Al-Maarif University

College of Pharmacy
Biochemistry (1)
1st Semester 2024/ 2025

ENZYMES

Ass. Prof. Dr. Abdulrahman Al-Bazzaz

Assoc. Prof. of Biochemistry
 Enzymes
▪ They are protein catalysts that increase the rate of reactions without
being changed in the overall process.

▪ The reaction must be thermodynamically (energetically) possible.

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 Enzyme major classes

Lactate dehydrogenase EC number 1.1.1.27

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 Properties of enzymes
1. Protein in nature: heat liable, large molecular weight
2. Active sites: substrate binding sites
3. Catalytic efficiency: enzymes are highly efficient, 103–108 times faster than
uncatalyzed reactions
4. Specificity: enzymes are highly specific, interacting with one or a few substrates and
catalyzing only 1-type of chemical reaction (Key& Lock theory or Induced Fit Theory)
5. Holoenzymes:

a) Protein part: Apoenzyme but is inactive alone

b) Non-protein part:

1. Cofactor: metal ion Fe and Zn

2. Coenzyme or prosthetic group: small organic molecule (vitamins, NAD, FAD)
6. Most of enzymes are intracellular
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 How enzymes work
❑ Two theories describe the mechanism of enzyme action

1. Energy changes occurring during the reaction

▪ All chemical reactions have an energy barrier separating the reactants

and the products.
▪ This barrier, called the free energy of activation, is the energy
difference between that of the reactants and a high-energy
intermediate that occurs during the formation of product.
▪ For molecules to react, they must contain sufficient energy to overcome
the energy barrier of the transition state.
▪ Enzyme accelerates the rate of reaction by lowering the free energy of
activation.
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 How enzymes work

❑ Turnover (rate or velocity): number of

moles of substrate converted to product
per second (mol/s).
❑ 1 Katal: amount of enzyme required to
increase the turnover by 1 mol/s
❑ IU: amount of enzymes that catalyze
conversion of 1 μmol of substrate to
product per minute
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 How enzymes work
2. Chemistry of the active site

▪ Active site is a complex molecular machine employing a

diversity of chemical mechanisms to facilitate the
conversion of substrate to product by:
a) Stabilizing the transition state
b) Providing catalytic groups that enhance the probability that
the transition state is formed.

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 Factors affecting reaction velocity or rate
Rate or velocity of a reaction (v) is the number of substrate
molecules converted to product per unit time. (μmol/min)
1. Enzyme concentration (with excess substrate)

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 Factors affecting reaction velocity
2. Substrate concentration

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Factors affecting reaction velocity

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 Factors affecting reaction velocity
3. Temperature

The optimum temperature for

most human enzymes is
between 35 and 40°C.
Human enzymes start to
denatur at temperatures above
40°C.

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 Factors affecting reaction velocity

4. Effect of pH:

a) Effect of pH on the ionization of the

active site

b) Effect of pH on enzyme denaturation

c) The pH optimum varies for different

enzymes

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 Enzyme kinetics
❑ MICHAELIS-MENTEN equation

▪ The enzyme reversibly combines with its substrate to form

an ES complex that subsequently yields product, regenerating
the free enzyme.

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 Enzyme kinetics

▪ The [S] is much greater than the [E], so that the percentage of total
substrate bound by the enzyme at any one time is small.
▪ [ES] does not change with time (the steady-state assumption), that is,
the rate of formation of ES is equal to that of the breakdown of ES (to
E+S and to E+P).
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Enzyme kinetics

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 Enzyme kinetics
▪ Km is characteristic of an enzyme & its particular substrate,
and reflects the affinity of the enzyme for that substrate.

▪ Km is numerically equal to the substrate concentration at

which the reaction velocity is equal to ½ Vmax.

▪ Km does not vary with the concentration of enzyme

Low Km ? ? ?
High Km ? ? ?
▪ Glucokinas (has high km for glucose)
▪ Hexokinase (has low km for glucose)
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 Enzyme kinetics
▪ Order of reaction:

1. When [S] is much less than Km, the velocity of the reaction is
approximately proportional to the substrate concentration
(first order).

2. When [S] is much greater than Km, the velocity is constant

and equal to Vmax. The rate of reaction is then independent of
substrate concentration, and is said to be (zero order).

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 Lineweaver-Burk plot
▪ When V0 is plotted against [S], it is not always possible to determine
when Vmax has been achieved, because of the gradual upward slope of
the hyperbolic curve at high substrate concentrations.

▪ However, if 1 / V0 is plotted versus 1 / [S], a straight line is obtained

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Lineweaver-Burk plot

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 5- Regulation of enzyme activity
1) Allosteric Regulation (non-covalent modification):
Effectors that bind non-covalently at a site other than the active site.
a) Homotropic effectors: substrate positively feed back the enzyme

b) Heterotropic effectors: product negatively feed back the enzyme

2) Covalent modification: phosphorylation and dephosphorylation (by the addition or

removal of phosphate groups from specific serine, threonine, or tyrosine residues of
the enzyme)

3) Induction and repression of enzyme synthesis: cells can regulate the amount of enzyme
present by altering the rate of enzyme degradation (repression) or, the rate of enzyme
synthesis (induction).

NB. 1 and 2 affect the enzyme activity (rapid) while 3 affects the amount of enzyme itself
(slow)
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 6- Inhibition of enzyme activity

▪ Any substance that can diminish the velocity of an enzyme

catalyzed reaction is called an inhibitor.

▪ They are of 2- types:

a) Irreversible inhibitors: bind to enzymes through covalent bonds.

b) Reversible inhibitors: bind to enzymes through non-covalent

bonds, thus dilution of the enzyme–inhibitor complex results in
recovery of enzyme activity (Competitive and Non-competitive).

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 Inhibition of enzyme activity ( Reversible inhibitor)
A. Competitive inhibitors: substrate and inhibitor bind to the same site
and, therefore, inhibitor competes with the substrate for that site
e.g: Statins drugs, sulfa drugs.

▪ Effect on Vmax: no change

▪ Effect on Km: ↑ Km (in the presence of a competitive inhibitor, more

substrate is needed to achieve ½ Vmax)

Dr. Yasser I. Kandil September 8, 2024

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 Inhibition of enzyme activity
B. Non-competitive inhibitors: inhibitor and substrate bind at different sites
on the enzyme: e.g. heavy metals and oxidizing agents.

▪ Effect on Vmax: ↓ Vmax

▪ Effect on Km: no change (do not interfere with the binding of substrate to
enzyme

Dr. Yasser I. Kandil September 8, 2024

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Competitive and Non- competitive inhibitors
Competitive

Non-Competitive

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 Enzymes in clinical diagnosis
▪ Plasma enzymes can be classified into two major groups:
1) Functional Enzymes: they are enzymes that act on substrate normally
present in plasma (coagulation enzymes, LCAT, LPL)
2) Non-functional Enzymes: they are enzymes that act on substrate within
the cell (intracellular) and have no physiologic use in the plasma, these
enzymes may released from cells to plasma during normal cell turnover
e.g: ALT, AST, ALP, amylase

Dr. Yasser I. Kandil September 8, 2024