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University of Mauritius

Doctor of Medicine (MD) Yr 1

BLOCK MODULE A: BMED 1101From Molecules to Cells:Biochemistry and Metabolism

Session 4

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principles of catalysis of chemical reactions byenzymes

• principles of enzyme kinetics

• quantitative assessment of enzyme kinetics

• nomenclature and classification of enzymes

• the nature of the active site of enzymes

• models of enzyme-substrate interactions

What we covered in session 3

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Contents:

factors affecting enzyme activity; allostericenzymes, coenzymes, redox coenzymes,

coenzyme A.4.1 Factors affecting enzyme activity

4.2 Allosteric enzymes

4.3 Co-enzymes

Session 4: Enzymes II

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• Temperature• pH

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4.1 Factors affecting enzyme activity

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Protein denaturation mostly results in an irreversible

and non-specific inhibition of enzyme activity.

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Protein denaturation

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Enzyme inhibitors are molecules that interfere with

catalysis, slowing down or halting enzymaticreactions.

Uses:

• Pharmaceutical agents

• Solving enzyme kinetics

• Resolving metabolic pathways.

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Enzyme inhibitors

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There are 2 classes of enzyme inhibitors:Reversible inhibitors:

• Competitive, uncompetitive, non-competitive, mixed

Irreversible inhibitors• Covalently attached

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Enzyme inhibition

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One common type of reversible inhibition is calledcompetitive. A competitive inhibitor competes withthe substrate for the active site of the enzyme.

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Reversible inhibition

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Many competitive inhibitors are structurally similar tothe substrate and combine with the enzyme toform an EI complex.

This association leads to a reduction in the enzymeefficiency.

Can be analysed by steady-state enzyme kinetics.

Can be relieved by increasing [S].

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Competitive inhibition

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One common type of reversible inhibition is calledcompetitive. A competitive inhibitor competes withthe substrate for the active site of the enzyme.

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Reversible inhibition

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What does low KM indicate in terms of affinity and

capacity?High affinity, low capacity.

What does high KM indicate in terms of affinity andcapacity?

Low affinity, high capacity.

Michaelis-Menten Model

BindingSite Binding of a glucose molecule

induces a conformational

change.

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From this reaction path, we see that:

1. There is a limit to the amount of S that a single Emolecule can process in a given time;

2. An increase in [S] increases the rate at which P isformed, up to a maximum value.

At the maximum value (Vmax), E is saturated with S.

Vmax depends ONLY on how rapidly E can process S.

This maximum rate divided by [E] is called theturnover number.

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Structural analogues of substrates. Increase KM, donot change Vmax.

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Reversible competitive inhibitors

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Cyanide is a competitive inhibitor of cytochromeoxidase.

Cytochrome oxidase is essential to one of the laststages of cellular respiration.

Without COX activity, pulmonary respiratorymovements stop.

Zyklon-B used in Nazi Death camps: hydrogen cyanide

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Ethanol is catabolised by oxidation in the liver in two

stages: ?a) to acetaldehyde b) to acetic acid

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Antabuse: competitive inhibitor of acetaldehyde

dehydrogenase. What would accumulate here?Results in undesirable effects

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Here the inhibitor and substrate can bindsimultaneously. The inhibitor decreases theturnover. Cannot be overcome by increasing [S].

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Noncompetitive inhibition

Examples: metals like Cu, Hg, Ag

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What does low KM indicate in terms of affinity and

capacity?High affinity, low capacity.

What does high KM indicate in terms of affinity andcapacity?

Low affinity, high capacity.


BindingSite Binding of a glucose molecule

induces a conformational

change.

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From this reaction path, we see that:

1. There is a limit to the amount of S that a single Emolecule can process in a given time;

2. An increase in [S] increases the rate at which P isformed, up to a maximum value.

At the maximum value (Vmax), E is saturated with S.

Vmax depends ONLY on how rapidly E can process S.

This maximum rate divided by [E] is called theturnover number.

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Reversible noncompetitive inhibitors

The inhibitor does not look like the substrate. Do not

change KM, decrease Vmax.

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Lineweaver-Burk plots

The inhibitor does not look like the substrate.

Does not change KM, decreases Vmax.Increases KM, does not change Vmax.

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The inhibitors bind covalently with or destroy afunctional group that is essential for the enzyme’sactivity.

Useful for studying reaction mechanisms and foridentifying key amino acid residues in enzymes.

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Irreversible inhibition

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• Metal ions (Ca, Mg, etc).• Limited proteolysis – a protease can cleave a

protein to activate its enzymatic activity.

• Reversible covalent modification e.g.

phosphorylation/dephosphorylation

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Activators

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The Michaelis-Menten model cannot account for thekinetics of many enzymes.

For example, the allosteric enzymes.

Consist of multiple subunits and multiple active sites.

These parts co-operate.

