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BB10003 Biochemistry 1
Proteins
Introduction to Biochemistry
o DNA (genetic information) → RNA → protein (functional macromolecule)
o Complexity of organisms:
o Genome – all genes encoded in genetic material, complete DNA sequence
...

o C-value – quantity of DNA in haploid nucleus related to genome size, not a good indicator of
complexity due to non-coding DNA
...

o Model systems in genomics:
o E
...
elegans, Mouse
o E
...

o Genomics – comprehensive study of whole sets of genes and their interactions, use microarray to
detect target molecules in ssDNA (deposit ssDNA, labelled target molecules bind)
...

o Analysis of proteomes
o More complex than genome: alternative splicing of pre-mRNA – 25000 genes to 100000
proteins; post-translational modification – proteolytic cleavage, glycosylation,
phosphorylation etc
...

o Hemagglutinin and neuraminidase on surface of flu virus are important for cell recognition
→ look at protein, electron density, subunits, ligand binding sites → by looking at 3D
structure can see the active site
...

o -amino acids
o Hydrophobic – side chains mostly carbon and hydrogen so have small dipole moments so
are repelled by water (Ala, Val, Leu, Ile, but also Phe, Pro, Trp, Cys, Tyr, Thr, Gly, Lys, Arg)
...

o Polar (Ser, Thr, Tyr, His, Cys, Trp, Asn, Gln)
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o Aromatic (Phe, Tyr, Trp)
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o Ionisation – at least two ionising groups (some side chains have extra)
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o Amino acids mostly classified according to polarity of side chains: non-polar R groups
(variety of shapes and sizes), un-charged polar R groups (hydroxyl, amide or thiol groups),
and charged polar R groups (may be positive or negative)
...

o Operational classification – optical rotation; shine polarised light into solution, optically
active molecules change direction, rotating the plane of plane-polarised light to different
angles, rotated to left is levorotatory, to right is dextrorotatory (Pasteur made tartaric acid
but wasn’t optically active as both isomers were present – only L isomer in nature), light
source → polariser → sample tube → analyser → viewer, optical rotation can only be

determined empirically (by experiment), problem with this is it provides no indication of
the absolute configuration (spatial arrangement) of chemical groups around a chiral centre
...


o

o
o

o

o Cahn-Ingold-Prelog System – absolute nomenclature, prioritise side chain groups by atomic
number and view chiral centre towards lowest priority substituents (lowest behind chiral
carbon), if priority decreases clockwise it’s the R enantiomer and if priority decreases anticlockwise it’s the S enantiomer, if CORN goes clockwise its Levorotatory and if it goes
anticlockwise its Dextrorotatory, D and R plus L and S forms are often but not always the
same, all naturally occurring amino acids have (S) configuration (except cysteine because of
its C-S side chain which has higher priority than CO2H), threonine and isoleucine have a
second asymmetric carbon centre at C3 along their chains
...

Secondary structure – each C is in regular arrangement with respect to its neighbours, stabilised
by hydrogen bonds
...
6 residues per turn (13 main chain atoms), rise of
5
...
5 Å per residue), dipole moment across peptide bonds (- CO, + NH), discovered by
Linus Pauling 1948 (predicted triple helix)
...


o Alpha-pleated sheet – hypothetical secondary structure in proteins first proposed by Linus Pauling,
could be an intermediate stage in conformation (in  all amino acids are the same way up)
...

o Beta turn – link in -sheet held by a hydrogen bond between two corner amino acids
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o Secondary – regular, local arrangement of polypeptide chain
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o Quaternary – association of multiple chains
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o Structure of a complete polypeptide chain: segments of secondary structure linked by less
regular segments (turns – loops) results in compact globular structure
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o Disulphide bonds – may stabilise conformation of a protein (covalent), covalent bond between
cysteine side chains (-CH2SH), not all proteins have cys residues, not all cys residues are involved in
disulphides (free cys), 2-mercaptoethanol is a reagent used to reduce disulphide bonds and
produces free –SH groups (cysteine side chains) which allows disulphide bonds to be broken in a
lab
...

o Insulin – synthesised as inactive precursor pre-proinsulin which is a random coil on membraneassociated ribosomes, leader sequence transports polypeptide through membrane, pre-sequence
(leader sequence) removed by proteolysis, proinsulin folds and disulphides form so an inactive
precursor is formed (pre-sequence required for correct folding), activation involves removal of prosequence, connecting sequence is cleaved to form insulins two polypeptide chains bonded
together
...
g
...

