH-­‐kinase  
§ HIF-­‐1  increases  synthesis  of  PDH  kinase  à  
Phosphorylation  of  PDH  à  Inactive  
§ Regulates  entry  into  ETC  during  hypoxia  
§ Slows  NADH  and  FADH2  production,  slowing  supply  of  
electrons  to  the  respiratory  chain  
o Regulation  of  complex  IV  (cytochrome  oxidase)  
§ HIF-­‐1  increases  synthesis  of  a  protease  that  degrades  a  
subunit  of  complex  IV  (COX4-­‐1)  
§ Triggers  synthesis  of  alternate  subunit  (COX4-­‐2)  which  is  
specifically  suited  to  hypoxic  conditions  
o Also  up-­‐regulates  glucose  transporters  and  glycolytic  enzymes  
 
Summary  
• BE  ABLE  TO  DRAW  GLUCOSE,  G6P,  PYRUVATE  AND  LACTIC  ACID  
• PDH  complex  regulated  allosterically  and  by  phosphorylation  
• Products  of  the  TCA  cycle  (NADH  and  ATP)  affect  all  regulated  enzymes  in  
the  cycle  
• Coupling  can  be  observed  using  an  oxygen  electrode  and  by  measuring  
ATP  production  
• Oxidative  phosphorylation  can  be  uncoupled  in  vitro  by  various  
uncoupling  reagents  and  in  vivo  by  uncoupling  proteins  
• Uncoupling  protein  1  (UCP1)  is  found  in  some  animals,  human  infants  
• In  hypoxia  there  is  an  imbalance  between  electron  supply  and  transfer  to  
O2  leading  to  ROS  production  
 
 

Gluconeogenesis  
• Glucose  can  be  synthesised  from  non-­‐carbohydrate  precursors  via  
gluconeogenesis,  using  steps  shared  with  glycolysis  and  alternate  
enzymes  to  bypass  the  irreversible  reactions  
o 3  irreversible  steps  in  glycolysis  which  must  be  bypassed  in  
gluconeogenesis  
• Different  tissues  will  utilise  gluconeogenesis  differently    
• Gluconeogenesis  is  coordinated  with  the  regulation  of  glycolysis  
o To  avoid  futile  cycling  
• Occurs  when  
o Glucose  supplies  are  limited  (during  meals,  fasts,  exercise)  
o Tissues  that  almost  completely  rely  upon  glucose  for  metabolic  
energy  
§ Brain,  nervous  system,  erythrocytes,  testes,  renal  medulla,  
embryonic  tissues  
§ Brain  alone  requires  120g  glucose  per  day  (60%  of  the  
glucose  stored  as  glycogen  in  muscle  and  liver)  
• Supply  of  glucose  from  glycogen  stores  is  not  always  sufficient  à  Need  a  
way  to  synthesise  glucose  
o Glycogen  stores  depleted  during  fasts/vigorous  exercise  
 
Gluconeogenesis  
• Formation  of  new  sugar  
• Process  that  converts  pyruvate  (and  related  3&4C  compounds)  to  glucose  
• Other  intermediates  may  also  serve  as  precursors  
o Glycolytic  intermediates  involved  in  gluconeogenesis  
• Occurs  mainly  in  the  liver  (kidney  and  small  intestine  too)  
o Liver  can  make  glucose  under  certain  conditions  
• Shares  many  steps  with  glycolysis  
• The  synthesis  of  glucose  from  non-­‐carbohydrate  precursors  
o Occurs  in  all  plants,  animals,  fungi  and  microorganisms  
• Important  precursors  of  glucose  in  animals  are  
o Pyruvate  
o Lactate  (During  vigorous  exercise,  the  lactate  produced  is  
converted  in  the  liver  back  to  glucose  via  TCA  cycle)  
o Certain  AA  (Feed  into  TCA  cycle)  
o Glycerol  
 
Glycolysis  vs
...
g  regulation  by  insulin  
o Insulin  released  in  the  fed  state  à  Glucose  taken  up  and  stored  
o Insulin  also  regulates  several  pathways  transcriptionally  
(participates  in  gene  regulation)  
o Up-­‐regulate  glycolytic  enzymes  
• Enzymes  involved  in  glycolysis/gluconeogenesis  regulated  by  insulin  
o Hexokinases  II  &  IV  
o PFK-­‐1  
o Pyruvate  kinase  
o PFK-­‐2/FBPase-­‐2  
 
Glycolysis  and  gluconeogenesis  are  reciprocally  regulated  by  allosteric  
modulators  
• F6Pà  F16BP  (glycolysis)  
• F16BP  à  F6P  (gluconeogenesis  via  FBPase-­‐1)  
o AMP  is  inhibitory  of  FBPase-­‐1  in  gluconeogenesis  (ensures  the  
pathways  do  not  occur  at  the  same  time)  
o ATP  is  a  negative  allosteric  regulator  of  glycolysis  
o ADP/AMP  are  positive  regulators  of  glycolysis    
• Not  opposite  reactions  
 
Glycolysis  and  gluconeogenesis  are  reciprocally  regulated  by  fructose-­‐2,6-­‐
bisphosphate  (F26BP)  
• Allosteric  regulation  of  F6P  (F6P  à  F26BP  via  PFK2)  

•
•
•

•
•

F26BP  not  a  glycolytic  intermediate  but  is  essential  for  regulation  
A  regulator  specifically  produced  to  regulate  glycolysis  and  
gluconeogenesis  
Levels  of  F26BP  important  for  regulating  glycolysis  
o Inhibit  FBPase-­‐1  
o Upregulates  PFK1  
F26BP  is  synthesised  from  F6P  by  PFK-­‐2  
Broken  down  by  fructose-­‐2,6-­‐bisphosphate  (FBPase-­‐2)  

 
Regulation  by  F26BP  
• Has  opposite  regulatory  effects  on  glycolysis  and  gluconeogenesis  
• F26BP  allosterically  regulates  fructose-­‐1,6-­‐bisphosphatase  in  a  similar  
manner  
o Activates  phosphofructokinase  (glycolysis)  
o Inhibits  fructose-­‐1,6-­‐bisphosphatase  (gluconeogenesis)  
• When  F26BP  binds  to  its  allosteric  site  on  FBPase-­‐1  
o Decreases  the  enzymes  affinity  for  F6P  
o Inhibited  by  as  little  as  1uM  F26BP  
o Increases  FBPase-­‐1  sensitivity  to  AMP  
• When  F26BP  binds  to  its  allosteric  site  on  PFK-­‐1  
o Increases  the  enzymes  affinity  for  F6P  
o Decreases  the  enzymes  affinity  for  ATP  
 
Regulation  of  F26BP  levels  
• PFK2  and  FBPase-­‐2  are  enzymes  on  the  same  protein    
o Make  the  same  non-­‐glycolytic  intermediate  F26BP  
• High  BG  à  Insulin  binds  to  cell  surface  à  De  phosphorylation  à  
Activates  PFK2  à  F26BP  à  Activates  PFK-­‐1  à  Glycolysis    
• Low  BG  (want  to  shut  down  glycolysis)  à  Glucagon  à  Signalling  cascade  
resulting  in  phosphorylation  of  cAMP  à  FBPase-­‐2    
• Increased  F26BP  stimulates  glycolysis  inhibits  glucongeogenesis  
• Decreased  F26BP  inhibits  glycolysis  and  stimulates  gluconeogenesis  
 
Summary  
• Gluconeogenesis  is  the  synthesis  of  glucose  from  non-­‐carbohydrate  
precursors  
o Pyruvate,  lactate,  amino  acids  and  glycerol  (but  not  fatty  acids  in  
animals)  
• Gluconeogenesis  and  glycolysis  share  7  common  steps,  but  not  those  that  
are  irreversible    
o Hexokinase  (step  1)  
o PFK-­‐1  (step  3)  
o Pyruvate  kinase  (step  10)  
§ These  reactions  are  bypassed  by  4  different  enzymes    
• Pyruvate  carboxylase  
• PEP  carboxykinase  
• Glucose-­‐6-­‐phosphatase    
• Frcutose-­‐1,6-­‐bisphosphatase    

Conversion  of  pyruvate  to  PEP  has  two  alternate  pathways,  
pyruvate  or  lactate,  depending  on  tissue  
Gluconeogenesis  is  energetically  expensive  but  necessary  for  specific  
tissues  
Regulation  of  gluconeogenesis  is  coordinated  with  glycolysis  to  prevent  
wasteful  cycling  of  ATP  
Glycolysis  and  gluconeogenesis  are  reciprocally  regulated  by  fructose-­‐2,6-­‐
bisphosphate  
§

•
•
•

 
Glycogen    
• Glycogen  is  a  glucose  storage  molecule  
• Cells  triggered  by  insulin  to  take  up  glucose  à  Stimulates  synthesis  of  
glycogen  
• Polymer  of  alpha(1-­‐4)  linked  subunits  of  glucose  with  alpha(1-­‐6)  linked  
branches  
• Each  chain  has  12-­‐14  glucose  residues  
• Glycogenin  protein  in  the  centre  
o Glycogenin  has  sugars  attached    
 
