ENZYMOLOGY

TEAM TEACHING BIOKIMIA

(EMA QURNIANINGSIH, EDHI RIANTO, TRI MARTINI)
Biochemistry

“Definition”
Biochemistry is the scientific discipline dealing with
the chemical nature of living matters
and the chemical reactions they undergo

Biochemistry seeks to understand the structure,

organization, and function of living matter in chemical
terms
INTRODUCTION
FOOD

DIGESTION

METABOLIZED
METABOLISM
• Metabolism:
Chemical reactions carried out by and
in living systems
• Breakdown of organic matter
(eg food) to release energy
(catabolism)
• Construction of cell
components (eg
carbohydrates, proteins, lipids,
nucleic acids, other
macromolecules) using energy
(anabolism)
• Carried out by enzymes (+ co-
factors)
• Essential to life
• No metabolism = no life
 Organism cells molecules

enzymes
Reticulum endoplasmic

rybosom
cytoskeleton

nucleus mitochondrion

Golgi app.

cytosol lysosome

peroxisome
5

Eucaryote cell
• ENZYME :
PROTEIN  BIOCATALYST

• METABOLIC PATHWAY
E1 E2 E3 E4 E5 E6 E7
A B C D E F G P

• A = substrate
• P = product
• B,C,D,E,F,G = intermediates
• E1 catalyzes one-way reaction
Key /Regulatory enzyme

6
• Enzyme’s location within the organel :
• Is related to the organel’s function
• MITOCONDRIAL ENZYMES  catalyze energy production
reactions
Oxidative reaction  Energy
Respiratory chain  inside mitocondrion
• RIBOSOMAL enzyme  PROTEIN synthesis
• CATALYST  enhance the rate of reactions
• Participate in chemical reaction, enhance the rate of reaction,
may undergotransient modification during catalysis, but are
neither consumed nor permanently altered.
• Is required in small amount.

CELAH AKTIF (active site)

E
+ S
+
P
KOMPLEKS [ES] E 7
• INORGANIC CATALYST ENZYME
1. H+, OH-, Pt 1. PROTEIN  biocatalyst
2. ACTIVATION ENERGY  2. Activation energy 
3. - 3. SPECIFIC / extremely
selective
4. - 4. Heat-labile

Exception :
 Ribozyme :
 RNA molecule  ribonucleic acid

 Protein (-)

 Functions in : protein synthesis, RNA processing and gen

expression
 Catalyzes ligation reaction or the breakdown of
phosphodiester bond. 8
 Enzyme that is used in PCR  thermostable polymerase
 kead. transisi

G tanpa katalisator

E. level
Ea

=
dgn katalisator inorg
E. bebas
Ea'
dgn enzim

Ea''
kead. awal G = Perubahan
E. bebas

kead. akhir

Perjalanan
reaksi

 In order for reaction to occur, the substrates undergoing

the reaction need to be activated.
 The difference in energy between the substrate and the
transition state complex is called activation energy.
 Other definition : the amount of energy required to carry all
molecules in one mole of material under certain
temperature from initial state into transitional state to
overcome energy barrier.
 Enzymes increase the rate of reaction by
decreasing activation energy.
 For enzyme-catalyzed reactions, the
transition state is :
a condition in which bonds in substrate
are maximally strained, or
 electronic configuration of substrate
becomes very strained and unstable as it
enters transition state.
 The enzyme does not change the initial
energy level of the substrates or the final
energy level of the products  thus, enzyme
does not influence ΔG (change in free Gibbs
energy).
 Enzyme is highly selective / specific
 Enzyme will only catalyze specific
substrate or a small set of closely related
substrates.
 Example :
 Lactose glucose + galactose
Lactase
 Hexokinase :
Glucose
Fructose
Different affinity
• Enzyme specificity :
• Absolute specificity
• Relative specificity
• Optic specificity  Maltase  α
• Group specificity  alcohol dehydrogenase
• Influenced by :
• Binding of substrate
• Characteristic of catalytic group
• Cofactor
x
• A+B C
2x
2A + 2B 2C
3x
3A + 3 B 3C
• Enzyme as catalyst does not stoichiometrically related with
substrate / product
Classification and Nomenclature of
Enzymes
 Earlier days of Biochemistry : simple (but persist in use
to this day), exp. Pepsin, trypsin, amylase.
 Suffix - ase to a descriptor for the type of reaction
catalyzed
 Dehydrogenase is enzyme that removed the
element of hydrogen, H2, or H+
 Protease is enzyme that hydrolize protein
 Isomerase is enzyme that catalyze rearrangement
 Many cases : descriptors were supplemented with
terms indicated particular substrate, its source,
regulation, etc
• As more enzymes were discovered  International Union of
Biochemistry (IUB) developed system of enzyme nomenclature.
• Each enzyme has unique name and code number  identify
reaction catalyzed and its substrate.
• Example : EC. 2.7.1.1
• 2 : class of enzyme  tansferase
• 7 : phospate transfer
• 1 : alcohol group as acceptor
• 1 (last number) : the enzyme name  hexokinase

Heksokinase/Glukokinase
α-D-glucose α-D-glucose-6-P
Mg++
ATP ADP
 Enzymes are grouped into following
seven classes :
1. Oxidoreductase
• Enzymes that catalyze oxidations and reductions (the
addition/release of electron or hydrogen)
• Enzymes catalyzed biological oxidation.
• Exp. Lactate dehydrogenase
2. Transferase
• Enzymes that catalyze transfer of moieties such as glycosyl,
methyl or phosphoryl groups.
• exp. Hexokinase
3. Hydrolases
• Enzymes that catalyze hydrolytic cleavage of C-C, C-O, C-N
and other covalent bonds.
• Exp. Chymotrypsin  hydrolase thet cleaved peptide bonds
in protein.
4. Lyases
• Enzymes that catalyze cleavage of C-C, C-O, C-N and other
covalent bonds by atom elemination, generating double
bonds.
• Enzymes that catalyze cleavage of C-C, C-O, C-N by means
other than hydrolysis or oxidation.
• exp. Aldolase, decarboxylase, thiolase, etc.
5. Isomerases
• Enzymes that catalyze geometric or structural changes within
a molecule.
• Exp. