Basic Enzymology (Part I)

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basic enzymology part 1

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Lecture 6
Dr. Mohamed Kotb El-Sayed
Associate Professor of Pharmaceutical Biochemistry & Molecular Biology
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Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology
Basic Enzymology (Part I)

Objectives:
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By the end of this lecture you should be familiar with:
Definition, properties and nomenclature of enzymes.
Classification of enzymes.
Mechanism of enzyme action.
Factors affecting enzyme activity.
Enzyme kinetics.
Types of enzyme activation.
Mechanism of enzyme activation.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Definition & Properties of the Enzymes:
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En = inZyme = yeast.
Enzymes: they are protein biocatalyst that accelerate the rate of
chemical reactions (103–108 times faster) at low or normal temperature
and low concentrations of reactants.
They have the following common features:
1. All are produced by living cells and can act outside these cells.
2. They are needed in very small amounts.
3. Proteins of high molecular weight and affected by heat.
4. Catalyze only one type of chemical reaction.
5. They accelerate the reaction without affecting its equilibrium (decrease
the energy needed for activation).
6. They are not changed chemically by the end of the reaction.
7. Highly specific (e.g., act on a specific substrate).
8. Most of enzymes are intracellular therefore, measuring of some
enzymes in plasma is useful for diagnosis of diseases.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Enzyme Nomenclature:
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1-Trivial name: such as trypsin, pepsin, and chymotrypsin
2-Recommended name: short and convenient for everyday use by
adding the suffix ase to the name of substrate;
Substrate + -ase (hexokinase, glucokinase, Maltase, lactase and
sucrase).
Action performed + -ase (dehydrogenase, oxygenase).
3-Systematic name: the reaction specificity + the substrate specificity.
Each enzyme is entered in the Enzyme Catalogue with a four-digit Enzyme
Commission number (EC number).
The first digit →class number (6 major classes).
The second digit →functional group upon which the enzyme acts
(subclass).
The third digit →coenzyme (sub-subclass).
The fourth digit →substrate (order of enzyme in sub-subclass).
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Classification of Enzymes:
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1-Oxido-reductases:
catalyzes oxidation-reduction reaction between two substrates. The
mechanism of oxidation is either addition of oxygen (oxidase)or
removal of hydrogen (dehydrogenase).
Oxidase catalyzes transfer of electron or hydrogen from substrate to
oxygen e.g.:-glucose oxidase that convert glucose to gluconate and H2O2
Oxygenase: catalyze incorporation of oxygen molecule into substrate.
Mono-oxygenase:catalyze the incorporation of one oxygen atom into
substrate.
2-Transferases:
Catalyzes the transfer of a group other than hydrogen (as acyl, amino, and
phosphate) between two substrates.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Classification of Enzymes:
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3-Hydrolases:
Catalyzes hydrolysis of substrate (i.e. breakdown of the chemical bond by
addition of water) such as digestive enzyme, and peptidases.
4-Lyases:
Catalyzes removal of group from substrate by mechanism other than
hydrolysis e.g.: aldolase enzyme which convert fructose-1,6-diphosphate
into glyceraldehydes-3-phosphate and DHAP .
5-Isomerases:
Catalyzes the interconversion of one isomer to the other e.g. glucose 6-p
is converted into fructose -6-p by isomerase.
6-Ligases:
Catalyzes the joining of 2 substrates using high energy released by
hydrolysis of high energy bond of ATP e.g.: Pyruvic acid is joined to CO2
forming oxaloacetic acid by aid of carboxylase enzyme.
.Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Classification of Enzymes:
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.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Classification of Enzymes:
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.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Classification of Enzymes:
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Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Mechanism of Enzyme Action:
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Enzymes are large protein molecules. Each
contains a small specific region, which
interacts with the substrate, termed the
active site, substrate site or catalytic site.
Enzymes increase the rate of chemical
reactions by decreasing the energy needed
for substrate activation.
Generally certain amino acid side chains
have important role in enzyme catalysis e.g.
SH of cysteine, OH of hydroxy-amino acids,
carboxylic group of acidic amino acids and
amino group of basic amino acids.
They help in binding of the enzyme with its
substrate and with the groups undergoing
transfer (added or removed).
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Mechanism of Enzyme Action:
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The Energy changes 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.
The theories of enzyme action can be explained by the
following two models:
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Mechanism of Enzyme Action:
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1-The key and lock (Fisher model)
theory:
The active site of the enzyme is
complementary in conformation to the
substrate, so that enzyme and substrate
recognize each other. This theory postulates
that active site has fixed shape.
The substrate binds to a specific site on the
enzyme to from enzyme substrate complex,
this is followed by activation of the substrate,
then formation of the reaction products and
the enzyme sets free to catalyze a new
reaction.
The only disadvantage of this model is the
rigidity of active site.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Mechanism of Enzyme Action:
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2-The induced fit theory (Khoshland model):
The enzyme changes its shape upon binding the substrate, so that catalytic
site is suggested to be pre-shaped to fit with substrate.
In this model, the substrate induces a conformational changes in the
enzyme, which make the catalytic site (or groups) more fit or more suitable
for both binding of substrate and catalysis.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Factors Affecting Enzyme Activity:
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The enzyme activity is measured by the rate of the reaction.
Rate of reaction (Turnover or velocity): is the 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 πmol of
substrate to product per minute.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Factors Affecting Enzyme Activity:
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1-Temperature:
The rate of an enzyme catalyzed reaction increases by raising temperature
till it reaches a maximum activity at the optimum temperature (around
40°), after that the activity of the enzyme decreases.
At first, the rise of temperature increases the kinetic energy of the
molecules, then above optimum temperature, gradual denaturation of the
enzyme occurs with complete loss of catalytic activity at 70°C.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Factors Affecting Enzyme Activity:
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2-pH:
Any enzyme has an optimum pH, at which it acts maximally. This pH
usually ranges between 5 to 9. Marked changes in pH produces
conformational changes in protein structures that result in decreased
activity.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Factors Affecting Enzyme Activity:
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3- Reaction Products:
For a reversible reaction, accumulation of the reaction products stimulates
the reversal of the reaction (increases V2) and produces a net decrease in V1
till equilibrium is reached (V1=V2).
Also removal of reaction products (conversion of C + D into E) accelerates
the initial reaction and increasesV1 to maximum.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology
4- Enzyme Concentration:
The velocity of the reaction increases
as the enzyme concentration increases
up to a certain point.
Above this point the concentration of
the substrate is a limiting factor.

