Enzymology

rules of catalysis

1. a catalyst can’t catalyse a thermodynamically unfavourable reaction

2. a catalyst can’t change the course of a reaction

3. a catalyst can’t change the equilibrium of a reaction, only the rate at which equilibrium is reached- it lowers the activation energy for the reaction

4. a catalyst may exert a directing influence. If 2 reactions are thermodynamically possible and a catalyst only catalyses one of them then that reaction will be favoured

5. a catalyst is recoverable, so only small amounts are necessary

specificity

• enzymes are often specific for 1 reaction only

• can distinguish between optical isomers

• 200 enzymes are commonly found in all organisms

• catalysis occurs at the active site of the enzyme

• the tertiary protein structure that determines the 3D shape of the enzyme is essential for its specific binding of substrates and its catalytic function

chemical identity

• the properties of enzymes are associated with the structure of proteins (their function depends on their shape which is determined by the tertiary structure and held together by non-covalent bonds)

• denaturation leads to loss of enzyme catalytic activity

mechanism of action

• enzymes increases the rate of the reaction they catalyse more than simple chemical catalysts do

• before a substrate can achieve this in its formation of products it must first pass through a higher energy state called the transition state. To do this it must acquire activation energy

mechanism of action of enzymes

lowers the activation energy for the reaction by binding the substrate(s) in its active site. This may have 1 or combination of effects:

1. the enzyme may position substrates in the active site and line up molecules precisely for reaction so that bonds can be broken or made more easily (‘orbital steering’)

2. some enzymes combine with the substrate to form an unstable covalent intermediate in a ‘ transition state’ that more readily undergoes reaction to form products. Substrates in the active site are oriented such that a transition state is readily formed

3. binding substrates in the active site increases the effective concentration of the reactants

4. binding substrates in the active site may bring them into close proximity to a catalytic group. enzymes may provide functional groups capable of acting as a portion donor or acceptor and an enzyme may bring about general acid- base catalysis

5. the enzyme may induce strain or distortion in the susceptible bond. these changes of shape are called conformational change

the active site

• the enzyme protein has 3D shape from its tertiary structure that determines its active site

• the amino acids forming the active site are few and my be far apart in the primary sequence but are spatially close due to the 3D structure

• the functional groups in the side chains of the amino acids interact with the substrate through weak non-covalent forces such as ionic bonds and position the substrate within the active site

• a transition state is formed between the enzyme and substrate (ES) which will then regenerate free enzyme plus products

Acetylcholinesterase

found in the synapse at the neuromuscular junction where its job is to destroy the neurotransmitter, acetylcholine, after it has medicated a nerve signal (action potential) across the synapse to the muscle. Acetylcholinesterase terminated the stimulatory signal

what is the active site of acetylcholinesterase made up of

• the anionic subsite binds the positive quaternary amine of acetylcholine. 14 aromatic amino acids line the gorge leading to the active site- tryptophan 84 is essential

• the esteratic subsite is where acetylcholine is hydrolysed to choline and acetate. a catalytic triad of 3 amino acid side chains: serine 203, histidine 447 and glutamic acid 334 attack the ester bond through the formation of a transition state in which the substrate is covalently bound to the serine prior to hydrolysis of the ester bond

mechanism of acetylcholinesterase

the enzyme has an anionic site in which negatively charged glutamic acid attracts the positively charged amine group of acetylcholine (A)

a specially activated serine in the active site is thus positioned next to and in line with the carbonyl of the ester group in the substrate. A covalent transition state intermediate is formed with this serine. Choline is released. (B)

This bond is then hydrolysed by a water molecule to release acetate (C)

nomenclature of enzymes

substrate + reaction + ase

type of reaction the enzyme catalyses

• oxidoreducatases (or dehydrogenases)

• transferases

• hydrolases

• lyases

• isomerases

• ligases

oxidoreductases (or dehydrogenases)

catalyse oxidation and reduction reactions

e.g. lactate dehydrogenase

transferases

transfer functional groups from donor to acceptor

e.g. aspartate aminotransferase

hydrolases

hydrolysis of C-O, C-N and C-C bonds

e.g. esterases, proteases

lyases

break bonds such as C-C, C-O, C-N

e.g. pyruvate decarboxylase '

isomerases

geometric or structural changes within a molecule

e.g. alanine racemase

ligases

joins molecules together, forms bonds, requiring ATP energy

e.g. glutamine synthetase

2 ways to measure enzymatic activity

1. the disappearance of substrate

2. the appearance of product

Factors affecting enzyme activity

1. Time

2. Enzyme concentration

3. Temperature

4. pH

5. Presence or absence of cofactors

6. Substrate concentration

Time

• the velocity of an enzyme reaction is the change in reactant or product concentration with time

• the initial velocity is the tangent to velocity at time 0

• the slowing down of the reaction is due to:

  • the concentration of substrate decreases

  • the concentration of product increases and this may:

    • inhibit the reaction

    • increases the velocity of the reverse reaction

    • cause a change in pH and slow the reaction

• initial velocity = change in product/ change in time

enzyme concentration

• the more enzyme present the faster the reaction goes

• if the product inhibits the enzyme then there may be a limit

• the amount of enzyme present in a tissue sample can be calculated from an assay of activity assuming that this linear relationship holds. This is important in clinical measurements

temperature

• the rate of an enzyme catalysed reaction increases with temperature

• high temperature denatures most proteins

• most enzymes are inactivated between 60-70 degrees

• initial velocity increases with temperature, the actual amount of product decreases above a certain temperature

pH

• enzymes are only active over a limited range of pH

• enzymes exist in a whole series of different states of ionisation which depend on pH

• extremes of pH will denature the enzyme and so destroy activity

Presence or absence of cofactors

• coenzymes are complex organic molecules derived from water soluble vitamins

• cofactors take part in reactions catalysed by the enzyme often as carriers of a particular chemical group

• cofactors could be:

  • organic coenzymes like coenzyme A, NADH

  • inorganic ions or activators e.g. Mg²⁺, Mn²⁺, etc.

Substrate concentration

• the value of V_max will vary with the concentration of enzyme used

Michaelis-Menten equation

K_m

• a dissociation constant for a given enzyme

• the concentration of substrate which allows the catalytic reaction to proceed at 0.5 V_max

• 1/K_m is a measure of the affinity between enzyme and substrate

V_max

• a constant for a given enzyme

• a quantitative assessment of the extent of catalysis

• the theoretical maximal rate of the reaction but it will never be achieved

line weaver-burk equation

turnover number (K_cat)

• gives the rate constant for the catalytic reaction

• enzyme turnover numbers range from 10-10⁷ per sec

• an turnover number of 1000 sec^-1 will catalyse the conversion of 1000 molecules of substrate to product per second

equation for turnover number

assumptions when calculating Michaelis-Menten enzyme kinetics

1. enzyme-substrate complex is formed

2. that the equilibrium where the rate of ES formation is equal to its breakdown is reached instantaneously

3. that the concentration of substrate in ES is negligible- enzyme concentration will probably be about 0.001% of the substrate concentration

4. there is no k_-2 i.e. there is no chance of E + P forming ES- it’s an irreversible reaction

allosteric enzymes

distinct regulatory (allosteric) and catalytic sites

binding of an effector (allosteric modulator) can either stimulate or inhibit the enzyme activity

how do allosteric enzymes work

• these enzymes also have an allosteric regulatory site distinct from the catalytic active site

• this allows the binding of an activator or inhibitor molecule (effector) which is structurally unrelated to the substrate and which can either stimulate or inhibit the enzyme activity by changing the shape of the active site and thus changing its affinity for substrate

• the effector could be another molecule in the metabolic pathway or even an ion such as Ca²⁺

Co-operative binding: aspartate transcarbamoylase (ATCase)

• enzyme that catalyses an early step in synthesis of pyrimidines

• ATCase is negatively regulated by CTP

• ATCase is positively regulated by ATP

  • If ATP abundant, DNA and RNA synthesis active

  • If ATP abundant and [ATP] > [CTP], need for pyrimidine synthesis to match the availability of purines

isozymes

an enzyme which has multiple molecular forms catalysing the same reaction

an example of an isozymes

• lactate dehydrogenase- can occur in 5 possible forms in organs of vertebrates

• the enzyme is a tetramer made from 2 types of monomer- M (muscle) and H (heart) and these 2 in various combinations give the 5 isozymes

• the various combinations have different kinetic properties depending on the physiological roles they perform

combinations of lactate dehydrogenase

LDH-1 (4H)- in the heart

LDH-2 (3H1M)- in the reticuloendothelial system

LDH-3 (2H2M)- in the lungs

LDH-4 (1H3M)- in the kidneys, placenta and pancreas

LDH-5 (4M)- in the liver and striated muscle

multifunctional enzymes

multiple active sits on a single polypeptide chain

example of multifunctional enzymes

mammalian fatty acid synthase has 7 different catalytic activities in 1 protein molecule. this allows a metabolic pathway to operate within a single enzyme which has advantage in terms of speed and control aa well as absence from interference

multienzyme complexes

there is an association of separate enzymes in separate subunits. the enzyme are organised physically so that the product of 1 becomes the substrate of another, without leaving the complex. this ensures a highly efficient progression from reactants to products

enzyme inhibition

• many drugs exert their action by inhibition of enzyme activity in the body

• inhibition may lead to death of the cell or organism

• you can design new drugs which are enzyme inhibitors once a target enzyme has been identified

reversible inhibitors

the effect of the inhibitor is instantaneous and it can be removed from the enzyme by dialysis or dilution so that the enzyme activity is returned to normal. Such inhibitors react with the enzyme by weak noncovalent bonds to form an enzyme inhibitor complex

E + I ←> EI

irreversible inhibitor

bind very tightly to the enzyme sometimes by formation of covalent bonds to form an enzyme inhibitor compound rather than a loose complex. the effect is therefore progressive, reaching a maximum when all the enzyme has reacted. this is not easily reversible by simple physical treatments such as dialysis

