Doctor of Medicine (MD) Yr 1

BLOCK MODULE A: BMED 1101

From Molecules to Cells:
Biochemistry and Metabolism
Session 4

University of Mauritius

What we covered in session 3

principles of catalysis of chemical reactions by
enzymes
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

Session 4: Enzymes II
Contents:
factors affecting enzyme activity; allosteric
enzymes, coenzymes, redox coenzymes,
coenzyme A.
4.1

Factors affecting enzyme activity

4.2

Allosteric enzymes

4.3

Co-enzymes

4.1 Factors affecting enzyme activity

Temperature
pH

Protein denaturation
Protein denaturation mostly results in an irreversible
and non-specific inhibition of enzyme activity.

Enzyme inhibitors
Enzyme inhibitors are molecules that interfere with
catalysis, slowing down or halting enzymatic
reactions.
Uses:

Pharmaceutical agents
Solving enzyme kinetics

Resolving metabolic pathways.

Enzyme inhibition
There are 2 classes of enzyme inhibitors:
Reversible inhibitors:

Competitive, uncompetitive, non-competitive, mixed

Irreversible inhibitors

Covalently attached

Reversible inhibition
One common type of reversible inhibition is called
competitive. A competitive inhibitor competes with
the substrate for the active site of the enzyme.

Competitive inhibition
Many competitive inhibitors are structurally similar to
the substrate and combine with the enzyme to
form an EI complex.
This association leads to a reduction in the enzyme
efficiency.
Can be analysed by steady-state enzyme kinetics.
Can be relieved by increasing [S].

Reversible inhibition
One common type of reversible inhibition is called
competitive. A competitive inhibitor competes with
the substrate for the active site of the enzyme.

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Michaelis-Menten Model
What does low KM indicate in terms of affinity and
capacity?
High affinity, low capacity.
What does high KM indicate in terms of affinity and
capacity?
Low affinity, high capacity.

Binding
Site

Binding of a glucose molecule

induces a conformational
change.

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Michaelis-Menten Model
From this reaction path, we see that:
1. There is a limit to the amount of S that a single E
molecule can process in a given time;
2. An increase in [S] increases the rate at which P is
formed, 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 the
turnover number.
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Reversible competitive inhibitors

Structural analogues of substrates. Increase KM, do
not change Vmax.

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Reversible competitive inhibitors

Cyanide is a competitive inhibitor of cytochrome
oxidase.

Cytochrome oxidase is essential to one of the last

stages of cellular respiration.
Without COX activity, pulmonary respiratory
movements stop.
Zyklon-B used in Nazi Death camps: hydrogen cyanide

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Reversible competitive inhibitors

Ethanol is catabolised by oxidation in the liver in two
stages: ?

a) to acetaldehyde b) to acetic acid

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Reversible competitive inhibitors

Antabuse: competitive inhibitor of acetaldehyde
dehydrogenase. What would accumulate here?

Results in undesirable effects

16

Noncompetitive inhibition
Here the inhibitor and substrate can bind
simultaneously. The inhibitor decreases the
turnover. Cannot be overcome by increasing [S].

Examples: metals like Cu, Hg, Ag

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Michaelis-Menten Model
What does low KM indicate in terms of affinity and
capacity?
High affinity, low capacity.
What does high KM indicate in terms of affinity and
capacity?
Low affinity, high capacity.

Binding
Site

Binding of a glucose molecule

induces a conformational
change.

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Michaelis-Menten Model
From this reaction path, we see that:
1. There is a limit to the amount of S that a single E
molecule can process in a given time;
2. An increase in [S] increases the rate at which P is
formed, 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 the
turnover number.
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Reversible noncompetitive inhibitors

The inhibitor does not look like the substrate. Do not
change KM, decrease Vmax.

20

Lineweaver-Burk plots
The inhibitor does not look like the substrate.

Increases KM, does not change Vmax.

Does not change KM, decreases Vmax.

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Irreversible inhibition
The inhibitors bind covalently with or destroy a
functional group that is essential for the enzymes
activity.
Useful for studying reaction mechanisms and for
identifying key amino acid residues in enzymes.