o Catalysis – in purely chemical reaction a heterogeneous catalyst uses its surface to bring things
together, activate them, and allow them to react
...

o Activation energy – energy barrier that prevents reactants becoming products instantaneously, so
some reactions with a negative G occur slowly or not at all, need energy input to convert
reactants into unstable molecular forms called transition-state species, size of activation energy
determines rate of reaction and catalyst reduces size of energy barrier, can be overcome with heat
and pressure (reduces entropy, increases chance of collision between molecules) but in biological
systems heat can denature proteins and high pressure can damage cells without defined cell walls
...

o Enzyme may put ‘strain’ on existing bonds, making them easier to break
...

o Sometimes the active site of the enzyme itself is directly involved in the reaction during the
transition states (i
...
a different reaction path)
...

o Induced fit model – the active site will interact with the substrate and adapt to it to make a perfect
fit, enzyme in native state has residues which will recognise substrate but isn’t rigid and folds
around it, creates suitable environment for the reaction
...

o Amino acids in the active site interact specifically with the substrate (electronic)
...

o Stereospecificity – enzymes are highly specific in binding chiral substrates and in catalysing their
reactions (Enantioselective), due to the enzymes active site (stereospecificity is a description of the
reaction path, not the selectivity for substrates or products)
...

o Geometric specificity (regiospecificity) – selective about chemical groups of the substrate (more
stringent requirement than stereospecificity), varying degrees in different enzymes, a few enzymes
are absolutely specific for one substrate and some work on a group of related molecules (e
...
yeast
ADH oxidises primary and secondary alcohols but ethanol is the most efficiently converted), many
enzymes are very permissive, some are not specific in the types of reactions catalysed (exceptions)
so can be used in industry to use intermediates biosynthetically and are flexible in what they do
...
g
...
2
...
20), EC stands for enzyme
classification, there are six classes:
1
...

2
...

3
...

4
...

5
...

6
...

o Coenzymes – some enzymes require small molecules during catalysis called cofactors, which can be
metal ions or organic molecules called coenzymes, some coenzymes are transiently associated with
the enzyme and known as cosubstrates, cofactors associated with the enzyme are known as
prosthetic groups, a catalytically active enzyme-cofactor complex is called a holoenzyme, the
inactive protein is called an apoenzyme
...

o Enzyme assays:
o Direct assay – has a way of detecting product or substrate directly due to some property of
either which can be measured continuously (if P is coloured then colour change, P might
fluoresce or be a gas
...
g
...

o Coupled assays – sometimes neither P nor S can be measured but P can be consumed in
another reaction and the product of that reaction can be measured (2nd enzyme must be in
excess so it doesn’t limit the rate), e
...
assay of -glycerokinase (GK) where production of
NADH can be monitored at 340nm
...


o When the substrate concentration becomes high enough to entirely convert the enzyme to
the enzyme substrate complex, the second step of the reaction becomes rate limiting and
the overall reaction becomes insensitive to further increases in substrate concentration
...


o Steady state assumption – under the physiologically common condition that substrate is in
great excess over enzyme, with the exception of the transient phase (initial stage, usually
over within milliseconds), [ES] remains approximately constant until the substrate is nearly
exhausted, so the rate of synthesis of ES must be equal to its rate of consumption, therefor
[ES] maintains a steady state, therefore it can be assumed that [ES] is constant and d[ES]/dt
= 0
...

o The Michaelis constant has a simple operational definition: when [S] = KM, vo = Vmax/2,
therefore KM is the substrate concentration at which the reaction rate is half the maximal
rate, it is different between enzymes and with different substrates and it alters with
temperature and pH, when [S]<[S], and when [S]>>KM (high) vo = Vmax and the initial rate is independent of [S], the higher
the KM the higher [S] needed to reach Vmax
...

o Lineweaver-Burk Plot – linearises the equation vo = Vmax[S] / KM + [S] by using the reciprocal of the
equation:

The disadvantage of this plot is most measurements of [S] are at high values, and the 1/[S] values
get crowded on the left side of the graph making drawing a straight line difficult and inaccurate,
and for small [S] small errors in vo lead to large errors in 1/ vo and hence large errors in KM and Vmax
...

o Eadie-Hofstee plot – slope is –KM, y-axis intercept is Vmax, can be subject to large error since both
coordinates contain dependent variable v but there is less bias on points at low [S]
...


