ENZYMES

Rondang R. Soegianto

2017
Textbooks Biochemistry, 3rd ed
Lippincott’s Illustrated Reviews
2005

Medical Biochemistry
Master Medicine
Brownie, A.C. and Kernohan, J.C.
2005

Harper’s Illustrated Biochemistry

29th ed, 2012
Enzymes are: Biocatalysts
Protein catalysts
Organic catalysts

Properties: Efficient
Potent
Specific
• Biocatalyst:
• - substrate specific
• - reaction specific
• - can be regulated in the body

• Inorganic catalyst
• - resistant to heat
• - not specific to certain substrate
• or reaction
• - Pt, Zn, H+
Enzymes direct all metabolic events

Efficient = Active under physiological

conditions

Potent = High yield of product per second

Specific = A different enzyme for almost every

biochemical reaction
RECOMMENDED NAME

- Substrate + ase
Protease, Lipase, Urease

- Action + ase
Lactate dehydrogenase

- Trivial name: Trypsin, Pepsin

Systematic name:
Developed by International Union of
Biochemistry and Molecular Biology
(IUBMB)
Enzymes are divided into 6 major classes
based on enzyme activity

D-glyceraldehyde 3-phosphate: NAD

oxidoreductase
• Why are enzymes essential to body
Homeostasis?

• At body temp. biochem. processes

have to proceed fast enough to
meet needs of the body

• Enzymes (unlike inorganic catalysts)

can be regulated in the body
• Inborn Error of Metabolism is caused

by enzyme abnormality
Enzyme Classification
• Active site, substrate site, catalytic site

• Specific area on enzyme surface that

binds
to substrate to form E-S complex, before
enzyme reaction takes place
General Enzyme Reaction

E + S  ES  E + P

Reaction is slightly reversible

• Mechanism of interaction ;
• 1. Emil Fischer
• Lock and Key Model
•
• 2. Koshland
• Induced fit model
• Enzyme undergoes conformational
• change
Lock and Key Model
and
Induced Fit Model
Lock and Key Model
Emil Fischer (1894)
Induced Fit Model
Daniel Koshland
 What is enzyme activity?

• Enzyme activity is measured as velocity of

enzyme reaction (v)

• v = amount of product per time unit

(sec. minute)
Also

• v = remaining substrate per time unit

Units of Enzyme Activity

International Union of Biochemistry

and Molecular Biology (IUBMB)
Katal (kat) = amount of enzyme that
converts 1 mol of reactant to product
in 1 second under standard conditions.
Health Sciences uses
International Unit (U).
1 U = amt of enzyme that forms 1µmol
of product per minute under standard
conditions. For body fluids: U/ml or
U/L
Standard conditions usually optimal.

Specific activity
Number of U or kat per mg of enzyme
protein. Specific activity is a measure of
purity in enzyme preparation.
• Cellular enzymes exist in compartments

• Carry out specific activities in organelles,

membranes and cytosol

• Enzymes in blood for coagulation process

• Enzymes in mitochondria for bioenergetic
process thru oxydative phosphorylation
REGULATION

Enzymes can be activated or inhibited

Product formation responds to needs of cell

FACTORS AFFECTING REACTION
VELOCITY
Enzymes can be isolated from cells.

E properties can be studied in test tubes

(in vitro)
A . SUBSTRATE CONCENTRATION

Max velocity (Vm) reached when enzymes

already saturated with substrate molecules

Hyperbolic shape of curve follows

Michaelis-Menten kinetics
The Michaelis-Menten equation describes
how reaction velocity varies with [S].

Vmax [S]
v = ---------------
Km + [S]
Characteristics of Km
(Michaelis constant)
- Reflects affinity of E for S
- Numerically equal to [S] at which
V equal to ½ Vm

Small Km = High affinity of E for S

Large Km = Low affinity of E for S
B. TEMPERATURE

1 Increase of velocity with

increasing temperature

2 Decrease of velocity with too high

temp (above optimal temp)
C. pH
Effect of pH on ionization of active site.