Glycogen  as  a  source  of  fuel  in  animals  
• Source  of  fuel  in  animals  (starch  in  plants)  
• Skeletal  muscle  
o <2%  wet  weight    
o Major  source  of  glucose  for  contraction  
• Liver  
o <10%  wet  weight  
o Plays  a  role  in  maintaining  blood  glucose  levels  
o Provides  glucose  to  other  tissues  between  meals  or  fasting  
(especially  the  brain)  
• The  total  amount  of  energy  stored  is  less  than  that  of  triacylglycerols  
(fatty  acids)  
• Muscle  glycogen  can  be  exhausted  in  less  than  1  hour  during  vigorous  
activity  (depleted  relatively  quickly;  the  liver  is  richer  in  glycogen)  
• Liver  glycogen  can  be  depleted  in  12-­‐24  hours  
o After  24  hours  when  glycogen  stores  are  depleted  you  begin  to  
break  down  proteins  and  eventually  fatty  acids  (which  cannot  be  
converted  to  glucose,  which  is  required  for  nervous  function)  
• Fatty  acids  cannot  be    
o Converted  to  glucose  in  mammals  
o Metabolised  anaerobically  
o Used  as  fuel  for  the  neurons  of  the  brain  
 
Why  not  store  monomeric  glucose?  
• Glycogen  stored  in  cells  at  0
...
4M  (places  osmotic  stress  on  cells)  
• Glucose  would  dominate  the  osmotic  properties  of  the  cell  leading  to  
osmotic  entry  of  water  into  cell  and  subsequent  cell  lysis  

•

Prohibits  glucose  uptake  (as  extracellular  glucose  is  5mM)  

 
Glycogen  beta-­‐particles  
• Elementary  glycogen  particles  (or  granules)  
• Often  associated  with  endoplasmic  reticulum  tubules  
• Each  glycogen  molecule  consist  of    
o 55000  glucose  molecules  
o 2000  non-­‐reducing  ends  
 
Glycogen  degradation  
• Requires  4  enzyme  activities  
• Glycogen  phosphorylase  à  Degrades  glycogen  
• Glycogen  debranching  enzyme  
o Catalyses  two  reactions  
o Remodels  glycogen  for  further  degradation  
• Phosphoglucomutase  à  Converts  the  breakdown  product  into  a  form  
suitable  for  further  degradation  
 
Reducing  sugars  
• Interconversion  between  alpha  and  beta    
• Glucose  is  an  aldose  (aldehyde  group)  
• Have  a  free  aldehyde  or  ketone  in  the  open  chain  form  
• Undergo  oxidation  by  oxidising  substances  
• Non-­‐reducing  sugars  lack  a  free  aldehyde  or  ketone  and  are  unable  to  
undergo  oxidation  (sucrose)  
 
Water  as  a  reactant  
• Water  can  participate  directly  in  chemical  reactions  
• Condensation  reaction  à  Formation  of  a  bond  between  OH  and  OH,  
expelling  water  
• Hydrolysis  reaction  à  Cleavage  of  this  bond  accompanied  by  addition  of  
water  
 
Glycogen  phosphorylase  
• Catalyses  the  breakage  of  the  alpha(1-­‐4)  linkages  between  glucose  
subunits  at  a  non-­‐reducing  end  
• Uses  Pi  and  incorporates  phosphate  into  the  glucose  molecule  so  it  is  
liberated  from  the  molecule  (G1P  released)  
• Phosphorylase  stops  when  within  4  subunits  of  an  alpha(1-­‐6)  link  due  to  
hindrance  
 
Dealing  with  branch  points  
• Glycogen  phosphorylase  acts  repeatedly  to  remove  glucose  subunits  à  
Produces  glucose-­‐1-­‐phosphate  
• Stops  when  within  4  subunits  of  an  alpha(1-­‐6)  linkage  
• Debranching  enzyme  catalyses  two  successive  reactions  
o Transfer  three  glucose  subunits  to  a  nearby  non-­‐reducing  end  
(move  a  branch  point  along  the  chain)  

o Cleaves  the  remaining  alpha(1-­‐6)  linked  glucose  

 
Glucose-­‐1-­‐phosphate  must  be  isomerised  to  glucose-­‐6-­‐phosphate  for  metabolism  
• Phosphoglucomutase  
o Catalyses  the  reversible  conversion  of  G6P  à  G1P  
o Last  step  of  glycogen  breakdown  (G1P  à  G6P  which  is  the  2nd  
intermediate  in  glycolysis  and  can  be  used  to  make  pyruvate)  
o First  step  of  glycogen  synthesis  (G6P  à  G1P)  
 
Metabolism  of  glucose-­‐6-­‐phosphate  
• Muscle  glucose-­‐6-­‐phosphate  enters  glycolysis  to  serve  as  an  energy  
source  
• In  a  liver  cell,  G6P  à  Glucose  (can  export  glucose  by  hydrolysing  the  Pi  on  
G6P  to  make  glucose)  
• Liver  and  kidney  glucose-­‐6-­‐phosphate  is  dephosphorylated  to  glucose  
and  released  into  the  blood  
 
Glycogen  synthesis  requires  three  enzyme  activities  (not  including  G6P  à  G1P  
conversion  by  mutase)  
• UDP-­‐glucose  phosphorylase  à  Forms  activated  glucose  (UDP  glucose)  
o Activated  glucose  used  to  make  glycogen  
• Glycogen  synthase  à  Adds  activated  glucose  to  growing  glycogen  
polymer  
o Grows  a  chain  of  alpha  (1-­‐4)  molecules  
• Glycogen  branching  enzyme  à  Introduces  alpha  (1-­‐6)  branch  points  
 
Glycogen  synthesis  
• Glycogen  degradation  and  synthesis  uses  different  pathways  
o Different  enzymes  allows  us  to  regulate    
• Glycogen  synthesis  uses  the  glucose  of  UDP  glucose,  the  activated  form  of  
glucose  
o UDP  also  seen  in  RNA  
o Contains  a  ribose  molecule  
o UDP  acts  as  a  handle  on  glucose  
 
Activated  sugar  nucleotides  
• Most  reactions  in  which  hexoses  are  transformed/polymerised  involve  
sugar  nucleotides  
o The  anomeric  carbon  of  sugar  is  activated  by  attachment  to  a  
nucleotide  
• Sugar  nucleotides  are  the  substrates  for  the  polymerisation  of  
monosaccharides  into  many  different  polysaccharides  
• Suitability  for  biosynthetic  reactions  
o Formation  metabolically  irreversible  
o Reactivity  of  the  nucleotide  group  
o Nucleotidyl  group  is  a  good  leaving  group  
o Tagging  of  sugars  allows  for  dedication  to  specific  purposes  
 
Formation  of  activated  sugars  

•
•

G1P  made  from  G6P  via  phosphoglucomutase  
G1P  +  UTP  à  UDP-­‐glucose  +  PPi  (via  UDP-­‐glucose  phosphorylase)  

 
Glycogen  synthase  
• Sugar  unit  transferred  onto  non-­‐reducing  end  of  growing  glucose  polymer  
to  make  a  new  alpha  1-­‐4  linkage  
• UDP  used  as  carrier  for  glucose  and  can  be  recycled  
• Catalyses  the  addition  of  UDP  glucose  to  a  non-­‐reducing  end  of  glycogen  
to  make  a  new  alpha  (1-­‐4)  linkage  
 
Glycogen  branching  enzyme  
• Takes  alpha  1-­‐4  units  and  shifts  them  to  generate  branch  points  
• Branch  points  useful  for  quick  breakdown  (lots  of  non-­‐reducing  ends  
available)  
• Catalyses  the  transfer  of  a  terminal  fragment  of  glucose  residues  from  the  
non-­‐reducing  end  of  a  chain  to  the  C6  OH  group  of  a  more  interior  glucose  
residue  of  the  same  or  another  glycogen  chain  to  form  a  new  branch  
 
Glycogenin  facilitates  the  formation  of  new  glycogen  chains  
• Glycogen  synthase  cannot  initiate  a  new  chain  
o Requires  a  preformed  alpha  (1-­‐4)  polyglucose  chain  (>8  residues)  
• Glycogenin  is  both  an  enzyme  and  a  primer  
o Catalyses  addition  of  glucose  residues  onto  itself  (used  by  glycogen  
synthase)  
• New  glycogen  chains  begin  with  the  autocatalytic  transfer  of  UDP-­‐glucose  
to  glycogenin,  followed  by  several  additions  of  glucose  residues  to  form  a  
primer  that  can  be  acted  upon  by  glycogen  synthase  
• Each  chain  has  12-­‐14  glucose  residues  
• Primer  à  Short  oligomer  of  sugars  (or  nucleotides)  to  which  an  enzyme  
adds  additional  monomeric  subunits  
 