Enzyme Regulation
o pH
o Usually only active in a narrow pH range (usually 5-9) due to pH sensitivity of substrate
binding, reduced catalytic efficiency of the enzyme, ionisation of substrate, protein
structural changes (usually at pH extremes)
...

o Tend to get a bell-shaped curve when looking at pH and reaction rate, different enzymes
have different optimum pH suited to environments where they work
...

o pH affects the state of the groups in the active site by protonation, as well as the substrate
...

o Relatively small impact from increased number of collisions as temperature increases
...

o Can be positive (activation) or negative (inhibition)
...

o Can be reversible (covalent and non-covalent) or irreversible (post translational
modifications, protein cleavage, irreversible regulatory molecule binding), from point of
view of cell economy it doesn’t make sense to have many reactions completely irreversible
...

o Competitive Inhibition – structure similar to substrate, occupies active site, inhibitor (I) and
substrate (S) compete, binding of I and S is mutually exclusive, effect is reversed by increasing
substrate concentration
...


It is assumed that I binds reversibly to the enzyme and is in rapid equilibrium with it so that
KI = [E][I]/[EI], then vo = Vmax[S] / KM(1 + [I]/KI) + [S], and Vmax = k2[E]T
...

vo = Vmax[S] / (KM + [S])(1 + [I]/KI)

o Uncompetitive Inhibition – occurs when the inhibitor binds only with ES to make ESI, cannot yield
products, not reversed by increasing substrate concentration, usually found in enzymatic reactions
with two or more substrates, appears to decrease KM (apparent increase in affinity of substrate to
enzyme) but actually inhibits substrate release, increasing [I] diminishes Vmax and KM but KM/ Vmax
remains constant, vo = Vmax[S] / KM + (1 + [I]/KI)[S]

o Allosteric Inhibition
o Simple inhibition is insufficient for metabolic needs (to alter enzyme activity by 80% would
need to alter inhibitor concentration by 80x which is unrealistic, requires activation as well
as inhibition, and effector needs to be structurally unrelated to S), solution is allosteric
regulation
...

o Negative feedback is very common
...

o V against [S] gives a sigmoidal curve – does not follow Michaelis-Menten kinetics
...

o Respond to relatively small changes in concentration but have large effect on rate
...


o Covalent Regulation – even allosteric regulation is insufficient (e
...
where enzyme activity needs to
be limited to certain locations it is produced in an inhibited state, or where activity must be
switched off reversibly), covalent modifications can alter activity (dramatically activating or
inactivating) although don’t usually switch off enzyme completely but changes rate of reaction,
covalent modifications can be reversible (enzyme may exist in an enzymatically inter-convertible
form) and irreversible (enzyme may be activated enzymatically by cleavage)
...
g
...

o Irreversible Covalent Regulation – some enzymes are potentially hugely harmful (e
...
proteases
could break down host), these enzymes are synthesised as inactive precursors called zymogens
which are activated by protease to form the active enzyme and a small peptide
...

Thermodynamics and Bioenergetics
o Thermodynamic laws used in bioenergetics:
o A system consists of matter at a given temperature, pressure and volume and is the part of
the Universe under study, there are three types: i
▪ isolated, which do not exchange material or energy with their surroundings
▪ closed, which do not exchange material but do exchange energy across their
boundaries leading to a change in internal energy by transfer of heat (q) (random
motion) or work (w) (organised motion)
▪ open, which exchange energy and material with their surroundings, all biological
systems are open
...

o The system and surroundings are separated by a boundary
...

o The first law of thermodynamics - energy can neither be created nor destroyed (but it can be
changed from one form to another or transported from one region to another), this suggests the
reaction can occur in either direction
...


(B) For a process occurring under conditions of
constant volume, the heat transferred to the
system is given by the change in energy, U, of
the system
...

(A) Fiji
(B) j
(C) For a process occurring under constant
pressure conditions, the heat transferred is
given by the change in enthalpy, H, of the
system
...

