ENZYMES

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

LEARNING OUTCOMES
• Discuss the role of enzymes and co-enzymes in the catalysis of
biological reactions.
• Describe enzymatic reactions quantitatively and mechanistically using
kinetic parameters.
• Apply the concepts of enzyme inhibition in the treatment of clinical
conditions.
• Recognize the role of enzymes as crucial points in metabolic
regulation.

READINGS: Harpers Biochemistry Chapters 7,8, and 9

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT ARE ENZYMES?
• proteins that catalyze metabolic reactions
• Speed up chemical reactions in living organisms without themselves
being chemically changed at the end of the reaction
• The rate of metabolic processes is determined largely by the amount
of enzyme proteins and their intrinsic activities

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT ARE THE ENZYME GENERAL
CHARACTERISTICS ?

● Large protein molecule with catalytic

→ Increase rate of reaction
● Specific
→ Can distinguish one substrate from another similar compound
→ Have active sites where substrate binds
→ Interactions: polar-polar, positive-negative
● Regulatory
→ Provide means of regulating metabolic processes
Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
ARE ALL ENZYMES PROTEIN?
• Not all enzymes are proteins
E.g., ribozymes - RNA with catalytic activity
• Not ribosomes
• Functions of Ribozymes:
• synthesis of RNA, membranes, amino acids
• Properties of Ribozymes:
• Catalytic behavior ( enhances rates to more than 20x )
• Genetically programmed
• Naturally occurring ( 60-90 bases )

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT ARE THE FACTORS
AFFECTING ENZYME ACTIVITY?

• Temperature
• Ph
• salt concentration
• the concentration of coenzymes, cofactors, and allosteric effectors
• covalent modification
• variations in gene expression.

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

HOW IS THE ACTIVITY OF THE
ENZYME MEASURED?

• expressed as µmol/min/mg of protein or IU/mg of protein

• The specific activity of enzymes varies greatly among tissues, depending
on the metabolic function of the tissue.
• Ex. The enzymes for cholesterol synthesis have a higher specific activity (IU/mg
tissue) in liver than in muscle, consistent with the role of the liver in the
biosynthesis of cholesterol
• The specific activity of an enzyme is useful for estimating its purity - the
higher the specific activity of an enzyme, the higher its purity, or
homogeneity.

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT IS THE ENZYME MECHANISM
OF ACTION?
1. Binding → As substrate binds
to the enzyme,
conformational changes
happen. From there,
transition complex is
achieved.
2. Release → Reaction products
released after the formation
of a transition complex
3. Return → Enzyme returns to
its original state (without a
substrate)

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

HOW SPECIFIC ARE ENZYMES?
• highly specific for both the type of
reaction catalyzed and the nature of the
substrate(s)
• Lock and Key Model/Induced Fit Model

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT ARE THE COMPONENTS OF
ENZYME ACTIVE SITE ?
• Substrate specificity is determined by
the size, structure, charges, polarity,
and hydrophobicity of the substrate-
binding site.
• This is because the substrate must
bind in the active site as the first step
in the reaction, setting the stage for
catalysis.

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

EXAMPLE OF ENZYMES WITH
BROADER SUBSBTRATE SPECIFICITY
• In chymotrypsin, a hydrophobic pocket binds
aromatic amino acid residues such as
phenylalanine (Phe).
• In trypsin, the negative charge of the aspartate
residue in the substrate-binding site promotes
cleavage to the carboxyl side of positively
charged lysine (Lys) and arginine (Arg) residues.
• In elastase, side chains of valine and threonine
block the substrate-binding site and permit
binding of amino acids with small or no side
chains, such as glycine (Gly). , site of hydrolysis
by enzyme.

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT ARE THE GENERAL FEATURES
OF ENZYME ACTIVE SITE?
● Different parts of amino acid sequence
→ E.g., lysozymes (important groups in active
site contributed
by 5 residues)
● Relatively small part of total volume of an
enzyme
→ Other amino acids serve as:
▪ Scaffold to create structure
▪ Regulatory sites
▪ Channels to bring substrate to active site
● Active sites are clefts or cervices
→ 3D cleft formed from folding proteins

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT ARE THE GENERAL FEATURES
OF ENZYME ACTIVE SITE?
● Bound to enzymes by multiple weak attractions:
→ Electrostatic – positive to negative
→ Hydrogen bond – H to N, O, or F
→ Van der Waals
▪ Can be a weak London dispersion force or a dipole-dipole
force
▪ Most common bond
→ Hydrophobic interactions

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT ARE THE GENERAL FEATURES
OF ENZYME ACTIVE SITE?
● Specificity
→ Precisely differentiated
→ Lock and Key
▪ Emil Fischer (1980)
▪ Specific interaction between two biological
molecules
→ Induced Fit
▪ Daniel Koshland
▪ Favored model
▪ Primary interaction between enzyme and substrate
− relatively weak and induces conformational
change
− E.g., glucokinase
Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
WHAT ARE THE COMPOSITIONS OF
ENZYMES?

