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Enzymes
28 January 2015

12:24

Enzymes
(1)

Michaelis-Menten equation
• Describes the relationship between reaction rate and substrate
concentration
• At equilibrium there's no net change in [S] and [P]
• At the steady state [ES] is constant
• Vo is a measure of the initial velocity
• At low [S] Vo increases with [S]
• At high [S] S has little effect on V 0
• Though lowered by E, activation energy is still present
• Product tends to accumulate
• 4 assumptions
1
...
K-2 is negligible
3
...
[E] < [S]

KM is affinity between [S] and [E]
Low KM = high affinity

• If Vmax is reached faster, KM is lower
• M-M gives a current line
...
nature
...
gif

Metabolism Page 4

http://www
...
com/gim/journal/v15/n2/images_
article/gim2012104f1
...
5ATP
○ FADH2 = 1
...
42ATP/g
• Glucose gives 32ATP/molecule
○ 0
...
42ATP/g
• Glucose gives 32ATP/molecule
○ 0
...

○ Fatty acid produced
○ Beta oxidation inhibited again
• Fatty acid synthesis from acetyl CoA and Melonyl CoA is catalysed by 1 large polypeptide (in
mammals)
○ Fatty acid synthase complex
Product release
• When the fatty acid is 16 carbons in length, a thioesterase domain catalyses hydrolysis of the
thioester linking the fatty acid to phosphopantetheine
• The 16C saturated fatty acid palmitate is the final product of the fatty acid synthase complex
• Acetyl CoA + 7malonyl-CoA + 14NADPH -------> Palmitate + 7CO2 + 14NADP+ + 8CoA
Berg Biochemistry chapter 20, 22
...
low levels
○ People with disorders show very different pattern
 Much higher concentrations
• Malate
○ Same pattern
• Reliable testing can improve quality of life for those affected
Phenylketonuria
• Autosomal recessive trait
• Deficiency of liver enzyme phenylalanine hydroxylase
○ Converts phenylalanine to tyrosine
• Prevents normal Phe metabolism
○ Essential amino acid
○ Obtained from food
• Untreated individuals
○ Toxic build-up of Phe - mental retardation
• Problem from before birth
○ New-borns screens
○ High Phe shows problem
• Treatment is low Phe diet
○ Less meat, fish, eggs, legumes, bread
• Artificial protein supplements without any Phe

Galactosemia
• Galactose metabolism
• Unable to convert galactose to glucose-1-phosphate
Gout
•
•
•
•

Metabolic disease
Largely affects middle-aged to elderly men and postmenopausal women
Elevated uric acid in bloodstream
Bodily response to crystalline deposits in joints
○ Under- or over-excretion of uric acid
• Uric acid produced by xanthine oxidase from xanthine or hypoxanthine
Metabolism Page 39

• Uric acid produced by xanthine oxidase from xanthine or hypoxanthine
• Increase in uric acid may be due to
○ Enzyme defects
○ Metabolic defects
○ Chronic anaemia
○ Kidney disease
• Causes
○ Dietary excess
○ Trauma
○ Surgery
○ Alcohol
○ Serious illness
 e
...
stroke
• Treatment
○ Controlled weight loss
○ Lower alcohol consumption
○ Low purine diet
○ Higher proportion of carbohydrates in diet
○ High fluid intake
Diabetes
• Insulin is produced in the pancreas
○ It plays a role in carbohydrate and fat metabolism
○ Role to play in blood glucose regulation
○ Promotes uptake of glucose by glycogen
• Wrong hormone balance can give problems
• Blood sugar levels can impact a number of pathways
• Causes
○ Genetic
○ Obesity
○ Pregnancy

Other Disorders
• Lesch-Nyhan Syndrome
○ Purine salvaging pathway
• Tay-Sachs disease
○ Phospholipid metabolism
• Favism
○ Glucose-6-phosphate dehydrogenase deficiency
• Maple Syrup Urine Disease
○ Problems with amino acid metabolism

