ENZYMES

Lecture Learning Outcomes

By the end of the lecture students will be able to:

• Define enzyme structure and function, emphasizing the role of active

sites in catalyzing biochemical reactions.

• Explain the factors influencing enzyme activity.

• Describe the mechanisms of enzyme catalysis.

• Discuss enzyme kinetics concepts such as Michaelis-Menten kinetics

• Explore enzyme regulation mechanisms.

 • Enzymes are biological catalysts produced by
living cells.
What are
Enzymes: • Enzymes are Biological substance that
accelerate the reaction without being
consumed or produced from the reaction.
 NOMENCLATURE
A. Recommended name:
1. Enzyme names have the suffix “-ase” attached to the substrate of the
reaction (for example, glucosidase and urease)
2. Substrate name and description of the action performed (for example,
lactate dehydrogenase and Pyruvate carboxylase).
B. Systematic name:
Enzymes are divided into six major classes each with numerous subgroups.
The suffix “-ase” is attached to a complete description of the chemical
reaction catalyzed, including the names of the substrates (for example,
Lactate: nicotinamide adenine dinucleotide (NAD+) oxidoreductase).
CLASSIFICATION OF ENZYMES
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
 Enzymes Properties
1. The active site:
• A special pocket or cleft ,which is concerned with the
binding of the substrate and the process of catalysis

• This small area consists of certain chemical groups [such

Active Site
as — OH, — SH, — NH3, — COO-], found in the side
chains of the amino acid residues of the enzyme protein.

• In most cases substrates are held in the active site by

weak interactions
 Enzymes Properties
2. Efficiency:

The turnover number is the number of substrate molecules converted to product

per enzyme molecule per second.

3. Specificity:

Enzymes are highly specific, interacting with one or a few substrates and
catalyzing only one type of chemical reaction.

The set of enzymes made in a cell determines which reactions occur in that cell
 Enzymes Properties
4. Nature of Enzyme:
❑Virtually all enzymes are proteins:
• Some are simple proteins e.g. pepsin while others are conjugated proteins.
• Others are conjugated proteins “holoenzyme” which consists of:
1. Protein part, the apoenzyme
2. Non-protein part, coenzyme or cofactor
• If the nonprotein part is a metal ion, such as zinc (Zn2+) or iron (Fe2+),
it is called a cofactor
• If it is a small organic molecule, it is termed a coenzyme.
• Transiently associate with enzyme cosubstrates.
• Permanently associated with the enzyme prosthetic group.
Enzymes Properties

5. Regulation:

Enzyme activity can be increased or decreased

according to the body needs

6. Location within the cell:

Enzymes are often localized in specific organelles

within the cell.
 MECHANISM OF ENZYME ACTION
(HOW ENZYMES WORK)
❑ The mechanism of enzyme action can be viewed by:

A. Treating catalysis in terms of energy changes that occur during the

reaction. That is, enzymes provide an alternate, energetically
favorable reaction pathway different from the uncatalyzed reaction.

B. Describing how the active site chemically facilitates catalysis.

Chemical reactions
• Chemical reactions need an initial
input of energy THE ACTIVATION
ENERGY.

• During this part of the reaction the

molecules are said to be in a
transition state.
 Making reactions go faster:
• Temperature increase molecules movement

• Biological systems are very sensitive to

temperature changes

• Enzymes can increase the rate of reactions

without increasing the temperature

• They do this by lowering the activation energy

• They create a new reaction pathway “a short cut” An enzyme-controlled pathway

 A. Free energy of activation Transition state: is an unstable
structural state of substrate
• This peak of energy before converting to product
represents the transition
state in which a high- energy Energy of activation: is the
intermediate is formed energy required for substrate to
during the conversion of reach the transition state
reactant to product.
• Because of the large
activation energy, the rate of
uncatalyzed chemical • There is no difference In the
reactions is often slow.
free energy ∆G of the
overall reaction between the
catalyzed and uncatalyzed
reactions.
• Free energy ∆G: energy of
reactants minus energy of
products
 B. Active site chemistry
1.Transition-state stabilization: Enzymes stabilize transition
states, increasing reactive intermediate concentration and
reaction rates.

2.Catalysis: Enzymes provide catalytic groups for transition state

formation, including general acid–base and covalent catalysis.

3.Transition-state visualization: Enzymes guide reactions akin to

removing a sweater from an infant, facilitating reaction
pathways
Factors affecting Enzymes
A. Temperature B. PH C. Substrate concentration
• In studying the effect of any of these factors, only one factor has to be
varied at a time, all other factors being kept constant.

• The rate of the reaction should always be measured at the very

beginning of the reaction, the so-called initial velocity (vo).

• The rate or velocity of a reaction (v) is the number of substrate

molecules converted to product per unit time (µmol/mint.).
• Maximal velocity reaction can be reached expressed by (Vmax).
 A. The effect of temperature
❑ Velocity increase with temperature:
• The reaction velocity increases with temperature until a peak
velocity is reached, as a results of increased number of
substrate having sufficient energy to form the products.
❑ Velocity decrease with higher temperature:
• Further elevation of the temperature causes a decrease in
reaction velocity, as a result of temperature-induced
denaturation of the enzyme.