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

 WHAT ARE CO FACTORS?
● Substances required in many enzyme-catalyzed reactions
● Prosthetic group – cofactor very tightly (or covalently) bound to the enzyme protein
● Type:
→ Small organic molecules, Also known as coenzymes
▪ Vitamins we get from our diet are transformed into active coenzyme form E.g., panthothenic
acid is transformed to coenzyme A
→ Metal cofactors
▪ Metalloenzyme – enzyme with a metal cofactor
● Apoenzyme – inactivated form of an enzyme
→ Apoenzyme + cofactor → holoenzyme
▪ Crucial for different metabolic processes
● Ethanol
→ Anti-vitamin; Decreases cellular content of almost every coenzyme; Inhibits the absorption
of thiamine (Vitamin B1); Acetaldehyde from ethanol oxidation displaces pyridoxil
Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
EXAMPLES OF CO FACTORS
Magnesium
→ Needed in phosphate transfer
→ Stabilizes the highly negative charge of
phosphate
→ Involved in ATP production and reactions
involving
phosphate

Selenium
→ Comes with glutathione (for skin whitening)

Molybdenum
→ Recently connected to a lot of enzymes

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

EXAMPLES OF CO ENZYMES

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

ENZYMATIC AND NON ENZYMATIC
REACTIONS
Reaction profile for enzymatic and nonenzymatic
reactions.
The basic principles of an enzyme-catalyzed reaction are
the same as those for any chemical reaction. When a
chemical reaction proceeds, the substrate must gain
activation energy to reach a point called the transition
state of the reaction, at which the energy level is
maximum. Because the transition state of the enzyme-
catalyzed reaction has a lower energy than that of the
uncatalyzed reaction, the reaction can proceed faster.

ES complex, enzyme–substrate complex; EP complex, enzyme–product

complex.

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT ARE THE MECHANISMS OF
ENZYME CATALYSIS?
● Catalysis by proximity
→ Enzyme binds substrate molecules at its active site
→ The closer the substrate to the enzyme, the faster the reaction
→ Substrate oriented in a position suitable for catalysis
● Acid-Base catalysis
→ Acid donates protons, base accepts proton
→ Types:
▪ Specific: H+ /OHsensitive to pH
▪ General: Any acid or base
− Can be an amino acid
− Amino acids catalyze the cleavage of peptide bond
− Aspartate can act both as an acid and as a base depending on its neighboring amino
acid Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
WHAT ARE THE MECHANISMS OF
ENZYME CATALYSIS?
● Catalysis by bond strain
→ Change in conformation of enzyme causes a bond strain
● Covalent catalysis
→ Covalent intermediate forms between the enzyme or coenzyme and
the substrate
• → Catalytic triad
▪ Serine, histidine, and aspartate
→ E.g., proteolysis by serine proteases which includes
• digestion of trypsin, chymotrypsin, and elastase
Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
WHAT ARE THE CLASSIFICATION OF
ENZYMES?

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

ENZYME USE FOR CLINICAL
DIAGNOSIS

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

WHAT ARE ISOZYMES?
Isozyme profiles are often performed in the clinical laboratory for diagnostic purposes
The definition of isozymes is often operational - that is, based on simple and reproducible
substrate-specific assay methods that sometimes do not require precise knowledge of
enzyme structure.
The term isozyme is commonly used to refer to
(1) Genetic variants of an enzyme;
(2) genetically independent proteins with little homology;
(3) heteropolymers of two or more noncovalently bound polypeptide chains;
(4) unrelated enzymes that catalyze similar reactions, such as enzymes conjugated with
different
prosthetic groups or requiring different coenzymes or cofactors;
(5) different forms of a single polypeptide chain
For example - varying in carbohydrate composition, deamination of amino acids,
or proteolytic modification.
Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
TISSUE SPECIFICITY OF LACTATE
DEHYDROGENASE ISOZYMES
A 56-year-old female was admitted to an intensive care unit. The patient had
suffered from a slight fever for 1 week and reported some chest pain and
difficulty breathing for the past 24 h. No abnormality was found on chest X-ray or
by electrocardiography. However, a blood test showed white blood cells
12,100/mm3
(normal: 4000–9000/mm3 ), red blood cells 240 × 104 /mm3 (normal: 380–500 ×
104 /mm3
), hemoglobin 8.6 g/dL (normal: 11.8–16.0 g/dL), lactate dehydrogenase (LDH)
1400 IU/L (normal:
200–400 IU/L). Levels of other enzymes were normal. Based on the blood tests,
the LDH isozyme profile, and other data, the patient was eventually diagnosed
with malignant lymphoma
Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
TISSUE SPECIFICITY OF LACTATE
DEHYDROGENASE ISOZYMES
LDH is a tetrameric oxidoreductase composed of two different
35-kDa subunits. The heart contains mainly the H-type subunit,
and skeletal muscle and the liver mainly contain the M type, which
are encoded by different genes. Five types of tetrameric isozymes
can be formed from these subunits: H4 (LDH1), H3M1 (LDH2), H2M2
(LDH3), H1M3 (LDH4), and M4 (LDH5). Because isozyme distributions
differ among tissues, it is possible to diagnose tissue damage by
assaying total LDH activity and then isozyme profiling