Binding of E to S and catalytic process

requires specific chemical groups.

Extreme pH (too acid or alkaline) can

denature enzyme proteins

pH optimum varies for different enzymes

INHIBITION OF ENZYME ACTIVITY

A Competitive inhibition
Inhibitor competes with substrate to
occupy active site on enzyme molecule

Effect is reversed by increasing [S]

At sufficiently high [S], normal Vm value
can be reached

Apparent Km value is increased

Statin drugs as examples of competitive
inhibitors

This antihyperlipidemic agents competitively

inhibits

Hydroxymethylglutaryl CoA reductase (HMG

CoA)

the first committed step in cholesterol

synthesis
B Noncompetitive inhibition

Inhibitor and substrate bind on different

sites on the enzyme

Cannot be overcome by increasing [S]

Inhibitor does not interfere with binding of S

on enzyme. Thus same Km value as
without inhibitor
Enzyme inhibitors as drugs

Antibiotics such as penicillin and amoxicillin

inhibit enzyme involved in bacterial cell wall
synthesis

ACE Angiotensin-converting enzyme

inhibitors
Inhibits cleavage of angiotensin I 
angiotensin II (potent vasoconstrictor)
REGULATION OF ENZYME ACTIVITY

Allosteric binding sites

Ligand to these sites can be

- Negative effectors

- Positive effectors
Feedback inhibition

End-product binds to allosteric site of

the regulating enzyme.

Avoids overproduction in biosynthesis

process
Regulation by covalent modification

Phosphorylation and dephosphorylatio

by protein kinases that use ATP and
dephosphorylation by phosphatases

Ex. Glycogen phosphorylase

Glycogen synthase
Enzymes in clinical diagnosis

Plasma enzymes as diagnostic tools

Isoenzymes and diseases of the heart

Isozymes of Lactate Dehydrogenase
used to detect Myocardial Infarctions
LDH is tetrameric = 4 subunits in two
isoforms : H (heart)
M (muscle)
Five combinations of isozymes:
Isozymes Subunits
I1 HHHH
I2 HHHM
I3 HHMM
I4 HMMM
I5 MMMM
Functional plasma enzymes

Lipoprotein lipase
Proenzymes of blood coagulation
Synthesized and secreted by liver

Nonfunctional plasma enzymes

Arise in blood from routine normal
destruction of blood- and other cells.
Increased in blood due to tissue
damage and necrosis in disease.
Recent analysis of enzymes to aid
diagnosis

Catalytic amplification of DNA in biologic

samples by polymerase chain reaction
(PCR).

Recombinant DNA. Involves isolation of an

enzyme, gene cloning of the enzyme and
Produce large quantities in E. coli or yeast.
• COFACTORS

• Nonprotein cofactors needed for enzyme activity

• - Metal ions: Zn2+, Fe2+, Mg2+

• - Organic molecules = Coenzymes

• Derivatives of vitamin B

• - Holoenzyme = Enzyme + Cofactor

• (Coenzyme)

• - Apoenzyme = Protein portion of

• holoenzyme
• Coenzyme Vitamin Activity

• NAD Nicotinic acid ox-red

• FAD Riboflavin ox-red

• ThiaminPP Thiamin Oxidative

decarboxylation
Tetrahydrofolate Folic acd Transport of
1-C unit
• Coenzyme A Pantothenat Activation
of FA
• Biotin Biotin CO2
fixation

• Lipoate Lipoic acid ox-red

Nicotinamide Adenine
Dinucleotide (NAD)
Flavin Mononucleotide (FMN)
Flavin Adenine Dinucleotide
(FAD)
Coenzyme A (SH)
Thiamine Pyrophosphate (TPP)
(Vitamin B1)
Pyridoxal Phosphate
(Vitamin B6)
Lipoic Acid (2 SH groups)
Biotin and Tetrahidrofolate
Pernicious anemiB12 dependent enzymes:
Methyl malonyl Coa mutase, Methionine
synthase a caused by Vit B12 deficiency
(impaired erythropoiesis