ENZYME

NI WAYAN TIANING
DEPT. BIOCHEMISTRY
FACULTY OF MEDICINE
UDAYANA UNIVERSITY

© 2007 Paul Billiet ODWS

OUTLINE:
1. ENZYME STRUCTURE
2. ENZYME DISTRIBUTION
3. ENZYME REACTIONS
4. ENZYME KINETICS
5. APPLICATION IN MOLECULAR
MEDICINE
 ENZYME
DEF: PROTEIN, CATALYZES, SPECIFIC.
CATALYZE: To accelerates, rate of chemical reaction.
a. Protein catalyze.
b. Non protein catalyze: H+, OH-, Cu++, etc
Isozyme: iso-enzyme
Zimogen: Pro-enzyme/Pra enzyme
Apoenzyme: a protein that combines with coenzyme
 active enzyme.
Holo-enzyme: apo-enzyme and co-enzyme (enzyme
complex)
Coenzymes/cofactors: Non protein
 COENZYME/COFACTOR
Non-protein molecule,
needed by some enzymes
to help the reaction (Part
of the enzyme)
Cofactors = coenzymes?

Many vitamins are

coenzymes (Vitamins B).
The latter are called 
prosthetic groups.
CLASSIFICATION OF COENZYME:

1. CoE that transfer H: NAD+, NADP+, FMN, FAD,

Lipoic acid, Co.Q

2. CoE that transfer non H: ATP, Sugar phosphate,

CoA, Thiaminpyroposphate, B6-phosphate, Folate,
Biotin, Cobamid CoE, Lipoic acid
3. Coenzyme not B vitamin: C and K vitamin
4. Coenzyme not vitamin: Q10 and ion group (Ca, Mg,
Zn, Na, ect)
COENZYME THAT TRANSFER H
 Vitamine Coenzymes Enzymes
B1 (thiamine) TPP Decarboxylase
B2 (riboflavine) FAD/FMN Dehydrogenase aerob

Niasine NAD/NADP Dehydrogenase anaerob

(Nicotinamide)
Pantothenic acid CoA Transamilase
B6 (piridoksine) Phosphate Pyridoxal Transaminase
Biotin Biotin Carboxylase
Folic acid a.Tetrahydrofolate Transformilase,
transmetilase
B12 CoE-Cobamide Transmetilase, Isomerase
SUBSTRATE
Substrates : reactants that
are activated by the enzyme
Enzymes : specific to their
substrates
Specific : determined by the
active site
Active site: specific region in
enzyme which interacts with its
substrate.
 ENZYME STRUCTURE

Proteins
globular
 ENZYME SPECIFICITY
SPECIFICITY TO:
a. ISOMER:
L-amino acid, cis-trans: fatty acid

b. CHEMICAL BOUND:
Cleave at hydrogen bound (hydrolase)
Cleave at peptide bound (peptidase), esterase,
phosphatase, ect.

c. Functional group: for the substrate with certain residues

(trombin: cleave peptide bound between arginine-glycine
residue)
d. Substrate: lactase, maltase, nucleotidase,
proteinase
e. Reaction: oxidase, dehydrogenase, transferase,
hydrolase
 NOMENCLATURE OF ENZYMES
Enzyme: En=In, Zyme =yeast
Firstly : depend on the founder (ptyalin)
Then : ………ase, after substrate (amylase, lipase, protease)
IUBS : International Union of Biochemistry System
1. Enzyme classified into 6 mayor class.
(1. oxido reductase, 2 transferase, 3 Hydrolase, 4. lyase,
5. Isomerase 6.ligase)
2. The enzymes name consist 2 part: the first is the substrate,
second part ending by ase which confirm type of reaction
catalyzed (glutamat dehydrogenase)
3. Systematic code number = Enzyme Code (E.C) consist of 4 digit
(EC 2.7.1.1)
 ENZYMES CLACIFICATION
Class Enzyme Reaction catalyzed
1 Oxidoreductases catalyze redox
(dehydrogenase) Processes
2 Transferases transfer chemical groups
(Transaminase) from one molecule to another or to another
part of the same molecule
3 Hydrolases (Trypsin) Hidrolysis reaction: cleave a bond using
water
to produce two molecules from one.
4 Lyases (carbonic Remove a group from or add a
anhidrase) group to double bonds.
5 Isomerases Isomeration reaction: Interconvert isomeric
(phosphoglucomutase) structures by molecular rearrangements.
6 Lygases (DNA ligase) Synthesis reaction
 ENZYME DISTRIBUTIONS
1. Oxidative phosphorylation (OXPHOS) in
mitochondria DNA (mtDNA)
2. TCA cycles
3. GI-Tract
4. Lysosome
5. Ribosome
 TEORIES OF THE ENZYMES
I. The Lock and Key Teory (Emil Fisher):
Fit between the substrate and the active site of the
enzyme is exact
The key is analogous to the enzyme and the
substrate analogous to the lock.
 enzyme-substrate complex formed
Product  released from the active site.
 II. INDUCED FIT TEORY (Koshland)

