General Enzymology

Janelle Calalang, MD
Topics:
• Overview of Enzymes
• Classes of Enzymes
• Properties of Enzymes
• Enzyme kinetics
• Regulation of Enzyme Activity
• Rate limiting Enzymes
• Clinical correlates
Overview of Enzymes
• Generally, enzymes are protein catalysts that increase the velocity of a
chemical reaction and not consumed during the reaction they catalyze
enzymes lower their activation energy of a chemical reaction ->
increases the velocity of the chemical reaction
enzymes do not change the energy of the reactants and products; it
has no effect in the equilibrium( it only hastens both forward and
reverse reaction, thus reaching equilibrium faster)

*Exception to the proteinaceous nature of enzymes are ribozymes (RNA

with catalytic activity)
Definition of terms:
SUBSTRATE refers to the substance on which an enzyme act

PRODUCT refers to the substance produced by the action of

enzyme on the substrate
LOWERING OF ACTIVATION
ENERGY
Definition of terms:
• Isozymes: are physically distinct versions of a given enzyme, each of
which catalyzes the same reaction
• they are structurally different, but they perform the same function.
For example, glucokinase and hexokinase both catalyze the
phosphorylation of glucose to glucose-6-phosphate but have different
structures. Hence, they are considered isozymes
Classes of Enzymes:
Oxidoreductases (Class 1) • Add oxygen or remove hydrogens (oxidases,
dehydrogenases)
• add hydrogens (reductases) to substrates
Transferases (Class 2) • Move chemical groups (glycosyl, methyl,
phosphoryl) from one substrate to another
Hydrolases (Class 3) • Cleave substrate bonds using water (adding H+ or
OH-) to the cleavage products
• Subtypes include peptidases, lipases, etc
Lysases (Class 4) • Catalyzes the cleavage of C-C, C-S, and certain C-N
bonds
Isomerases (Class 5) • Rearrange substrate molecules to form a different
isomer
Ligases (Class 6) • Catalyze the formation of bonds between C and
O,S, and N coupled to hydrolysis of high-energy
phosphates (e.g., ATP hydrolysis)
Other notable enzymes:
Phosphatases Remove phosphate groups
Phosphorylases and Kinases Add phosphate groups to substrates
Properties of enzymes:
• contain an active site for the substrate, held together by hydrogen
bonds
• highly efficient
• highly specific (Lock and key vs Induced fit model)
• some enzymes require nonproteins for enzymatic activity (cofactors,
coenzymes)
• compartmentalized on different areas of the cell (cytoplasm,
mitochondria, SER, RER, Golgi apparatus)
• can be regulated or inhibited
Lock and key model vs. Induced
Fit Model
LOCK and KEY MODEL INDUCED FIT MODEL
• the enzyme and • Binding of a substrate
substrate FIT to a specific part of
TOGETHER the enzyme ->
INDUCES
CONFORMATIONAL
CHANGES in the active
site of the enzyme
• enzyme changes
shape during or after
binding the substrate
• failed to explain the • more accepted model
dynamic changes that
accompany the
catalysts
• less accepted model • more accepted model
Holoenzymes, Apoenzymes, Cofactors,
Coenzymes
• Simple enzymes: enzymes consisting only of proteins
• Complex enzymes: enzymes consisting of a protein part and
(apoenzyme)non protein component
• Some enzymes require nonproteins for enzymatic activity
Holoenzymes, Apoenzymes, Cofactors,
Coenzymes
Holoenzymes refers to the active enzyme + nonprotein
•
component
Apoenzyme • refers to the enzyme without its nonprotein
component
• rendered INACTIVE
Cofactor • refers to the nonprotein component that is a metal
ion (eg., minerals)
Coenzyme • refers to the nonprotein component that is a small
organic molecule
• usually derived from vitamins
• can be a COSUBSTRATE or PROSTHETIC GROUP

Cosubstrate • refers to coenzymes that are TRANSIENTLY

associated with the enzyme
Prosthetic group • refers to coenzymes that are permanently
associated with the enzyme
Effectors
• are not required for the function of an enzyme