Metabolism Page 40

Oxidative Phosphorylation
18 March 2015

12:46

Mitochondria
• Outer membrane very permeable to small molecules and ions
• Inner membrane almost completely impermeable to ions and polar
molecules
○ Specific transporters move metabolites

•

• The respiratory chain is on the inner membrane
○ Spontaneous electron transfer through complexes 1, 3, and 4 is
coupled to H+ ejection from the matrix into the intermembrane space
○ Free energy of spontaneous e- transfers conserved as H+
electrochemical gradient

Metabolism Page 41

ATP Synthase
• Actin filament fluorescent labelled
• ATP is added, causing counterclockwise rotation
○ Hydrolysis of ATP drives rotation of gamma subunit
Complexes
• Complex I
○ NADH-coenzyme Q reductase
○ >30 subunits
○ 850kDa
○ 1 molecule of flavin
○ 7 Fe-S clusters
○ 4H+ per 2e○ 2e- goes to coenzyme Q (CoQ)
• Complex II
CoQ
○ Succinate coenzyme Q reductase
• Transfers e- from I and II to III
○ Succinate dehydrogenase
• Good at diffusion
○ 100-140kDa
○ 4 subunits
Cytochrome c
○ H+ and e- transferred to FAD
• Loosely associated with membrane
○ e- directly to F-S centres, then to CoQ
• Electron carrier
○ No proton transfer
• Complex III
○ Coenzyme Q cytochrome c reductase
○ 248 kDa
○ 11 subunits
○ 3 cytochromes
○ 1 Fe-S protein
○ e- transferred to cytochrome c
○ 4H+ per 2e• Complex IV
○ Cytochrome c oxidase
○ 162 kDa
○ >10 subunits
○ 4 cyte (Fe2+) +4H+ + O2 ---> 4cyt c (Fe3+) + 2H2O
○ 2H+ per 2e-

Metabolism Page 42

Integration of Metabolism
13 April 2015

11:06

• Pathways not isolated
○ Recurring motifs of regulation
○ Major control sites
○ Three key crossroads
 Glucose-6-phosphate
 Pyruvate
 Acetyl-CoA
• Highly interconnected pathways

Interconnected Pathways
• Catabolism - shared features
○ Role of ATP
○ Reducing power
 NADH
 NADPH
 FADH
○ Biosynthetic precursors
• Central themes
○ ATP is universal energy currency
 Coupled reactions
 Energy released by ATP hydrolysis drives substrate to product conversion
○ ATP generated by oxidation of fuel molecules
○ NADPH electron donor in reductive biosynthesis
 Nicotinamide Adenine Dinucleotide
 Electron acceptor
 Nicotinamide accepts 2e- and H+ when reducing to NADH
NADPH has additional Pi
□ e- donor in synthetic pathways
○ Biosynthetic precursors
○ Biosynthetic and degradative pathways distinct
Metabolic Regulation
• Anabolism and catabolism must be precisely regulated
○ Allosteric interactions
○ Covalent modification
○ Enzyme levels
○ Compartmentalisation
○ Specialisation of organs
Major Control Sites
• Glycolysis
○ Phosphofructokinase (F6P --> f1,6BP
 ATP lower activity - allosteric
 AMP reverses action
• Citric Acid Cycle and oxidative phosphorylation
○ Electron donors are oxidised and recycles back into TCA only if ADP simultaneously goes
to ATP
○ ATP inhibits isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase
○ Citric acid cycle also has anabolic role
• Gluconeogenesis
Glycolysis and gluconeogenesis reciprocally regulated
Metabolism Page 43

○ Glycolysis and gluconeogenesis reciprocally regulated
• Glycogen synthesis and degradation
○ Hormonally controlled
○ Phosphorylation and allosteric control
○ 90% liver, 10% kidney
○ Glycogen phosphorylase activated by AMP, inhibited by ATP and G6P
○ Glycogen synthase stimulated by G6P
○ Both enzymes regulated by covalent modification - phosphorylation
Glucagon vs

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