When the
substrate bind
with an enzyme, it
induces a change
in the enzyme to
conformation.

Simultaneous
 ENZYME KINETICS
Definition: rate of a reaction:
For an enzyme acting on its substrate, (ordinary
chemical reaction)
The rate of the reaction depends on the concentration
of substrate,
A reaction leading to formation of product:
S P

Rate = change in [P] / change in time or

rate = v = Δ[P]/Δt
Enzyme can control
of reactions, faster
than non-enzyme
reactions (100 to
1000 times faster).
 low energy.
Chemical reactions:
The rate is proportional to reactant [S].

rate = v = Δ[P]/Δt
= k [S] rate

[S]
In contrast, found empirically for enzymes:

Rate depends on [S]

 hyperbolic curve

rate

[S]
 Michaelis-Menten Model

E + S  E●S  E + P

Where E●S  enzyme-substrate complex,

(an intermediate complex) rate stops increasing
because the complex E●S becomes filled
at high [S]
Constants to MM model:
k1 k3
E + S  E●S  E+ P
k2

Michaelis-Menten:
Vmax[S]
V= [S] + Km

 hyperbolic curve .
Vmax: the maximum rate (plateau) is k3 x [total enzyme]
Km = (k2 +k3)/k1, almost a binding constant
Vmax

Vmax/2
v
Km

[S]
V = velocity = μmoles/min×mg protein
Km = [S], where the velocity/v = Vmax /2,
is called the Michaelis constant.

Km : units of concentration

Km : estimate for the optimum
concentration of substrate.
 FACTORS AFFECTING ENZYME REACTION

General reaction of enzyme:

E + S  ES  E + P
1. Substrate concentration
2. Enzyme concentration
3. Temperature
4. Acidity (pH)
5. Inhibition
 I. SUBSTRATE CONCENTRATION
(NON-ENZYMIC REACTIONS)

Reaction
velocity

Substrate concentration

The increase in velocity is proportional to the

substrate concentration
 II. ENZYME CONCENTRATION
(ENZYMIC REACTIONS)

Vmax

Reaction
velocity

Substrate concentration
 III. TEMPERATURE
Coefficient of temperature  increase in reaction rate.
Q10  2 to 3 increase the rate of reaction every 10°C rise in
temperature
Enzyme-controlled but at high temperatures proteins or
enzyme denature (most enzymes denatured at 70°C)
The optimum temperature for an enzyme controlled
reaction.
Most enzymes the optimum temperature is about 30°C-
37°C
A few bacteria have enzymes can stable up to 100°C (used
in PCR reaction  Taq polymerase)
 IV. ACIDITY (pH)

Extreme pH  enzyme denaturation

Structure of the enzyme is change
The active site is distorted and the substrate
molecules will no longer fit in it
pH values between 5.0 and 9.0
A few enzyme such as pepsine optimal pH <2
 V. INHIBITORS
Inhibitors are chemicals that reduce the rate of
enzymic reactions.
The are usually specific and they work at low
concentrations.
Many drugs and poisons are inhibitors of enzymes in
the nervous system.
Type of inhibitor:
1. Competitive Inhibitor (CI)
2. Non Competitive Inhibitor (NCI)

1. Competitive inhibitor = substrate analog inhibitor.

Bind to same site with substrate (bind at the active
site).
x
I EnzI Enz + P
E
S EnzS Enz + P
Competitive Inhibitors: block enzyme
reactions  potent chemotherapeutic agents
Sulfanilamide, structural analog of PABA will
block folic acid synthesis.
Folic acid is important for bacteria to divided
Pteridine + PABA + Glutamic acid  FA
Amethopterin (MTX) is C-I of dihidrofolate in
dihidrofolat reductase reaction.
Antagonist to B vitamin: pyrithiamin and
oxythiamin antagonist to B1.
Sulfonamide derivatives acetazolamid (Diamox) is
C-I of Carbonic anhydrase
2. Non Competitive Inhibitor (NCI)
Reversible:
ENz +/- I  EnzI + S  ENzIS  E + P
Enz +/- S  EnzS + I  EnzSI  E + P