Factors Affecting Enzyme Activity:
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5- Substrate Concentration:
The velocity of the reaction increases as the
substrate concentration increases up to a point
where the enzyme is saturated with the substrate.
The substrate concentration that produces half
maximal velocity termed Michaelis constant
(or Km).
Enzymes with high affinity to substrate have low
Km and vice versa.
E.g. enzyme-1 has lower Km-1 and higher affinity
to its substrate (high activity at low substrate
concentration) compared to enzyme-2, which has
higher Km-2 and lower affinity to its substrate
(low activity at high substrate concentration).
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Enzyme Kinetics:
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It is the study of the rate or velocity of reactions catalyzed by enzymes.
Initial reaction velocity (V0): The rate at which reaction proceeds and
it is measured as decrease in concentration of substrates or increase in
concentration of products with time.
If the enzyme is incubated with its substrate and the appearance of the
product is recorded as graph, the resulting line will have hyperbolic.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Enzyme Kinetics:
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If Km= [S] →the velocity will be ½
Vmax.
Thus, Km can be defined as substrate
concentration that produces half
maximum velocity.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Michaelis-Menten Equation:
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Km is numerically equal to the substrate concentration at which the
reaction velocity is equal to ½ Vmax.
Substrates are usually present in physiological fluids in amounts nearly
equal to Kmvalues.
Kmis constant, characteristic for each enzyme and particular substrate.
Kmreflects the affinity of the enzyme to substrate.
Small (low) Km=high affinity of the enzyme for substrate i.e. low
concentration of substrate is needed to half saturate enzyme.
Large (high) Km=low affinity of the enzyme for the substrate (high
concentration of substrate is needed to half saturate enzyme).
Km does not vary with the concentration of enzyme.
Examples; Hexokinase is more active than glucokinase because the amount
of glucose (substrate) needed to produce half Vmax in case of hexokinase is
less (2 mg) than in case of glucokinase (200 mg) i.e. Kmof hexokinase is
less than of glucokinase.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Determination of Km and Vmax for a given enzyme:
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Calculation of Kmand
Vmax to determine the
mechanism of action of
enzyme inhibitors.
The Km value is usually
expressed in units of
concentration
(moles/L).
1/V0 is plotted versus
1/[S], a straight line is
obtained (Line weaver-
Burk plot) and it
becomes more practical
to estimate both Km and
Vmax.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Types of Enzyme Activities:
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They are mainly proteins in nature, either in the form of simple protein
enzymes (Apoenzyme)or conjugated protein enzymes (Holoenzymes).
Holoenzymes are formed of a protein part (apoenzyme/inactive alone)
and a non-protein part (coenzyme or prosthetic group).
Coenzymes are non-covalently (loosely) bounded to the apoenzyme and
are low molecular weight organic molecules e.g. NAD+for lactate
dehydrogenases. They act as carriers for certain groups, which have to be
added to or removed from the substrate.
Prosthetic groups are covalently (firmly) bounded to the apoenzyme.
They are either organic e.g. heme in cytochromes, or inorganic e.g.
metal ions as zinc in carbonic anhydrase.
Cofactor: Metal ion Fe and Zn.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Coenzymes:
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Carriers are classified into two main groups:
1- Hydrogen and Electron Carriers: They include the following:
- NAD+and NADP+. - FMN and FAD.
- Heme in cytochromes. - Lipoate.
- L-ascorbic acid. - Glutathione.
- CoQ.
II- Other Group Carriers: They are vitamin derivatives and include the
following:
- PLP (Pyridoxal phosphate) (NH2). - Biotin &TPP (CO2).
- CoA (CoA-SH) (Acyl group). -Tetrahydrofolate (CH3).
- Folic acid = one carbon carrier.cobolamine= CH3carrier.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Mechanisms of Enzyme Activations:
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Include the following:
1- Activation of zymogens. 2- Activation by metal ions.
3- Allosteric activation. 4- Covalent modification.
1- Activation of Zymogens (Pro-enzymes):
Many enzymes are formed in the form of proenzymes or zymogens.
In this form they are inactive. Activation requires proteolysis (removal of a
part of the polypeptide chain which masks the active site or substrate site).
A good example is the formation of digestive proteolytic enzymes as
zymogens inside the cells to prevent digestion of cellular proteins.
When these zymogens are released to the gut, they are activated to digest
food proteins.
Many of these enzymes after activation can activate its zymogen in a process
termed autocatalytic activation (autocatalysis).
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Mechanisms of Enzyme Activations:
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Another good example is the activation of different blood clotting factors.
Many of these factors are formed as zymogens and activated by specific
proteases.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Mechanisms of Enzyme Activations:
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2- Metal Ion Activation:
There are three main possible mechanisms by which the metals interact with
the enzyme and substrate as follows:
A. The metal helps to maintain an active conformation of the enzyme e.g.
Mg2+ in glutamine synthase.
B. The metal binds with the substrate, then it helps binding of the substrate
to the enzyme as in phosphotransferase reactions.
C. The metal is associated with the active center of the enzyme and helps in
binding of the substrate to the enzyme e.g. cytochromes.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