The second law of thermodynamics – any spontaneous process must cause the entropy of the
Universe to increase, entropy is a state function that measures the degree of disorder or
randomness of a system and is related to the number of equivalent arrangements or
configurations of it where all configurations of atoms have equal probability, systems of molecules
have a natural tendency towards randomisation, the entropy of an isolated system will tend to
increase to a maximum value (the total entropy of the universe is always increasing)
...

The combined entropy of the system and the surroundings increases in a spontaneous process –
the new equilibrium is at higher entropy
...

o G > 0 – endergonic process, forward process is energetically unfavourable, reverse
process proceeds spontaneously
...

o G < 0 – exergonic process, forward process is energetically favourable, forward
proceeds spontaneously
...
e
...


o Factors that affect G:
o Alter concentrations
...

o G = Gº + RT ln([products] / [reactants]), [products] / [reactants] is called the mass action
ratio (), when all concentrations are 1M then G = Gº, at equilibrium G = 0, and [C]c[D]d
/ [A]a[B]b = Keq, so:
Gº = -RT ln Keq
o Relationship between Gº and Keq
o Gº < 0, Keq > 1 (when Gº is large and negative, Keq is very large)
o Gº = 0, Keq = 1
o Gº > 0, Keq < 1 (when Gº is large and positive, Keq is very small)
...

o Standard quantities in biochemistry vs physical chemistry – in biochemistry the concentration of
water is taken to unity (1) for reactions in dilute solutions since the concentration of water is
essentially constant (55M – always in its standard state, pH 7 and [H+] = 10-7M)
...
constants are multiplicative so it is very difficult to alter the direction of reactions that
have very large or very small values of Keqm by changing the mass action ratio
...

o Phosphate compounds as free energy intermediates –nearly all chemical processes in cells
proceed by sequential reactions in which the product of one is the substrate for the next
(common intermediate principle), ATP is a common intermediate in energy transformations,
phosphate esters or nucleotide triphosphates (e
...
ATP) like shuttles of free energy in the
cell, hydrolysis of these compounds releases a large amount of energy and each is
theoretically capable of driving the phosphorylation of compounds with lower Gº (as long
as mechanisms exist)
...

o Phosphate transfer potentials – ranking of phosphorylated compounds according to Gº for
hydrolysis, each compound capable of driving phosphorylation of compounds lower on the scale via
suitable mechanism
...

o Active transporters can move molecules across cell membranes against concentration
gradients – maltose transporter hydrolyses two molecules of ATP for every molecule of
maltose moved across the membrane, an example of chemical work, work done is coupled
to hydrolysis of ATP by transporter proteins due to series of conformational changes
triggered by ATP and the maltose-binding protein, maltose transported initially has the
maltose binding site empty and open to the interior of the cell then ATP binds to the
cytoplasmic ATPase domains and a maltose-loaded binding protein binds to the exterior
face of the transporter so the transporter changes shape and the maltose can be moved
into the interior of the transporter, the catalytic centre of the ATPase domains are properly
configured for catalysis and ATP hydrolysis occurs converting the shape of the complex and
releasing ADP and maltose as it returns to its original conformation, free energy change of
hydrolysis of ATP limits extent to which molecules can be moved against a concentration
gradient
...

o Glucose – main carbohydrate used for energy, found as monosaccharide (fruits, vegetables, honey,
nuts), disaccharide (sugars and milk), and polysaccharide (starchy foods like potatoes, wheat and
rice), cellulose is made up of beta glucose (we can’t break it down, some animals recruit bacteria to
do it for them), starch and glycogen are stores of alpha glucose
...


o Simple sugars (monosaccharides) are either aldoses (glucose and galactose) or ketoses (fructose) –
aldehyde or ketone derivatives of straight-chain polyhydroxy alcohols containing at least 3 carbons,
they cannot be hydrolysed to form simpler saccharides (only specific sugars are sweet depending
on receptors
...

o Aldo-tetroses are four carbon sugars
...

o Aldo-hexoses are six carbon sugars
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o D-glucose – metabolised to provide ATP, D-mannose – C2 epimer of D-glucose (found in
carbohydrate polymers, particularly the carbohydrate portion of glycoproteins), D-galactose
- C4 epimer of D-glucose (found in lactose, the disaccharide in milk)
...

o Glucose – the C5 hydroxyl group acts as an alcohol, attacks C1 aldehyde group to form a 6membered pyranose ring, forwards and reverse reactions are in equilibrium, can also form 5
membered rings if C4 reacts but these are much less common in solution (pentoses more
readily form furanose rings, the linear form is dangerous as it can damage proteins, more of
the  form due to sterics
...