E + I → EI

competitive reversible enzyme inhibitor

molecules which closely resemble the substrate in size, shape and charge distribution may also fit the active site of the enzyme and this may lead to inhibition because the active site is blocked

both I and S compete for the same active site, if [S] is increased sufficiently while [I] is constant, the proportion of I bound to E will decrease hence formation of ES will increase and the rate of enzyme action will increase. If sufficient [S] is present then eventually the inhibition by I will be overcome

Michaelis-Menten equation for competitive reversible inhibitor

V_max is unchanged

[I]

inhibitor concentration

k_i

the dissociation constant for the equilibrium between E and I

the concentration of inhibitor required to slow the reaction to half the rate it shoes in the absence of inhibitor

line weaver burk plot for competitive reversible inhibitor

y-intercept = 1/V_max

line weaver burk plot equation

non-competitive inhibitor

reacts with the enzyme-substrate complex and slows the rate of reaction to form the enzyme-product complex

• when both the substrate and inhibitor are bound, the enzyme-substrate- inhibitor complex can’t form product and can only be converted back to the enzyme-substrate complex or the enzyme-inhibitor complex

• V_max is changed

• K_m is unchanged

reversible

timescale of the inhibition is similar to that of enzyme action

irreversible

the enzyme activity is inhibited for times significantly longer than the assay times for the enzyme

quasi-irreversible

in between reversible and irreversible

including tight-binding inhibitor, transition state analogues and slowly dissociating intermediate

potency

the inhibitor will need to be potent enough so that the dose require is in the order of milligrams to grams

specificity- enzyme inhibitors as drugs

if a compound is a nonspecific enzyme inhibitor it is more likely to be toxic and exhibit side effects

simple reversible inhibitor

binds to the enzyme and decreases activity instantaneously and reverses within the time of the enzyme action

the inhibitor binds noncovalently to the enzyme and the strength of binding us of a similar order to the substrate

k_i will be of similar size to k_m

tight binding inhibitors

binding is very tight (K_i in order of 10^-9 mol L^-1 to 10^-10 mol L^-1)

they are potent enough to act as drugs in vivo

transition state analogue

binds much tighter than an analogue of the substrate

the outcome is a potent in vivo and potentially specific inhibitor

competitive reversible inhibitors of acetylcholinesterase

Ki values of 10^-3 mol L^-1 to 10^-5 mol L^-1 so they are too weak to have much action in vivo

lower the Ki

the greater the affinity of the inhibitor and hence the greater its potency

examples of acetylcholinesterases

edrophonium

neostigtmine

physostigmine (eserine)

edrophonium

competitive reversible inhibitor of acetylcholinesterase used in the diagnosis of myasthenia gravis because in vivo its action is very short

has a quaternary amine giving it a positive charge and will react with the anionic site on acetylcholinesterase

edrophonium won’t react with the esteratic site because it has no ester bond

neostigmine

a more potent acetylcholinesterase

they bind to the enzyme with high affinity and remain on the enzyme for much longer

process of neostigmine

A. the quaternary (+) nitrogen of neostigmine is attracted to the anionic site positioning the ester portion of the molecule in close proximity to the catalytic site

B. the ester bond on neostigmine is cleaved, the enzyme is carbamylated, which prevents the enzyme from interacting with ACh and the remainder of the neostigmine molecule is released

C. hydrolysis of carbamylated enzyme occurs slowly casing reversible inhibition of the enzyme

physostigmine (eserine)

has parasympathomimetic properties and has been used in the treatment of glaucoma

example of irreversible anticholinesterases

organophosphates

organophosphates

a typical member is diisopropylfluorphosphate (DFP)

the active site serine reacts with organophosphates to form a covalent intermediate which is not hydrolysed and the inhibition is reversible

DFP causes severe in vivo symptoms resulting in death and is very toxic

although enzyme poisoned with organophosphates will not regenerate they can be reactivated using an antidote

3 levels of regulation of enzyme activity within cells

1. allosteric regulation

2. covalent modification

3. regulation of enzyme concentration

allosteric regulation of enzyme activity

some enzymes have binding sites for small molecules. the effectors can:

• stimulate enzyme activity

• inhibit enzyme activity

the allosteric binding sites are distinct from the active catalytic site of the enzyme

feedback inhibitor

allosteric inhibitors are often end-products of a pathway that feed=back and inhibit an early step in the pathway to prevent overproduction and wastage

feedforward activation

allosteric activators are often substrates for the reaction or the pathway that feed-forward to encourage the pathway to dispose of the substrate

mechanism of allosteric sites

the allosteric effector binds specifically and reversibly to the allosteric sites on the enzyme and is done rather than non-covalent bonding. Binding at the allosteric site induces a conformational change in the protein that alters the shape of the active site

allosteric regulation

many regulatory enzymes are multimeric with several copies of a single polypeptide chain each with its own active site forming the quaternary structure of the enzyme

cooperativity

binding of substrate to the active site of 1 subunit causes a conformational change that is transmitted through the quaternary structure to the other subunits and makes it easier for these bind substrate

allosteric activators

stabilise the conformation of the substrate bound form

allosteric inhibitor

stabilise the conformation of the no-substrate bound form