POSITIVE EFFECTOR NEGATIVE EFFECTOR

Will ↑ the rate of the reaction Will ↓ the rate of a reaction
Enzyme Kinetics
Michaelis-Menten Equation
• describes how reaction
velocity varies with substrate
concentration
• Assumes that:
[S] much greater than [E]
[ES] is constant
[P] is low
• enzymes that follow V1: initial reaction velocity
Vmax: maximal velocity
Michaelis-Menten kinetics Km: michaelis constant
have a hyperbolic curve [S]: substrate concentration
Enzyme Kinetics
• Maximal velocity (Vmax)
maximal number of substrate molecules converted to product per
unit time
• Michaelis Constant (Km)
The substrate concentration at which V1 is half the maximal velocity
(Vmax+2) attainable at a particular concentration enzyme
Enzyme kinetics: Factors that can affect the
rate of a reaction
• substrate concentration
• temperature
• pH
Enzyme Kinetics
Lineweaver-burk plot
• reciprocal of the Michaelis-
Menten equation
• used to calculate the Km and
Vmax as well as to determine
the mechanism of action of
enzyme inhibitors
Enzyme inhibitor
• any substance that can diminish
the velocity of an enzyme-
catalyzed reaction
• inhibition can be:
reversible
irreversible
Types of enzyme inhibitor
COMPETITIVE NONCOMPETITIVE

MECHANISM Inhibitor is shaped similar to Inhibitor binds to enzyme

substrate and competes for binding somewhere other than the active
site site and halts catalysis
REVERSAL Increase [S] Increase [E]

Km Increased Not changed

Vmax Not changed Lowered

Regulation of enzyme activity
CHANGE IN THE SUBSTRATE CONCENTRATION • The rates of most enzymes are
responsive to changes in substrate
concentration because the intracellular
level of many substrates in the range of
the Km
• Time required: IMMEDIATE

ALLOSTERIC ENZYMES • Allosteric enzymes are regulated by

molecules called effectors that bind non-
covalently at a site other than the active
site
• Positive effectors: increases enzyme
activity
• Negative effectors: decreases enzyme
activity
• Allosteric enzymes frequently catalyze
the commited step in a metabolic
pathway (often the rate limiting step)
• Time required: IMMEDIATE
Regulation of enzyme activity
COVALENT MODIFICATION • Phosphorylation vs Dephosphorylation
• Time required: IMMEDIATE to MINUTES

INDUCTION AND REPRESSION OF ENZYME SYNTHESIS • enzymes are often those that are needed at only
one stage of development or under selected
physiologic conditions
• Time required: HOURS TO DAYS
Rate limiting enzymes
• Rate-limiting enzymes are the slowest type of metabolic pathway
which determines the overall rate of a metabolic pathway
• ex. Glycolysis= Phosphofructokinase-1 (PFK-1)
Clinical Correlates:
Enzymes in Clinical Diagnosis
• the presence of elevated enzyme activity in the plasma may indicate
tissue damage that is accompanied by increased release of the
intracellular enzymes
• may be useful in evaluating the prognosis of the patient
• the lack of tissue specifically limits the diagnostic value of many
plasma enzymes
Principal Serum Enzymes used
in Clinical
SERUM ENZYME
Diagnosis
MAJOR DIAGNOSTIC USE
AMINOTRANSFERASES MYOCARDIAL INFARCTION (NON-SPECIFIC)
ASPARTATE AMINOTRANSFERASES (AST/SGOT) VIRAL HEPATITIS
AMYLASE ACUTE PANCREATITIS
CREATINE KINASE MUSCLE DISORDERS AND MYOCARDIAL INFARCTION
у-GLUTAMYL TRANSFERASE VARIOUS LIVER DISEASE
LACTATE DEHYDROGENASE ISOZYME 5 LIVER DISEASES
β-GLUCOCEREBROSIDE GAUCHER DISEASE
ALKALINE PHOSPHATASE (ISOZYMES) VARIOUS BONE DISORDERS, OBSTRUCTIVE LIVER
DISEASE
Thank you for listening! :)