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Activators

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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4.2 Allosteric enzymes

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

For example, the allosteric enzymes.

Consist of multiple subunits and multiple active sites.
These parts co-operate.

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Allosteric enzymes
Do not obey Michaelis-Menten kinetics display a
sigmoidal curve:

25

Allosteric enzymes
As [S] tends to infinity, vi = vmax

26

Allosteric enzymes
Vi accelerates as it nears Km (or K0.5), then brutally
slows down after Km.
The substrate interaction with the enzyme increases
the affinity of the enzyme for the substrate.

27

Allosteric enzymes
Concerted model of cooperativity

T-state:
weak affinity for S

T and R?

R-state:
High affinity for S

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Concerted Model
Here, a conformational change in one subunit is
necessarily transferred to all other subunits.

Thus, all subunits must exist in the same

conformation.
In the absence of S, the equilibrium favours one of
the conformational states, T or R.
Binding of an effector molecule shifts the equilibrium
to the R or T state.

29

Allosteric enzymes
Sequential model of cooperativity

T-state:
weak affinity
for S

R-state:
Increasing affinity for S

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Sequential Model

Subunits are not connected in such a way that a

conformational change in one induces a similar
change in the others.

Substrate molecules bind via the induced fit model.

Substrate-binding at one subunit only slightly

alters the structure of other subunits to increase
substrate affinity in adjacent subunits.

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Effectors
Activities of allosteric enzymes may be altered by
regulatory molecules that are reversibly bound to
specific sites other than active sites.
Hence allosteric (allos = different, Gr.)
Their catalytic activities can thus be adjusted to meet
the metabolic needs of a cell.
Key regulators of metabolic pathways.

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Effectors
Effectors can be positive = activators, or negative =
inhibitors.
Affinity towards substrate can either be increased or
decreased.

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Example: Phosphofructokinase in glycolysis

Activated by ADP.
Inhibited by ATP.

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4.3 Co-enzymes
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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36

Coenzymes
Can transfer the following transiently, from one
molecule to another:

Electrons

Atoms

Molecules

37

Coenzymes
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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Mechanism of coenzyme activity as cosubstrates

Weak interactions with enzyme through noncovalent 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 prosthetic groups

Covalently linked

Participate in one coupled double enzymatic

reaction in one operation, i.e.:

Get hold of factor X and transfer factor X in one

operation.

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Coenzymes involved in oxidoreduction

Operate by transferring reducing equivalents:

Electrons e-

Hydrogen atoms H

Hydride ions H-

1 reducing equivalent = 1 electron transferred in a

redox reaction (independent of transfer
mechanism)

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Coenzymes involved in redox reactions

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

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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Example:

Enzyme E dehydrogenase

Redox coenzyme NAD

Reduced metabolite AH2

Below, E has extracted reducing equivalents from AH2.

The reduced coenzyme can transfer its electrons
to another factor.
This results in both extraction and transfer of energy.

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

Niacin: refers to nicotinic acid and nicotinic amide

NAD and NADP play an essential role in electron

transfer in metabolic pathways.

Niacin is present in animal and plant sources of

nutrients.

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Coenzyme NAD(P)

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Coenzyme FAD

FAD: dinucleotide formed from FMN and AMP

(flavin mononucleotide and adenosine
monophosphate)

Derived from riboflavin (Vitamin B2)

Prosthetic group of some dehydrogenases.

Transfers reducing equivalents to the respiratory

chain.

Found in mitochondria.

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Coenzyme FAD

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Coenzyme A, or CoA

Transports acyl groups

Derived from vitamins (pantothenic acid)

Forms acetyl-CoA, which is coupled to exergonic

reactions

Acetyl-coA is central to metabolic pathways.

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Coenzyme A, or CoA

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What you should learn from this lecture

factors that affect enzyme activity
types of enzyme inhibitors and their kinetics
two models for allosteric enzyme function

non-Michaelis-Menten enzyme kinetics displayed by

allosteric enzymes
structures and functions of coenzymes
basic mechanisms of action of coenzymes