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

ENZYME KINETICS
• The Michaelis–Menten equation:
A simple model of enzyme catalysis
• In 1913 - Leonor Michaelis and Maud Leonora Menten developed a simple
model for the kinetics of enzyme-catalyzed reactions

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

MICHAELIS-MENTEN EQUATION

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

MICHAELIS-MENTEN EQUATION

Km is a useful constant for estimating the

affinity of an enzyme for its substrate.
Enzymes with a high Km require high
substrate concentration for efficient
activity, whereas those with low Km
operate efficiently on trace levels of
substrate

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

The Michaelis–Menten model is based on the
following assumptions:
■ E, S, and ES are in rapid equilibrium.
■ There are no forms of the enzyme present other than E
and ES.
■ The conversion of ES into E + P is a rate-limiting, irreversible
step. Although all enzyme-catalyzed reactions are theoretically reversible, initial
velocities are normally measured
when product concentration, and therefore the rate of the
reverse reaction, is negligible.

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

Use of the Lineweaver–Burk
Alternative graphical analyses permit more accurate
determination of the Km and Vmax of an enzyme

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

Lineweaver–Burk plot
• The Lineweaver–Burk, or double-reciprocal, plot is obtained by taking
the reciprocal of the Michaelis–Menten equation

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

Eadie–Hofstee plot

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

ENZYME INHIBITION

• competitive
• uncompetitive,
• noncompetitive inhibition.

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

COMPETITIVE INHIBITORS
• Competitive inhibitors cause an apparent
increase in Km
without changing Vmax
• the rate of the enzymecatalyzed reaction in
the presence of a competitive inhibitor can
be increased by increasing the substrate
concentration because substrate, at higher
concentration, competes more effectively
with the inhibitor.

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

UNCOMPETITIVE INHIBITORS
• Uncompetitive inhibitors cause an apparent decrease in Vmax

• An uncompetitive inhibitor binds only to the enzyme–substrate

complex and not to the free enzyme. The following equation shows
the reaction scheme for uncompetitive inhibition. In this case, the Ki is
the dissociation constant for the enzyme– substrate–inhibitor (ESI)
complex

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

NONCOMPETITIVE INHIBITOR

A noncompetitive (mixed) inhibitor can bind either to the free enzyme or to the
enzyme–substrate complex typically at a site outside the active site.
Noncompetitive inhibitors exhibit more complex effects and may alter both the
Km and the Vmax of an enzymatic reaction.

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

HOW TO REGULATEOF ENZYME
ACTIVITY
5 independent mechanisms
1. The expression of the enzyme protein from the corresponding gene changes
in response to the cell’s changing environment or metabolic demands.
2. Enzymes may be irreversibly activated or inactivated by proteolytic enzymes.
3. Enzymes may be reversibly activated or inactivated by covalent modification,
such as phosphorylation.
4. Allosteric regulation modulates the activity of key enzymes through reversible
binding of small molecules at sites distinct from the active site in a process that
is relatively rapid and, hence, the first response of cells to changing conditions.
5. The rate of degradation of enzymes by intracellular proteases in the
lysosome or by proteasomes in the cytosol also determines the half-life of
enzymes and, consequently, enzyme activity over a much longer period of time.
Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
Proteolytic activation of digestive enzymes
• Several digestive enzymes are stored as inactive
zymogens or proenzymes in secretory vesicles in the
pancreas.
• Trypsinogen is converted into trypsin by the action
of intestinal enteropeptidase.
• The active trypsin then digests other zymogens, such
as procarboxypeptidase, proelastase, and
chymotrypsinogen, as well as other trypsinogen
molecules Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA
Allosteric regulation of rate-limiting enzymes
in metabolic pathways
An allosteric (other-site) effector molecule binds to the enzyme at a site that is
distinct and physically separate from the substrate binding site and affects
substrate binding (Km) and/or kcat

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

Allosteric regulation of rate-limiting enzymes
in metabolic pathways
If the allosteric effector is different from the substrate, it is referred to
as a heterotropic effect

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA

CLINICAL CORRELATION

Rowena Magaway-Constantino, MD, FPPS, MHcA, CHA, FPCHA