Glycogen  synthesis  summary  
• Glycogenin  forms  a  primer  of  8-­‐12  alpha  (1-­‐4)  linked  glucose  units  
• UDP  glucose  pyrophosphorylase  synthesises  UDP  glucose  from  UTP  and  
G1P  
• Glycogen  synthase  catalyses  the  addition  of  UDP  glucose  to  a  non-­‐
reducing  end  of  glycogen  to  make  a  new  alpha  (1-­‐4)  linkage  
• Glycogen  branching  enzyme  catalyses  the  transfer  of  a  terminal  fragment  
of  glucose  to  a  non  reducing  end  of  a  chain  to  the  C6  OH  group  of  a  more  
interior  glucose  residue  of  the  same  or  another  glycogen  chain  to  form  a  
new  branch  (alpha  1-­‐6)  
 
Glycogen  breakdown  summary  
• Glycogen  phosphorylase  breaks  alpha  (1-­‐4)  linked  glucose  units  to  
produce  G1P  
o Important  for  regulation  
o Phosphorylates  the  alpha  1-­‐4  residues  
• De-­‐branching  enzyme  catalyses  two  successive  reactions  

•

o Transfer  three  glucose  subunits  to  a  nearby  non-­‐reducing  end  
o Cleaves  off  the  remaining  alpha  (1-­‐6)  linked  glucose  
Phosphoglucomutase  converts  G1P  to  G6P  

 
Glycogen  phosphorylase  is  regulated  allosterically  and  hormonally  
• Phosphorylase  a  and  b  (different  forms)  
o Phosphorylase  b  is  less  active  
o Phosphorylase  b  kinase  phosphorylates  phosphorylase  b  into  
phosphorylase  a  (more  active  form)  
o Phosphorylase  a  phosphatase  dephosphorylates  phosphorylase  a  
into  phosphorylase  b  
o Glucagon/epinephrine  signals  glycogen  breakdown  
o Regulate  glycogen  synthesis  and  breakdown  hormonally  
• Glucagon/epinephrine  signalling  pathway  
o Starts  phosphorylation  cascade  via  cAMP  
o Activates  glycogen  phosphorylase  
• Glycogen  phosphorylase  cleaves  glucose  residues  off  glycogen,  generating  
G1P  (which  is  then  converted  to  G6P  and  fed  through  glycolysis)  
 
Epinephrine  (adrenaline)  and  glucagon  induce  glycogen  degradation  
(glycogenolysis)  
• Epinephrine/glucagon  initiates  a  signalling  cascade  
• Adenylate  cyclase  à  Cyclic  AMP  à  Activates  protein  kinase  A  à  
Phosphorylates  phosphorylase  kinase  à  Phosphorylase  kinase  
phosphorylates  phosphorylase  b  à  a  (active)  
 
Glycogen  phosphorylase  in  liver  as  a  glucose  sensor  
• Regulated  allosterically  by  glucose  
• Binding  of  glucose  to  an  allosteric  site  on  phosphorylase  a  induces  a  
conformational  change  
o Exposes  phosphorylated  serine  side  chains  to  the  action  of  
phosphorylase  a  phosphatase  (PP1)  
• Phosphorylase  a  phosphatase  dephosphorylates  phosphorylase  a  à  
phosphorylase  b  (less  active)  
 
Carbohydrate  metabolism  differs  between  tissues  
• Liver  
o Liver  has  different  proteins  for  glycolysis  (specific  glucokinase  
isozyme)  
o Liver  able  to  release  glucose  into  bloodstream  (breaks  down  G6P  
à  glucose)  whereas  other  tissues  use  G6P  into  glycolysis  and  
make  pyruvate  
 
Epinephrine  (adrenaline)  and  glucagon  induce  glycogen  degradation  and  inhibit  
glycogen  synthesis  
• Protein  kinase  A  phosphorylates  glycogen  synthase  which  inactivates  it  
 
Insulin  induced  glycogen  synthesis    

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•
•
•

When  BG  levels  are  high  insulin  stimulates  glycogen  synthesis  by  
inactivation  of  glycogen  synthase  kinase  3  
Glycogen  synthase  kinase  phosphorylates  glycogen  synthase  (inactive)  
Phosphatase  activates  glycogen  synthase  
Inactivation  of  glycogen  synthase  kinase  allows  PP1  to  dephosphorylate  
and  activate  glycogen  synthase  

 
Phosphoprotein  phosphatase  1  is  central  to  glycogen  metabolism  
• PP1  can  remove  phosphoryl  groups  from  all  three  enzymes  
phosphorylated  in  response  to  glucagon/epinephrine  
o Phosphorylase  kinase  
o Glycogen  phosphorylase  
o Glycogen  synthase  
• PP1  Is  tightly  bound  to  its  targets  via  glycogen  targeting  protein  (Gm)  
o Binds  glycogen  and  above  3  enzymes  
• PP1  is  itself  subject  to  covalent  and  allosteric  regulation  
 
These  allosteric  and  hormonal  signals  coordinate  carbohydrate  metabolism  
globally    
 
Summary  
• Glycogen  is  a  polymer  of  alpha  (1-­‐4)  linked  subunits  of  glucose,  with  
alpha  (1-­‐6)  linked  branches  constructed  around  a  core  primer  based  on  
the  enzyme  glycogenin  
• Glucose  6  phosphate  is  converted  to  glucose-­‐1-­‐phosphate  by  
phosphoglucomutase  (reversibly),  before  activation  via  the  addition  of  a  
nucleotide,  forming  UDP  glucose  –  the  building  block  of  glycogen  
• Glycogen  branching  and  debranching  enzymes  are  responsible  for  
forming  and  degrading/remodelling  the  alpha  (1-­‐6)  linked  branches  
• Epinephrine  and  glucagon  induce  glycogen  breakdown  (via  
phosphorylase  kinase  which  phosphorylates/activates  phosphorylase)  
and  impair  glycogen  synthesis  (via  phosphorylating/inactivating  glycogen  
synthase)  
• Insulin  and  the  availability  of  glucose  induces  glycogen  synthesis,  with  
phosphoprotein  phosphatase  1  (PP1)  dephosphorylating/activating  
glycogen  synthase,  and  dephosphorylating/inactivating  phosphorylase  
 
Type  I  diabetes  
• Results  from  the  autoimmune  destruction  of  the  insulin  producing  beta  
cells  in  the  pancreas  
o Cells  destroyed  by  autoimmune  processes  
o Glucose  metabolism  limited  
• Manifests  itself  in  childhood  (juvenile  diabetes)  
• Metabolism  of  glucose  is  limited  by  the  rate  of  uptake;  glucose  uptake  is  
deficient  in  type  I  diabetes  
• Classical  symptoms  
o Polyuria  (frequent  urination)  
o Polydipsia  (increased  thirst)  

•
•

o Polyphagia  (increased  hunger)  
o Weight  loss  
BG  builds  up  to  high  levels  à  Increased  urination  à  Increased  thirst  
Cells  unable  to  take  up  glucose  à  Increased  hunger  

 
Glucose  uptake  
• Mediated  by  GLUT  transporters  
• In  some  cells  GLUT  transporters  are  always  present  
o GLUT1  (ubiquitous;  in  all  cell  types)  
o GLUT2  (liver)  
o GLUT3  (brain)  
§ Always  expressed  à  Brain  reliant  on  glucose  as  an  energy  
source  
§ Other  transporters  are  regulated  
• In  skeletal  muscle,  cardiac  muscle  and  adipose  tissue,  GLUT4  is  
sequestered  in  vesicles  and  moves  to  the  plasma  membrane  in  response  
to  insulin  
o Recruited  into  membrane  under  insulin  signal  
 
Effects  of  type  I  diabetes  on  CHO  and  fat  metabolism  
• Pancreas  fails  to  secrete  insulin  
• Insulin  receptor  not  activated  
• Failure  to  recruit  GLUT4  transporters  in  membrane  (in  skeletal  muscle,  
cardiac  muscle  and  adipose  tissue)  
• Glucose  not  taken  up  by  cells  
• Insufficient  glycolysis  (due  to  insufficient  glucose)  
• Insufficient  TCA  cycle  and  oxidative  phosphorylation  
• Triglyceride  breakdown  and  fatty  acid  oxidation    
 
Inborn  errors  of  metabolism  
• Rare  genetic  disorders  that  affect  enzymes  in  metabolism  
• Gene  mutations  à  Individual  enzymes  affected  
• Body  cannot  properly  turn  food  into  energy  (enzymes  defective)  
• Usually  caused  by  defects  in  specific  proteins  (enzymes)  that  help  break  
down  (metabolise)  parts  of  food  
 
Glycolysis  in  disease  
• Glycolytic  mutations  are  relatively  rare  due  to  importance  of  this  pathway  
(produces  pyruvate  for  TCA  and  precursors  for  other  biosynthetic  
pathways)  
• Majority  of  mutations  in  enzymes  à  Cells  cant  respire  à  Cell  death  
 