o Interconversion of - and -D-glucose – of solution of  anomer is prepared, composition
goes down into a mixture, but -D-glucose form is preferred (OH groups pointing away from
each other)
...

o D-fructose – 6-carbon ketose important in metabolism
...
2 moles, require hydrolysis of 100
to 150 moles daily, each equivalent of ATP is recycled 500-750 times in a day
...

o Glycolysis happens in the cytoplasm, its transported into the cell by glucose transporters which all
cells have (some tissues can also take up fructose)
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o Glucose → Glucose-6-phosphate (G6P)
o Keeps glucose in the cell, and keeps glucose concentration low so it continues to be
transported into the cell
o Hexokinase – keeps away from water which would hydrolyse by changing from open to
closed form by induced fit, regulated by allosteric inhibition (build-up of G6P), four types
...

o ATP → ADP – energy investment, coupled to make the reaction favourable and exergonic
...


o Hexokinase IV (glucokinase) – found in the liver, only works at high glucose concentrations
(which happens when other organs don’t need glucose and it needs storing) so is not
affected by allosteric inhibition
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o Isomerisation – conversion of aldose to ketose, base-catalysed rearrangement (two basic
residues that pull of two hydrogens)
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o Fructose-6-phosphate (F6P) → Fructose-1,6-bisphosphate (F1,6BP)
o Phosphofructokinase – adds another phosphate to fructose to make a symmetrical
molecule, reaction pushed forwards although technically reversible, exists as a
homotetramer in bacteria and mammals (where each monomer possesses two similar
domains) and an octomer in yeast (with four  and four  chains, the latter possessing two
similar domains), in mammals the tetramer is composed of a combination of three types of
subunits (muscle, liver, and platelet) depending on the tissue, allosteric inhibition in
presence of ATP by binding to allosteric sites between subunits on PFK, R-state is active, Tstate is inactive
...

o Mg2+ - important in coordinating reaction
...
), is an autosomal recessive trait, documented in over 100 English
Springer Spaniels, rare in humans
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Glycolysis (energy yielding phase)
o Glyceraldehyde 3-phosphate (G3P) ⇌ 1,3-Bisphosphoglycerate (1,3-BPG)
o Glyceraldehyde-3-phosphate dehydrogenase – adds phosphate group forming high energy
linkages which need to override energy ATP could deliver (to make ATP need higher energy
bond), a two-step reaction, homotetramer
o NAD → NADH + H+ (first step, favourable)
...

o Hydrogen taken up by NAD, enables cysteine side chain to attack carbonyl group aided by
basic residue (histidine), leads to covalent intermediate easily attacked by phosphate,
release of cystine, now sugar with phosphate on either end - really high energy bond, ready
to start generating ATP by donating phosphates
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o Mg2+ - important in coordinating phosphates
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o 2-Phosphoglycerate (2PG) ⇌ Phosphoenolpyruvate (PEP)
o Enolase – removes a water molecule forming a double bond, two active sites, binds two
metal ions, lysine and glutamine rearrange bonds
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o H2O produced
...


o Alcohol dehydrogenase – in yeast pyruvate can be turned to ethanol and CO2, producing
NAD
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o Required for: exporting from liver to maintain blood glucose, reacting to form pentoses (ribose,
deoxyribose, etc
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o Some of the reversible steps of glycolysis need to be overcome to send the reactions in the other
direction, regeneration of NAD+ is necessary
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o Malase turns oxaloacetate to malate to get it out of the mitochondria via the malate shuttle using
malate dehydrogenase with NAD then it is converted back with NADH and H+
...


o Fructose-1,6-bisphosphatase – converts fructose-1,6-bisphosphate to fructose-6-phosphate (the
reverse of the rate determining step in glycolysis), requites ATP, very highly regulated (reciprocal
regulation)
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Title: Biochemistry Introduction
Description: Biochemistry Introduction - Proteins (amino acids), Enzymology (mechanics, kinetics, regulation), Thermodynamics and Bioenergetics, Carbohydrate Structure and Metabolism (Glycolysis, Gluconeogenesis), 1st Year Uni of Bath BB10003, Jean Van den Elsen, Stefan Bagby

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