❖ The optimum temperature for most human enzymes is

between 35°C and 40°C. Human enzymes start to denature
at temperatures above 40°C
B. The effect of pH
• The active site is distorted and the substrate
molecules will no longer fit in it

• Extreme pH levels will produce denaturation of

enzyme

• At pH values slightly different from the enzyme’s

optimum value, small changes in the charges of
the enzyme and it’s substrate molecules will occur
 C. Substrate concentration:
• At low substrate concentration, only a few enzyme molecules
are in the form of E-S complex → As the substrate concentration
is increased more enzyme molecules are involved in the
formation of E-S complex and the velocity of the reaction
increases.

• This is true up to the point when all enzyme molecules are in the
Faster reaction but it reaches a
form of E-S complex at which the maximal rate of the reaction saturation point when all the
enzyme molecules are occupied.
Vmax is reached. Any further increase in the substrate
concentration will not increase the velocity of the reaction
 Michaelis-Menten equation
• The relation between the initial velocity of the
reaction, vo, and the substrate concentration [S] is
expressed by the Michaelis-Menten equation.

• The substrate concentration that gives 1/2 Vmax is

known as the ‘Michaelis constant” or “km”.

• The Km indicates the affinity between the enzyme

and the substrate, the lower the Km the greater is
the affinity.
 Enzyme Inhibitors
❖ Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an
inhibitor.
❑ Types of inhibitors
A. Irreversible inhibitors:
inhibitors bind to enzymes through covalent bonds. Lead, for example, forms covalent bonds
with the sulfhydryl side chain of cysteine in proteins.
1. lead forms covalent bonds with the sulfhydryl side chains of cysteine in proteins.
2. Heavy metals, Ag or Hg, combine with –SH groups.
B. Reversible inhibitors:
1. COMPETITIVE
2. NON-COMPETITIVE
Competitive inhibitors
• This type of inhibition is due to the structural
similarity between the inhibitor (I) and the
substrate.

• These compete with the substrate molecules for

the active site.

• Thus, the formation of E-S complex is decreased,

inhibiting the activity of the enzyme.
 Competitive inhibitors

• Effect on Vmax: The effect of a competitive inhibitor is

reversed by increasing the concentration of substrate.

• At higher substrate concentrations, the inhibitor is

completely displaced and the same Vmax can be reached.

• Effect on Km: A competitive inhibitor increases the

apparent Km for a given substrate, so more substrate is
needed to achieve one half Vmax.
Examples of competitive inhibitors:
Statin drugs is an example of competitive inhibitors
• This group of antihyperlipidemic agents competitively
inhibits hydroxymethylglutaryl CoA reductase enzyme (HMG
CoA reductase) the rate-limiting step in cholesterol
synthesis.

• Statin drugs, are structural analogs of the natural substrate

for this enzyme, and compete effectively to inhibit HMG CoA
reductase, By doing so, they inhibit de novo cholesterol
synthesis, thereby lowering plasma cholesterol levels.
Non-competitive inhibitors:
• Noncompetitive inhibition occurs when the inhibitor and
substrate bind at different sites on the enzyme.

• The noncompetitive inhibitor can bind either free enzyme

or the enzyme–substrate complex, thereby preventing the
reaction from occurring.

• This type of inhibition is recognized by its characteristic

effect on Vmax

• Examples:
 Non-competitive inhibitors:
• Effect on Vmax: noncompetitive inhibitors
decrease the apparent Vmax of the
reaction.

• Vmax cannot be overcome by increasing

the concentration of substrate.

• Effect on Km: since the inhibitor does not

interfere with substrate binding, it does
not change the Km.
Enzyme inhibitors as drugs

• Many drugs act as enzyme inhibitors.

For example:

• The widely prescribed β-lactam antibiotics, such as

penicillin and amoxicillin, act by inhibiting enzymes
involved in bacterial cell wall synthesis.
Regulation of enzyme action
• Since enzymes have great catalytic power, their activity should be strictly regulated.
Their action on metabolites should be allowed only where needed and when needed.

1. The rates of most enzymes are responsive to changes in substrate concentration,

because the intracellular level of many substrates is in the range of the Km. Thus, an
increase in substrate concentration prompts an increase in reaction rate, which tends to
return the concentration of substrate toward normal.

2. In addition, Metabolic pathways are usually controlled by regulating the activity of one
enzyme, called the rate-limiting or key enzyme, which usually catalyzes an irreversible
reaction in the pathway. Can be controlled by: Allosteric enzymes, Covalent
modification, and Enzyme synthesis.
 A. Allosteric enzymes
• Allosteric enzymes are regulated by molecules called effectors that bind
noncovalently at a site other than the active site.

• Effectors that inhibit enzyme activity are termed negative effectors,

whereas those that increase enzyme activity are called positive effectors.

• They can affect the affinity of the enzyme for its substrate or maximal
catalytic activity of enzyme (Vmax)
 B. Covalent modification
• Many enzymes are regulated by covalent modification,
most often by the addition or removal of phosphate groups
from specific serine, threonine, or tyrosine residues of the
enzyme.

• Phosphorylation reactions are catalyzed by a family of

enzymes called protein kinases that use ATP as the
phosphate donor. Phosphate groups are cleaved from
phosphorylated enzymes by the action of phosphoprotein
phosphatases
C. Enzyme synthesis
• Cells can regulate the amount of enzyme present by
altering the rate of enzyme degradation or the rate of
enzyme synthesis.

• Stimulation of enzyme synthesis by this mechanism is

called enzyme induction, while inhibition is called
enzyme repression.