Mechanisms of Enzyme Activations:
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3- Allosteric Activation (Non-Covalent Modification):
Certain enzymes contain specific site (away from the catalytic site). The
binding of an allosteric activator with the allosteric site produces
conformational changes in the protein structure of the enzyme which result
in increased velocity of the reaction.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology
4. Covalent modification: Phosphorylation and
dephosphorylation(by the addition or removal of
phosphate groups from specific serine, threonine, or
tyrosine residues of the enzyme).
5. 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).

Mechanisms of Enzyme Activations:
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4- Covalent Modification for Activation by Phosphorylation and
Dephosphorylation):
Many enzymes are activated by phosphorylation and inactivated by
dephosphorylation and vice versa.
This means that the enzyme is present in two interconvertible forms
(phosphorylated and dephosphorylated).
The phosphate groups are usually attached to the hydroxyl group of amino
acid residues (mainly serine or tyrosine) present in the polypeptide chain of
the enzyme.
Good example is the activation of glycogen phosphorylase and hormone
sensitive lipase by phosphorylation and activation of glycogen synthase by
dephosphorylation.
Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology

References:
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Dr. Mohamed I. Kotb –Associate Professor of Pharmaceutical Biochemistry and Molecular Biology
mohamed.kotb71524@gmail.com
WhatsApp: 00201140400767
https://www.facebook.com/moh.elsayed.925/
https://www.researchgate.net/profile/Mohamed_kotb_Kotb-El-Sayed2
•Lieberman M, Marks AD. Marks’Basic Medical Biochemistry: A Clinical Approach.
3rd Ed. Baltimore: LippincottWilliams &Wilkins, 2009,
•Pelley, John W. Rapid review biochemistry / John W. Pelley, Edward F. Goljan. –3rd
ed.p.;cm.–(Rapid review series) Rev. ed.of: Biochemistry. 2nd ed. c2007. ISBN
978-0-323-06887-1