Pyruvate  kinase  deficiency  
• Final  step  of  glycolysis  (phosphoenolpyruvate  +  ADP  à  Pyruvate  +  ATP)  
• Erythrocytes  (RBCs)  obtain  all  their  ATP  from  glycolysis  
• A  deficiency  in  pyruvate  kinase  (PK)  results  in  erythrocytes  with  
decreased  energy  

•

Build  up  of  glycolytic  intermediates  can  increase  the  level  of  2,3-­‐
bisphosphoglycerate  (23BPG)  à  Decreases  affinity  of  haemoglobin  for  O2  
à  Affects  tissue  oxygenation  

 
Phosphofructokinase-­‐1  (PFK1)  and  cancer  
• Altered  glycolytic  flux  is  a  hallmark  of  cancers  
• PFK1  proposed  to  have  important  roles  in  metabolic  reprogramming  in  
cancer  
• Cancer  mutations  of  PFK  alter  enzymatic  activity  and  allosteric  regulation  
o R48C  mutant  has  reduced  citrate  inhibition  (citrate  regulates  
PFK1)  
o N426S  mutant  partly  relieves  ATP  inhibition  
• Inhibition  of  glycolytic  flux  by  loss  of  function  mutations  may  confer  a  
selective  advantage  for  cancer  cell  growth  and  metastasis  by  redirecting  
carbon  flow  through  the  pentose  phosphate  pathway  
o Directing  glucose  through  PPP    
 
Mitochondrial  diseases  
• Where  Krebs  and  oxidative  phosphorylation  occurs  
• Mitochondria  has  its  own  DNA    
• Mutations  of  mitochondrial  proteins  à  Decrease  in  ATP  (as  mitochondria  
is  a  major  ATP  source)  
• Mitochondrial  or  nuclear  genetic  defects  involving  enzymes  used  in  
oxidative  phosphorylation  impair  cellular  respiration;  decreasing  the  
ATP:ADP  ratio  
• Tissues  with  high  energy  demand  are  particularly  vulnerable  (brain,  
nerves,  retina,  skeletal,  cardiac  muscle)  
• Red  ragged  fibres  à  Clumps  of  diseased  mitochondria  
 
Point  mutations  in  protein  coding  genes  
• Complex  I  mutations  à  Leber’s  hereditary  optic  neuropathy  
o Not  a  multisystem  disorder  but  a  highly  selective  degeneration  of  
the  optic  nerve  
• Cytochrome  b  (part  of  complex  III)  
o Cytochrome  b  is  an  electron  carrier  
o Exercise  intolerance  
o Myalgia  
• Complex  IV  (cytochrome  c  oxidase,  COX)  
o Only  a  few  patients  with  multisystem  disorders  
• Complex  V  (ATP  synthase)  
o Neuropathy  
o Ataxia  and  retinitis  pigmentosa  (decreased  ATP  synthesis  rate  and  
decreased  stability  of  the  F0F1  ATPase)  
o More  severe  phenotype  is  called  Leigh  syndrome  
 
Leber’s  hereditary  optic  neuropathy  
• Degeneration  of  retinal  ganglion  cells  and  their  axons  that  leads  to  loss  of  
central  vision  (predominantly  young  adult  males)  

•

•

Transmitted  through  the  mother  as  it  is  primarily  due  to  mutations  in  the  
mitochondrial  genome  (only  the  egg  contributes  mitochondria  to  the  
embryo)  
LHON  is  usually  due  to  one  of  three  pathogenic  mitochondrial  DNA  
(mtDNA)  point  mutations  in  the  ND4,  ND1  and  ND6  subunit  genes  of  
complex  I  of  the  oxidative  phosphorylation  chain  

 
Leigh  syndrome  
• About  20-­‐25%  of  sufferers  have  a  mutation  in  mtDNA  
• May  have  the  following  symptoms  à  Loss  of  appetite,  vomiting,  failure  to  
thrive,  irritability/continuous  crying,  delayed  motor  and  language  
milestones,  seizures,  generalised  weakness,  decreased  muscle  tone,  
tremor,  problems  with  muscle  coordination,  abnormal  eye  movements,  
vision  problems,  heart  problems  
• Lactic  acidosis  due  to  TCA  insufficiency  à  kidney  problems  (pyruvate  
converted  to  lactate  instead)  
• Some  patients  have  deficient  activity  in  pyruvate  dehydrogenase  
o Step  before  entry  to  TCAs  
o Produces  acetyl  CoA  which  is  imported  into  TCA    
• Knowledge  of  affected  genes  mean  you  can  bypass  the  pathway  as  
treatment  
 
MERRF  syndrome  (Myoclonic  epilepsy  with  ragged  red  fibres)  
• Symptoms  à  Muscle  twitches  (myoclonus),  weakness  (myopathy),  and  
progressive  stiffness  (spasticity)  
• Muscle  cells  have  red  ragged  fibres  (diseased  mitochondria)  
• Caused  by  mutations  in  mitochondrial  tRNA-­‐lysine    
 
Fructose  bisphosphate  deficiency  
• Deficiency  of  fructose-­‐1,6-­‐bisphosphate,  one  of  the  enzymes  in  
gluconeogenesis  (Opposite  to  PFK1  in  glycolysis)  
• In  FBP  deficiency  
o There  is  not  enough  fructose  bisphosphate  for  gluconeogenesis  to  
occur  correctly  
o As  three  carbon  molecules  cannot  be  used  to  make  glucose  they  
will  instead  by  made  into  pyruvate  and  lactate  
o May  cause  hypoglycaemia  and  lactic  acidosis  
o Glycolysis  will  still  work,  as  it  does  not  use  this  enzyme  
 
Glycogen  storage  diseases  
• Due  to  defects  in  glycogen  synthesis  or  breakdown  
• Enzymes  affected  may  include  glycogen  synthase,  G6P,  branching  enzyme,  
debranching  enzyme,  phosphorylase  kinase,  GLUT2  and  others  
• Type  la  (Von  Gierke  disease)  
o Most  common  type  of  glycogen  storage  disease  
o Caused  by  mutations  in  G6P  à  Enlarged  liver,  kidney  failure,  
shortage  of  white  blood  cells  (neutropenia)  
§ In  most  cell  types  G6P  can  enter  glycolysis  

§

Liver  must  be  able  to  export  glucose  

 
Altitude  sickness  
• Increased  altitude  is  coupled  with  decreased  atmospheric  pressure  
therefore,  for  every  breath  inhaled  there  is  less  O2  available  à  Bodies  are  
forced  to  work  harder  to  metabolise  
• Respiration  must  increase  so  that  our  muscles  and  brain  can  get  enough  
O2  for  electron  transport,  so  that  enough  ATP  can  be  made  for  energy  
usage  
• The  body  may  overcompensate  by  sending  too  much  blood  to  the  brain  à  
swelling  (cerebral  oedema)  à  Dilation  of  the  blood  vessels  leading  to  the  
head  and  neck  causes  headaches  and  brain  swelling  leads  to  imbalance,  
vomiting,  hallucinations  
• Increased  blood  flow/pressure  can  also  cause  leakage  from  the  blood  
vessels  into  the  alveolar  sacs  of  the  lung  causing  difficulty  in  breathing,  
gurgling  sounds  in  the  lung,  coughing,  exhaustion  à  Body  compensates  
by  increasing  HR  and  BP,  forcing  more  fluid  into  the  lungs  à  Drown  
 
Oxygen  deprivation  in  heart  attack  (myocardial  infarction)  
• Interruption  of  blood  supply  to  a  portion  of  the  heart  
• In  the  absence  of  O2  the  cell  must  rely  on  glycolysis  for  ATP  production  à  
glycolysis  does  not  produce  much  ATP  
• Stores  of  glycogen  and  phosphocreatine  are  rapidly  depleted  
o Phosphocreatine  is  a  backup  energy  storage  molecule  
o Creatine  kinase  catalyses  conversion  of  creatine  to  
phosphocreatine  
• ATP  becomes  too  low  to  maintain  membrane  ion  pumps  
• Osmotic  balance  is  upset  and  cellular  organelles  begin  to  swell  and  leak  
 
Creatine/phosphocreatine  
• Phosphocreatine  (creatine  phosphate)  is  a  readily  available  source  for  
replenishing  ATP  
• Acted  on  by  creatine  kinase  to  release  ATP  
 
Hypoxic/ischemic  injury  
• Accumulation  of  succinate  in  ischaemic  cells  
• Leads  to  loss  of  oxidative  phosphorylation  and  reduction  in  ATP  
production  which  causes  
o Failure  of  ATP  dependent  Na/K  pumps  and  Ca2+  pump  that  
normally  maintains  high  cell  K+  and  low  cell  Na+  and  Ca2+  
o K+  decreases  and  Na+/Ca2+  increase  in  cell  
o Na+  brings  water  into  cell  à  swelling  
o Ca2+  causes  activation  of  phospholipases  that  disrupt  the  
membrane  and  is  an  important  signalling  molecule  
• Decrease  in  ATP  à  Increased  glycolysis  à  Decreased  pH  from  lactic  acid  
build  up  within  the  cell  and  decreases  glycogen  stores  
• During  ischemia  succinate  accumulates  