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4.2 Allosteric enzymes

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Do not obey Michaelis-Menten kinetics – display asigmoidal curve:

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Allosteric enzymes

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As [S] tends to infinity, vi = vmax

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Allosteric enzymes

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Vi accelerates as it nears Km (or K0.5), then brutally

slows down after Km.The substrate interaction with the enzyme increasesthe affinity of the enzyme for the substrate.

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Allosteric enzymes

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Concerted model of cooperativity

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Allosteric enzymes

T-state:weak affinity for S

R-state:High affinity for S

T and R?

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Here, a conformational change in one subunit isnecessarily transferred to all other subunits.

Thus, all subunits must exist in the sameconformation.

In the absence of S, the equilibrium favours one ofthe conformational states, T or R.

Binding of an effector molecule shifts the equilibriumto the R or T state.

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Concerted Model

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Sequential model of cooperativity

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Allosteric enzymes

T-state:weak affinity

for S

R-state:Increasing affinity for S

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• Subunits are not connected in such a way that aconformational change in one induces a similarchange in the others.

• Substrate molecules bind via the induced fit model.

• Substrate-binding at one subunit only slightlyalters the structure of other subunits to increasesubstrate affinity in adjacent subunits.

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Sequential Model

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Activities of allosteric enzymes may be altered by

regulatory molecules that are reversibly bound tospecific sites other than active sites.

Hence allosteric (allos = different, Gr.)

Their catalytic activities can thus be adjusted to meetthe metabolic needs of a cell.

Key regulators of metabolic pathways.

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Effectors

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Effectors can be positive = activators, or negative =

inhibitors.Affinity towards substrate can either be increased or

decreased.

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Effectors

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Activated by ADP.

Inhibited by ATP.

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Example: Phosphofructokinase in glycolysis

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• Essential to activity of many enzymes.

• Are not proteins.

• Regain their initial state after the activity.

• Participate in the stoichiometry of the reaction.• Are generally vitamins.

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4.3 Co-enzymes

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Can transfer the following transiently, from one

molecule to another:• Electrons

• Atoms

• Molecules

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Coenzymes

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Two mechanisms:

• As co-substrates (free coenzyme) e.g. NAD

• As prosthetic groups (linked coenzyme) e.g. FAD

Two functions:

• Oxidoreduction

• Transfer of groups of atoms

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Coenzymes

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• Weak interactions with enzyme through non-

covalent interactions• Participate in two coupled sequential enzymatic

reactions:

– Get hold of factor X

– Transfer factor X

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Mechanism of coenzyme activity as cosubstrates

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• Covalently linked

• Participate in one coupled double enzymaticreaction in one operation, i.e.:

– Get hold of factor X and transfer factor X in oneoperation.

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Mechanism of coenzyme activity as prosthetic groups

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Operate by transferring reducing equivalents:

• Electrons e-

• Hydrogen atoms H

• Hydride ions H-

1 reducing equivalent = 1 electron transferred in aredox reaction (independent of transfermechanism)

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Coenzymes involved in oxidoreduction

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Transfer of reducing equivalents – only two types to

be considered here:• Pyridine-based: NAD and NADP

• Flavin-based: FAD and FMN

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Coenzymes involved in redox reactions

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These coenzymes act as cofactors for

oxidoreductases:• Oxidases (transfer from S to oxygen)

• Dehydrogenases (electron transfer to S)

• Hydroproxydases (degrade H2O2)

• Oxygenases (transfer of oxygen to S)

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Coenzymes involved in redox reactions

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• Enzyme E – dehydrogenase

• Redox coenzyme NAD

• Reduced metabolite AH2

Below, E has extracted reducing equivalents from AH2.

The reduced coenzyme can transfer its electronsto another factor.

This results in both extraction and transfer of energy.

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Example:

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• Niacin: refers to nicotinic acid and nicotinic amide

• NAD and NADP play an essential role in electrontransfer in metabolic pathways.

• Niacin is present in animal and plant sources ofnutrients.

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Coenzyme NAD(P), Vitamin PP(B3) or niacin

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Coenzyme NAD(P)

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• FAD: dinucleotide formed from FMN and AMP

(flavin mononucleotide and adenosinemonophosphate)

• Derived from riboflavin (Vitamin B2)

• Prosthetic group of some dehydrogenases.

• Transfers reducing equivalents to the respiratorychain.

• Found in mitochondria.

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Coenzyme FAD

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Coenzyme FAD

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• Transports acyl groups

• Derived from vitamins (pantothenic acid)

• Forms acetyl-CoA, which is coupled to exergonicreactions

• Acetyl-coA is central to metabolic pathways.

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Coenzyme A, or CoA

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Coenzyme A, or CoA

l l l

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•

factors that affect enzyme activity• types of enzyme inhibitors and their kinetics

• two models for allosteric enzyme function

• non-Michaelis-Menten enzyme kinetics displayed byallosteric enzymes

• structures and functions of coenzymes

• basic mechanisms of action of coenzymes

What you should learn from this lecture

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