Irreversible:
Poison such as Iodoacetamide, heavy metal
(Hg, Ag), Oxidizing agent  reduce enzyme
activity
Noncompetitive Inhibitor
 Effect of enzyme inhibition
Irreversible inhibitors: Strongly bind at active
site.
Examples: pesticides, containing
organophosphorus, bind with serine residues in
the enzyme acetylcholine esterase.
Reversible inhibitors: These can be washed out
of the solution of enzyme by dialysis.
 ISOZYME
Isozyme = iso-enzyme
Same catalytic activity with enzyme.
Medical interest in isozymes start in 1957: where
human contained several lactate de-hydrogenase
isozymes.
The active of lactate dehydrogenase mol consist of
4 subunits of 2 types H and M.
Only the tetrameric mol possesses catalytic activity
HHHH, HHHM, HHMM, HMMM, MMMM. (I1,
I2, I3, I4, I5)
 ENZYME IN CLINICAL DIAGNOSIS
A. Functional plasma enzyme
B. Nonfunctional plasma enzyme.

A. Functional plasma enzymes:

Present all time in the circulation.
Level activity in plasma always high
Its substrates are in the blood.
lipoprotein lipase, enzyme for blood clot and
blood clot dissolution.
B. Non Functional Plasma Enzymes:
No physiologic function in blood
Their substrate absent from plasma.
Its made in tissue/organ.
Its level activity in plasma much lower than in the
tissue.
High concentration in plasma suggest a tissue
destruction
TRANS-AMINASE:
Two kind of Transaminase have clinical interest
GOT (AST) and GPT (ALT)
GOT(AST) transfer of amino group of aspartic acid
to alphaketo glutaric acid forming glutamic and
oxaloacetate
GPT(ALT) transfer amino group of alanin to
alphaketo glutaric acid, forming glutamic and
pyruvic acid
Increase after organ destruction such as AMI.
LACTATE DEHYDROGENASE (LD/LDH)
Catalyze the reduction of pyruvate to lactate in the
presence of NADH
Increase in myocardial infarction, acute and chronic
leukemia, acute hepatitis.
There are 5 isozymes of LDH: I1, I2,I3,I4 and I5
Cardiac muscle contains LD I1 .
Liver rich of LD-I2 and LD-I5
 CPK (creatine phospho kinase) /CK (Creatine
Kinase)
The measurement of CK/CPK activity is a value in
diagnosis of disorder affecting skeletal and cardiac muscle
such as dystrophia musculurum familial (pseudo-
hyperthropy)
Non muscular tissues, except brain, do not contain high
level of CK
CK has 3 isozyme: CK-MM, CK-MB, CK-BB
CK-MB increase in cardiac muscle disorder
 CERULLOPLASMIN
Copper-containing globulin  oxidase activity
Cerruloplasmin elevated in:
cirrhosis hepatis
Bacterial infection
Pregnancy.
Lipase: Elevated in acute pancreatitis, pancreatic
Ca.
Amylase: Increase in high intestinal obstructions,
parotitis, acute pancreatitis and DM
Trypsine: elevated in acute disease of pancreas.
More sensitive compare to lipase and amylase
Cholinesterase: High level occurs in the nephrotic
syndrome. Some insecticide (phosphate
inorganic) depress cholinesterase activity.
Test of this enzyme in blood used as indicator for
over exposure to insecticide .
ALKALINE PHOSPHATASE:
Catalyzing the hydrolysis of various phosphat
ester at alkaline pH.
Increase in rickets, hyperparathyroidism, Paget
disease, osteoblastic sarcoma, obstructive
jaundice, metastatic Ca. Also increase in
congestive heart failure as result by injury of liver.
ACID PHOSPHATASE:
Catalyzing hydrolysis reaction of phosphate ester in
acidic pH
May be elevated in metastatic prostate carcinoma.
Specific test for prostate such as PSA (Prostate
Specific Antigen)
APPLICATION OF ENZYME IN MOLECULAR MEDICINE

Recombinant DNA: Vaccine

Analysis of genetic disease
Paternal cases: Restriction enzyme
 Restriction Fragment Length
Polymorphism (RFLP)
THAN
K YOU