•

During  reperfusion  succinate  is  consumed  à  Too  much  QH2  produced  à  
Drives  reverse  electron  transport  at  complex  I  à  Drives  ROS  production  
at  complex  I  à  Cell  damage  

 
Summary  
• Type  I  diabetes  results  from  inability  to  produce  insulin  
• Type  I  diabetes  impacts  on  glucose  uptake  by  skeletal,  cardiac,  muscle  
and  fat  cells  
• Inborn  errors  in  glycolytic  enzymes  are  rare  but  found  
• Mitochondrial  diseases  are  a  group  of  diseases  resulting  from  mutation  of  
proteins  important  in  electron  transport  and  other  processes  in  the  
mitochondria  
o Leber’s  hereditary  optic  neuropathy  
o Leigh  syndrome  
o MERRF  sundrome  
• Other  inborn  errors  in  metabolism  include  fructose  bisphosphate  
deficiency  and  glycogen  storage  diseases  
• At  high  altitude,  O2  deficiency  can  lead  to  increased  HR,  brain  swelling,  
leakage  of  blood  vessels  of  alveolar  sacs  of  lung  and  death  
• HR  leads  to  O2  deficiency,  rapid  depletion  of  ATP  and  energy  stores  and  
breakdown  in  osmotic  balance  of  cells  
 
Pentose  phosphate  pathway  
• Produces  NADPH  and  ribose-­‐5-­‐phosphate  
o NADPH  similar  in  chemical  properties  to  NADH  (source  of  
reducing  power)  
o NADH  occurs  in  oxidative  and  energy  metabolism,  NADPH  electron  
donor  in  biosynthesis  
• Required  for  reducing  power  and  nucleotide  precursors  
• NADPH  is  an  electron  donor  
o Reductive  biosynthesis  of  fatty  acids  and  steroids  
o Repair  of  oxidative  damage  
• Ribose-­‐5-­‐phosphate  is  a  biosynthetic  precursor  of  nucleotides  
o Used  in  DNA  and  RNA  synthesis  
o Used  in  synthesis  of  some  coenzymes  (eg  Coenzyme  A)  
o We  need  ribose  to  make  NAD  and  NADP  (cofactors)  
o Ribose  at  the  heart  of  ATP  
o Ribose  linked  to  phosphates  and  base  in  RNA  
§ Ribose  in  RNA  has  an  extra  OH  at  C2  
§ Deoxyribose  in  DNA  does  not  à  Increased  flexibility  
 
NADPH  vs
...
 PPP  
• Oxidative  phase  à  2  NADPH  +  loss  of  CO2  à  C5  sugar  produced  
• Carbons  shuffled  around  in  non-­‐oxidative  steps  provide  intermediates  for  
glycolysis  
• Glucose  à  G6P  (via  hexokinase)  
• G6P  à  Glycolysis  or  PPP  
• NADPH  negatively  feedsback  to  the  first  step  (G6P  à  6-­‐
phosphogluconolactone  via  glucose-­‐6-­‐dehydrogenase)  
o Product  inhibits  its  own  production  
 
G6P  dehydrogenase  deficiency  
• Can  be  fatal  in  cases  of  high  oxidative  stress  (certain  drugs,  herbicides,  
some  foods  cause  oxidative  damage)  
• Need  glutathione  and  NADPH  for  protection  from  oxidative  stress  
• Confers  resistance  to  malaria  due  to  high  oxidative  stress  in  RBCs  (as  can  
sickle  cell  anaemia  à  Deficiency  in  haemoglobin)  
o Selection  pressure  to  maintain  the  deficiency  in  the  population  
• Side  product  of  oxygen  metabolism  is  radicals  à  Hydrogen  peroxide  
• Glutathione  peroxidase  uses  glutathione  to  breakdown  hydrogen  
peroxide  à  If  deficient  in  G6P  dehydrogenase  à  Form  hydroxyl  radicals  
from  hydrogen  peroxide  à  Oxidative  damage  to  lipids,  proteins,  DNA  

•

•

•

Primaquine  and  compounds  found  in  fava  beans  lead  to  increased  
amounts  of  peroxides  and  other  reactive  oxygen  species  (cause  oxidative  
damage)  
If  people  with  G6PD  deficiency  eat  fava  beans  à  Deficient  in  G6P  
dehydrogenase  à  Not  enough  NADPH  à  Oxidative  stress  à  Hydroxyl  
radicals  
o Erythrocytes  lyse  
o Haemoglobin  is  released  into  the  blood  
o Jaundice  and  kidney  failure  can  result  
G6PD  deficiency  impairs  the  ability  of  red  blood  cells  to  maintain  their  
levels  of  reduced  glutathione  and  therefore  their  ability  to  deal  with  
oxidative  stress  

 
What  happens  in  the  erythrocytes?  
• Hemoglobin  (Fe2+)  à  oxidation  à  Methaemoglobin  (Fe3+)  
o In  order  to  carry  O2,  the  heme  iron  must  be  2+  
o Methaemoglobin  cannot  carry  oxygen  
o If  we  are  able  to  make  NADPH  we  can  reduce  Fe3+  to  Fe2+  
• Methaemoglobin  signifies  damage,  cells  destroyed  
 
Summary  
• The  PPP  is  a  process  by  which  cells  can  generate  reducing  power  
(NADPH)  that  is  needed  for  
o The  biosynthesis  of  various  compounds  
o Reduction  of  oxidised  glutathione    
• Produces  ribose-­‐5-­‐phosphate  
• The  non-­‐oxidative  phase  of  the  PPP  can  convert  pentose  phosphates  back  
to  G6P  and  other  glycolytic  intermediates  
• A  deficiency  in  the  first  enzyme  in  the  pathway  (glucose  6  phosphate  
dehydrogenase)  leads  to  a  decreased  amount  of  reduced  glutathione,  and  
increased  susceptibility  to  oxidative  damage,  particularly  in  erythrocytes  
(red  blood  cells)  
 
Revision  
• GSH  (glutathione)  
o Gamma-­‐glutamate  +  cysteine  +  glycine  
o SH  group  from  cysteine  
• Peroxidase  reaction  (H2O2  (hydrogen  peroxide)  à  H2O)  via  the  reducing  
power  of  glutathione  
o H2O2  +  H+  +  2e-­‐  à  2H2O    
• Glutathione  S-­‐transferase  takes  glutathione  and  conjugates  it  to  various  
toxins  with  nucleophilic  centres    
o By  conjugating  a  toxic  molecule  to  glutathione  it  becomes  more  
soluble  and  the  body  can  then  remove  compounds  conjugated  to  
glutathione  from  the  cell  
 
Nitrogen  metabolism:  Amino  acids  
• Roles  of  amino  acids  
o Energy  production  

o
o
o
o

Prophyrin  synthesis  
Neurotransmitter  and  hormone  synthesis  
Glutathione  synthesis  
Nitric  oxide  synthesis  

 
Importance  of  Nitrogen  in  biochemistry  
• Nitrogen  (with  H,  O  and  C)  is  a  major  elemental  constituent  of  living  
organisms  
• Mostly  in  nucleic  acids  and  proteins  
• Also  found  in  
o Several  cofactors  (NAD,  FAD,  biotin)  
o Small  hormones  (epinephrine)  
o Neurotransmitters  (serotonin)  
o Pigments  (chlorophyll)  
o Defense  chemicals  (amanitin)  
 
Biochemistry  of  molecular  nitrogen  
• N2  comprises  80%  of  the  Earth’s  atmosphere  but  is  virtually  unusable  by  
most  organisms  due  to  the  highly  resistant  N-­‐N  bond  (tightly  bound)  
o Resistant  to  modifications  so  needs  to  be  extracted    
o Hydrogen  molecules  added  à  Ammonium  (NH3)  
§ Need  N2  +  3H2  à  2NH3  (NH3  +  H2O  à  NH4+  +  OH-­‐)  
o Nitrogen  fixing  bacteria  and  archea  convert  N2  to  NH3  
(ammonium)  by  a  process  called  nitrogen  fixation  
o Some  N  fixing  microbes  live  in  symbiosis  with  plants  
(proteobacteria  with  legumes  such  as  peanuts,  beans)  
o A  few  live  in  symbiosis  with  animals  (spirochaete  with  termites)  
o Combined,  represents  60%  of  newly  fixed  N2  (fixed  from  the  air)  
• A  few  non-­‐biological  processes  convert  N2  to  biologically  useful  forms  
o N2  and  O2  à  NO  via  lightening  (represents  25%  of  newly  fixed  
N2)  
o NO  can  be  incorporated  into  biological  system  
o N2  and  H2  à  NH3  via  the  industrial  Haber  process    
§ Requires  T>400C,  P>200  atm  
§ Represents  15%  of  newly  fixed  N2  
 
The  Nitrogen  cycle  
• Chemical  transformations  maintain  a  balance  between  N2  and  
biologically  useful  forms  of  nitrogen  
o Fixation  à  Bacteria  reduce  N2  to  NH3/NH4+    
o Nitrification  à  Bacteria  oxidise  ammonia  into  nitrite  (NO2-­‐)  and  
nitrate  (NO3-­‐)  
o Assimilation  à  Plants  and  microorganisms  reduce  NO2-­‐  and  NO3-­‐  
to  
§ NH3  via  nitrite  reductases  and  nitrate  reductases  
§ NH3  (from  either  fixation  or  assimilation)  is  incorporated  
into  amino  acids  
§ Organisms  die,  returning  NH3  to  soil  
§ Nitrifying  bacteria  again  convert  NH3  to  nitrite  and  nitrate  

o Denitrification  à  Nitrate  (NO3-­‐)  is  reduced  to  N2  under  anaerobic  
conditions  
§ NO3-­‐  is  the  ultimate  electron  acceptor  instead  of  O2  
 
The  nitrogen  cycle  
•  Amino  acids  and  other  reduced  N-­‐C  compounds  degraded  by  animals  à  
NH4+  (ammonia)  
• NH4+  nitrification  by  bacteria  and  archea  à  NO2-­‐  (nitrite)  à  Nitrifying  
bacteria  à  NO3-­‐  (nitrate)  
• NO3-­‐  (nitrate)  can  be  denitrified  by  bacteria,  archea  and  fungi  to  N2  à  
Nitrogen  fixing  bacteria  and  archea  à  NH4+  ammonia)  à  NH4+  
incorporated  into  biological  system  (AA  etc)  
 
Ammonia  is  incorporated  into  biomolecules  via  glutamate  and  glutamine  
• Glutamate  and  glutamine  are  the  crucial  entry  point  for  the  NH4+  
• In  bacteria  and  plants  (but  not  mammals),  glutamate  synthase  catalyses  
the  incorporation  of  ammonia  (NH4+)  into  alpha-­‐ketoglutarate  to  
generate  glutamate  
• Alpha  ketoglutarate  +  NH4+  forms  glutamate  (driven  by  NADPH)  
o Reaction  does  not  require  ATP  (it  is  part  of  the  other  coupled  
reaction  in  the  equation)  
o Alpha-­‐ketoglutarate  +  NH4+  +  NADPH  +  ATP  à  Glutamate  +  
NADP+  +  ADP  +  Pi  
• In  mammals,  glutamate  concentrations  are  maintained  by  the  
transamination  of  alpha-­‐ketoglutarate  during  amino  acid  metabolism  
(from  diet)  
• Glutamate  serves  as  a  nitrogen  source  for  biosynthesis  reactions,  such  as  
the  synthesis  of  amino  acids  from  alpha-­‐ketoacids  
• Glutamine  (Gln)  is  made  from  glutamate  (Glu)  by  glutamine  synthetase  in  
a  two  step  process  
o This  reaction  is  driven  by  ATP  (phosphorylates  glutamate)  
o Glu  +  ATP  à  Gamma-­‐glutamyl  +  NH4+  à  Gln  +  Pi  
o Phosphorylation  of  glutamate  creates  a  good  leaving  group  that  
can  be  easily  displaced  by  ammonia  
 
Regulation  of  glutamine  synthetase  by  allosteric  inhibitors  
• Glutamine  synthetase  
o Found  in  nearly  all  organisms  
o Plays  a  central  role  in  the  conversion  of  toxic  free  NH3  to  
glutamine  and  in  the  metabolism  of  amino  acids  
o Converts  ammonia  and  glutamate  to  glutamine  which  can  be  used  
in  a  variety  of  processes  
o Undergoes  cumulative  regulation  by  six  end  products  of  glutamine  
metabolism  
§ AMP  
§ CTP  
§ Histidine  
§ Tryptophan  
§ Carbamoyl  phosphate  

§

Glucosamine-­‐6-­‐phosphate  

 
Glutamine  synthetase  is  also  inhibited  by  adenylylation  
• Adenylylation  is  a  post  translational  modification  where  AMP  binds  to  a  
hydroxyl  group  of  a  molecule  via  adenylyltransferase  (AT)  
• Adenylylation  inactivates  glutamine  synthetase  
• Deadenylylation  activates  glutamine  synthetase  (converts  glutamate  to  
glutamine)  
• Adenylylation  is  indirectly  
o Stimulated  by  glutamine  and  Pi  
o Inhibited  by  alpha-­‐ketoglutarate  and  ATP  
 
Revision  
• The  process  by  which  nitrogen  fixing  bacteria  and  archea  convert  N2  to  
NH3  is  nitrogen  fixation  
• The  four  chemical  transformations  that  maintain  a  balance  between  N2  
and  biologically  useful  forms  of  nitrogen  
o Nitrogen  fixation  
o Nitrification  (conversion  of  ammonia  to  nitrite  and  nitrate)  
o Denitrification  (nitrate  à  N2)  
o Assimilation    
• The  enzyme  in  bacteria  and  plants  which  catalyses  NH4+  +  alpha-­‐
ketoglutarate  à  glutamate  is  glutamate  synthase  
• Glutamate  serves  a  key  role  in  biosynthesis  à  Provides  an  entry  point  for  
ammonia  into  the  system  (N  source  in  the  synthesis  of  AA  from  alpha-­‐
ketoacids)  
• Glutamine  synthetase  is  a  primary  regulation  point  in  biosynthesis  
(Glutamate  +  ATP  +  NH3  à  Glutamine)  
• Two  main  mechanisms  by  which  glutamine  synthetase  is  regulated  
o Adenylylation  
o Allosteric  modulation  
 
Amino  acid  biosynthesis  
• Source  of  nitrogen  is  glutamate  or  glutamine  
o Form  key  entry  points  for  N  entry  
o The  N  can  be  used  in  various  ways  and  can  be  added  to  C  skeletons  
• Carbon  skeletons  come  from  intermediates  of  
o Glycolysis  
o Citric  acid  cycle  
o Pentose  phosphate  pathway  
• Bacteria  can  synthesise  all  20  but  mammals  require  some  in  diet  
(essential  AA)  
 
Amino  acids  made  from  intermediates  of  major  pathways  
• Substrates  (C  skeletons)  from  major  pathways  can  be  attached  to  N  and  
form  AA  
• Some  AA  can  be  made  from  other  AA  (eg  Glutamine  formed  from  
glutamate  via  glutamate  synthetase)  

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The  carbon  skeleton  comes  from  three  sources  
o Glycolysis  
o Citric  acid  cycle  
o Pentose  phosphate  pathway  
Nitrogen  enters  these  pathways  via  transamination  with  glutamate  and  
glutamine  
Non  essential  amino  acids  can  be  made  from  one  of  6  precursors  
produced  in  major  biochemical  pathways  (TCA,  glycolysis,  PPP)  
o Alpha-­‐ketoglutarate  
o 3-­‐phosphoglycerate  
o Oxaloacetate  
o Pyruvate  
o Phosphoenolpyruvate  
o Ribose-­‐5-­‐phosphate  
Essential  AA  must  be  supplied  in  the  diet  
A  deficiency  of  one  AA  effects  the  synthesis  of  all  proteins  required  for  life  

 
Amino  acid  biosynthesis  
• Non  essential  AA  (except  Arg)  are  synthesised  by  simple  reactions  
involving  1-­‐5  steps  
• Essential  AA  are  synthesised  by  more  complex  steps  involving  5-­‐16  steps  
(essential  AA  require  more  steps  for  synthesis)  
• Reactions  involve  either  
o Transfer  of  the  amino  group  from  glutamate  by  transamination  
reactions  (transamination)  
o Transfer  of  methyl  groups  from  tetrahydrofolate  (derived  from  
folic  acid/Vitamin  B9)  or  S-­‐adenosylmethionine  
• 5-­‐phosphoribosyl-­‐1-­‐pyrophosphate  (derived  from  ribose-­‐5-­‐phosphate)  is  
a  notable  intermediate  in  several  pathways  of  amino  acid  (Try,  His)  and  
nucleotide  synthesis  
 
Transamination  
• Alpha-­‐ketoglutarate  +  AA  (glutamate/glutamine)  à  AA  +  alpha-­‐keto  acid  
(intermediate)  
o Reversible  reaction  
o Transfer  of  amino  group  from  one  AA  to  another  by  
aminotransferases    
• Uses  the  pyridoxal  phosphate  (PLP)  cofactor  
• L-­‐glutamine  acts  as  a  temporary  storage  of  nitrogen  
• L-­‐glutamine  can  donate  the  amino  group  when  needed  for  AA  
biosynthesis,  thus  readily  reversible    
• Pyridoxal  phosphate  is  
o A  cofactor  in  transamination  reactions  
o Derived  from  pyridoxine  (vitamin  B6)  
o Used  to  transfer  an  amine  group,  primarily  from  glutamate  or  
glutamine,  to  a  carbon  skeleton  for  AA  synthesis  
 
Feedback  inhibition  regulates  AA  biosynthesis  

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Inhibition  of  the  first  irreversible  step  (or  committed  step)  in  linear  
pathways  
Feedback  inhibition  and  activation  of  branched  pathways  involves  the  
inhibition  of  common  initial  step  by  its  own  product  and  is  activated  by  
the  product  of  another  pathway  
Enzyme  multiplicity  in  branched  pathways    
o Inhibition  of  the  committed  step  catalysed  by  2  or  more  enzymes  
Cumulative  feed  inhibition  of  branched  pathways  
o Eg  glutamine  synthetase  
o Partial  inhibition  of  a  common  step  by  each  of  the  final  products  

 
Enzyme  multiplicity  regulated  amino  acid  biosynthesis  
• Inhibition  or  activation  keeps  AA  synthesis  in  balance  
• Aspartate  may  produce  3  different  products  (AA)    
o One  directly  inhibits  an  enzyme  and  another  may  inhibit  a  
different  enzyme,  one  may  have  no  feedback  effect  
 
Roles  of  AA  
• Energy  production  
o The  TCA  cycle  
o Gluconeogenesis  (Via  TCA)  
o Ketogenesis  (important  in  generation  of  ketone  bodies)  
• Synthesis  
o Proteins  
o Nucleotides  
o Porphyrins  
o Creatine  
o Glutathione  
o Neurotransmitters  
o Rigid  polymers  
o Hormones  
o Polyamines  
o Nitric  oxide  
• Some  AA  may  enter  TCA  (can  be  either  glucogenic  and  drive  
gluconeogenesis  or  ketogenic  and  generate  ketone  bodies)  
• Alpha-­‐ketoglutarate  +  glutamate  synthase  à  Glutamate  
• Glutamate  +  glutamine  synthetase  à  Glutamine  
 
Roles  of  AA  à  Porphyrin  synthesis  
• Porphyrins  are  large,  ring  like  protein  structures  
• Porphyrin  makes  up  the  heme  of  haemoglobin,  cytochromes  and  
myoglobin  
• In  higher  animals,  porphyrin  arises  from  reactions  of  glycine  with  
succinyl-­‐CoA  
• In  plants  and  bacteria,  glutamate  is  the  precursor  
• Pathway  generates  two  molecules  of  the  important  intermediate  delta-­‐
aminolevulinate  

o Two  molecules  of  delta-­‐aminolevulinate  condense  to  form  
porphobilinogen  (important  intermediate)  
o Four  molecules  porphobilinogen  combine  to  form  protoporphyrin  
o Fe  ion  is  inserted  into  protoporphyrin  with  the  enzyme  
ferrocheletase  
o Fe  ion  functions  as  both  an  electron  donor  or  acceptor  
o Fe  ion  binds  O2  in  haemoglobin  (erythrocytes)  and  myoglobin  
(myocytes)  
 
Heme  is  the  source  of  bile  pigments  
• Bilirubin  is  breakdown  product  of  heme  
• Green  compound  seen  in  bruises  
• Urobilin  gives  urine  its  yellow  colour  
• Stercobilnin  gives  red-­‐brown  colour  to  faeces  
 
Defects  in  porphyrin  synthesis  
• Most  animals  synthesise  their  own  heme  
• Mutations  or  misregulation  of  enzymes  in  heme  biosynthesis  pathway  
lead  to  porphyrias  
o Precursors  accumulate  in  red  blood  cells,  body  fluids  and  liver  
o Homozygous  individuals  also  suffer  intermitted  neurological  
impairment,  abdominal  pain  
 
Roles  of  AA  à  Neurotransmitter  and  hormone  synthesis  
• Tyrosine  à  Epinephrine  
• Glutamate  à  GABA  
• Tryptophan  à  Serotonin  
 
Roles  of  amino  acids  à  Glutathione  synthesis  
• 3  AA  form  glutathione  (glutamate,  cysteine,  glycine  form  a  reduced  
tripeptide)  
• Glutathione  (GSH)  is  present  in  most  cells  at  high  amounts  
• Reducing  agent/antioxidant  
o Keeps  proteins  and  metal  cations  reduced  
o Keeps  redox  enzymes  in  reduced  state  
o Removes  toxic  reactive  oxygen  species  
• Oxidised  to  a  dimer  (GSSG)  
 
Roles  of  amino  acids  à  Nitric  oxide  synthesis  
• Pollutant  NO  plays  an  important  role  in  neurotransmission,  BP  regulation,  
blood  clotting  and  immunity  
• Synthesised  from  Arginine    
o Arginine  à  Citrulline  +  NO  
o Mediated  by  NO  synthase  
o Driven  by  NADPH  
 
Revision  
• Two  amino  acids  which  provide  a  source  of  N  in  metabolism  are  

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The  three  pathways  which  yield  the  carbon  skeleton  for  AA  synthesis  
o Glycolysis  
o TCA  
o PPP  
Essential  AA  in  hosts  must  be  supplied  in  the  diet  
Reactions  used  to  transfer  an  amine  group,  primarily  from  glutamate  or  
glutamine  to  a  carbon  skeleton  for  AA  synthesis  are  transamination  
In  addition  to  biosynthesis,  AA  are  involved  in  energy  production;  the  
three  main  energy  production  pathways  they  are  involved  in  are  
3  examples  of  heme  proteins  which  contain  iron-­‐porphyrin  (heme)  
groups  are  haemoglobin,  cytochrome,    
The  enzyme  which  converts  arginine  to  NO  is  NO  synthase  

•
 
Protein  synthesis  and  degradation  
• Protein  synthesis  à  Dehydration  (or  condensation)  reaction  where  a  
peptide  bond  is  formed  between  amine  and  carboxylic  acid  with  the  loss  
of  a  water  molecule    
• Protein  degradation  à  Hydrolysis  reaction  where  a  water  molecule  is  
inserted  to  break  the  peptide  bond  and  reform  the  amine  and  carboxylic  
acid  
• Form  a  dipeptide  (with  a  peptide  bond)  with  an  N  and  C-­‐terminus  
 
Amino  acid  catabolism  in  mammals  
• AA  from  dietary  proteins  are  the  main  source  of  amino  groups  
(ammonium)  
• When  metabolised  AA  provide  carbon  skeletons  which  can  be  fed  into  the  
TCA  cycle  and  gluconeogenesis  pathways  
• Most  AA  are  metabolised  in  the  liver  
• AA  are  used  to    
o Produce  glucose  or  lipids  
o Produce  energy  
o Provide  carbon  skeletons  
o Produce  proteins  
• Excess  ammonium  (NH4+)  must  be  excreted  via  the  urea  cycle  
 
Dietary  protein  is  enzymatically  degraded  through  the  digestive  tract  
• Acute  pancreatisis  à  Obstruction  of  pancreatic  secretions  à  Proteolytic  
activation  of  zymogens  to  attack  the  pancreatic  tissue  à  Pain  and  damage  
• Stomach  à  pepsinogen  (zymogen)  à  Pepsin  (active  via  proteolysis)  
• Pancreas    
o Chymotrypsinogen  à  Chymotrypsin  
o Trypsinogen  à  Trypsin  
 
Digestive  enzymes  are  secreted  as  zymogens  or  proenzymes  
• Zymogens  often  work  in  cascades  
• Enteropeptidase  breaks  down  trypsinogen  à  Trypsin  
• Trypsin  has  many  actions  including  chymotrypsinogen  à  chymotrypsin  
 

The  digestion  and  absorbtion  of  proteins  
• Proteolytic  enzymes  break  proteins  down  to  single  amino  acids  or  
oligopeptides  (short  branches  of  AA)  
• AA  enter  intestinal  cell  directly  via  AA  transporters  then  transported  into  
blood  stream  
• Oligopeptides  too  large  to  enter  intestinal  cell  (broken  down  by  
peptidases)  into  tripeptides  or  dipeptides  (which  are  further  broken  
down  to  single  AA  in  the  intestinal  cell)  
 
Amino  acid  catabolism  in  vertebrates  
• Removal  of  the  amino  groups  is  the  first  step  of  degradation  for  all  AA  
• AA  group  transferred  to  alpha-­‐keto  glutarate  à  glutamate  
• Glutamate,  glutamine,  alanine  and  aspartate  are  central  in  nitrogen  
metabolism  as  they  are  readily  converted  into  the  TCA  cycle    
o Alanine  from  muscle  
o Glutamine  from  muscle  and  other  tissues  
o AA  from  ingested  proteins  
• Glutamine  and  alanine  are  important  in  the  transport  of  amino  groups  
from  extra  hepatic  tissues  and  skeletal  muscle  respectively  to  the  liver  
• Glutamate  plays  a  role  in  removing  excess  ammonia  via  the  urea  cycle  
• Aspartate  provides  the  second  amino  group  in  the  urea  cycle  
 
Ammonia  is  safely  transported  in  the  bloodstream  as  glutamine  
• In  the  tissues  
o Excess  NH4+  in  tissues  is  added  to  glutamate  à  glutamine  (via  
glutamine  synthetase)  
• Glutamine  is  transported  in  the  bloodstream  
• NH4+  is  released  in  the  liver  à  Release  glutamate    
• NH4+  enters  urea  cycle  à  urea  which  can  then  be  excreted  
 
Glucose-­‐alanine  cycle  
• Vigorously  working  muscles  operate  nearly  anaerobically  and  rely  on  
glycolysis  for  energy  
• Glycolysis  yields  pyruvate  and  if  not  eliminated  then  lactic  acid  build  up  
• This  pyruvate  can  be  converted  to  alanine  for  transport  to  the  liver  via  
transamination  reaction  (glutamate  +  pyruvate  à  alanine  +  alpha  keto-­‐
glutarate  (transamination  reaction))  
• This  alanine  in  the  liver  can  enter  the  urea  cycle  and  gluconeogenesis  
pathway  (Alanine  +  alpha  ketoglutarate)  à  glutamate  +  pyruvate  
o Pyruvate  used  by  liver  and  fed  into  gluconeogenesis  to  form  
glucose  which  is  exported  and  sent  to  tissues  where  required  
• NH4+  is  toxic  to  the  body  and  must  therefore  be  regulated  (must  be  kept  
out  of  the  bloodstream)  
 
Amino  groups  are  transferred  to  alpha-­‐ketoglutarate  by  aminotransferases  
• This  is  an  example  of  a  transamination  reaction  
• PLP  (pyridoxal  phosphate)  is  the  essential  cofactor  in  this  reaction  
(derived  from  vitamin  B)  

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Transfer  of  amino  groups  from  one  to  another    
Alpha  ketoglutarate  and  AA  (via  amino  transferase  +  PLP)  à  Glutamate  
and  alpha-­‐keto  acid  (Amino  group  transferred  to  glutamate)  
Alpha  keto  acids  contain  a  carboxyl  and  ketone  group  (eg  pyruvate)  

 
AA  catabolism  in  terrestrial  vertebrates  
• Removal  of  the  amino  group  (transamination)  is  the  first  step  of  
degradation  for  all  AA  
• In  most  cases  the  amino  group  is  transferred  to  alpha-­‐ketoglutarate  to  
form  glutamate  
• AA  in  liver  à  NH4+  attached  to  glutamate  à  Urea  cycle  (excretion  of  
ammonium  as  urea)  
• Glutamate  is  transported  from  the  liver  cytosol  to  the  liver  mitochondria  
to  release  NH4+  
• Left  over  carbon  skeletons  of  AA  are  converted  to  glucose  or  oxidised  by  
the  TCA  cycle  
 
Excretory  forms  of  nitrogen  
• AA  not  used  for  new  AA  or  other  products  are  removed  as  a  single  
excretory  end  product  
• Animals  excrete  nitrogenous  wastes  in  different  forms  à  Ammonia,  urea  
or  uric  acid  
• NH4+  is  highly  toxic  to  animal  tissues  
• Mammals  secrete  NH4+  as  urea  
• In  ureotelic  organisms  (many  terrestrial  vertebrates;  sharks  and  
mammals)  
o The  liver  of  mammals  and  most  adult  amphibians  converts  
ammonia  to  the  less  toxic  urea  
o The  circulatory  system  carries  urea  to  the  kidneys  where  it  is  
excreted  
o Conversion  of  ammonia  to  urea  is  energetically  expensive;  
excretion  of  urea  requires  less  water  than  ammonia  
• Uricotelic  animals  (birds,  reptiles)  
o Insects,  snails  and  many  reptiles  including  birds  excrete  uric  acid  
o Uric  acid  is  relatively  non-­‐toxic  and  does  not  readily  dissolve  in  
water  
o Can  be  secreted  as  a  paste  with  little  water  loss  
o More  energetically  expensive  to  produce  than  urea  
• Ammonotelic  animals  (aquatic  vertebrates  eg  fish  and  the  larvae  of  
amphibia)  
o Animals  that  excrete  nitrogenous  wastes  as  ammonia  need  access  
to  lots  of  water  
o They  release  ammonia  across  the  whole  body  surface  or  through  
gills  
• Ammonia  excretion  requires  high  amounts  of  water  (fish)  
o Low  energy  expenditure,  high  water  loss  
• Uric  acid  excretion  limits  water  loss  (excreted  almost  dry)  
o High  energy  expenditure,  low  water  loss  

  
The  urea  cycle  à  Excess  glutamate  is  metabolised  by  the  mitochondria  of  
hepatocytes  
• Glutamine  from  muscles  etc
...
 A  CO2  molecule  is  released  in  
photorespiration  

 
Improving  rubisco  
• Rubisco  is  rate  limiting  for  photosynthesis  
• Improved  efficiency  would  have  a  large  impact  on  CO2  reduction  in  the  
atmosphere  and  food  production  
o Improve  specificity  à  Replace  crop  gene  with  rubisco  from  red  
algae  or  purple  photosynthetic  bacteria)  
o Increase  catalytic  activity  à  Introduction  of  altered  rubisco  with  
higher  activity  
o Increase  levels  of  expression  
 
C3  and  C4  photosynthesis  
• C3  plants  use  the  conventional  pathway  of  CO2  uptake  in  which  the  first  
intermediate  produced  is  a  C3  compound  
o CO2  à  3-­‐phosphoglycerate  (C3)  
• C4  plants  use  an  alternative  pathway  of  CO2  uptake  in  which  the  first  
intermediate  produced  is  a  C4  compound  (In  order  to  avoid  
photorespiration)  
o CO2  à  Oxaloacetate  (C4)    
o Found  in  tropical  grasses  (eg  maize,  sugarcane)  and  is  more  
efficient  in  intense  sunlight  and  high  temperatures  
• Characteristics  of  C4  plants  
o High  photosynthetic  rates  
o High  growth  rates  
o Low  photorespiration  rates  (better  efficiency)  
o Low  rates  of  water  loss  
o Specialised  leaf  structure  
 
C4  photosynthesis  
• PEP  carboxylase,  the  enzyme  that  incorporates  atmospheric  CO2,  has  no  
affinity  for  O2  (Replaces  rubisco  which  has  the  affinity  for  O2)  
• C4  plants  have  different  anatomy  which  allows  them  to  concentrate  CO2  
and  overcome  photorespiration  
• Two  types  of  cells  
o Mesophyll  cells  
o Bundle  sheath  cell  
• CO2  from  air  à  Carbonate  

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PEP  à  PEP  carboxylase  à  Oxaloacetate  à  Malate  à  CO2  à  Enters  
normal  calvin  cycle  and  rubisco  converts  it  to  3-­‐phosphoglycerate  
Malate  à  Pyruvate  which  is  used  to  regenerate  oxaloacetate  for  the  cycle  
(requires  an  additional  2  ATP  than  the  C3  pathway)  
The  rubisco  in  the  bundle  sheath  cells  is  never  exposed  to  O2  in  the  
atmosphere  so  the  plant  cannot  undergo  photorespiration  
CO2  fixation  by  the  C4  pathway  requires  more  energy  (which  is  why  not  
all  plants  do  it)  
o The  C3  pathway  requires  3ATPs  per  CO2  fixed  
o The  C4  pathway  requires  5ATPs  per  CO2  fixed  
Concentrates  CO2  for  rubisco  in  the  calvin  cycle  but  requires  additional  
energy  
Used  when  the  gains  in  efficiency  outweigh  the  increased  energy  
requirement  
o The  C4  pathway  requires  high  sunlight  for  energy  production  
(increased  ATP  synthesis);  therefore  they  are  usually  plants  in  
high  sunlight  areas  
o Rubisco’s  affinity  for  CO2  decreases  with  increased  temperature;  
above  30  degrees  the  C4  pathway  provides  a  significant  gain  in  the  
efficiency  of  CO2  fixation  
o Concentration  of  CO2  allows  stomata  to  partially  close  during  the  
day,  conserving  water  
Therefore,  photosynthesis  in  C4  plants  is  more  efficient  in  hot,  arid  or  
tropical  climates  

 
Summary  of  C4  photosynthesis  
• CO2  fixed  into  organic  acid  in  mesophyll  cells  by  PEP  carboxylase  which  
has  no  O2  affinity  
• CO2  fixed  by  rubisco  in  bundle  sheath  cells;  [CO2]  maintained  at  high  
levels  in  these  cells  
• C4  photosynthesis  requires  more  energy  than  the  C3  pathway  (5ATP  vs
...
5kJ/mol)  
o G6P  à  Glucose  +  Pi  (∆G10  =  -­‐13

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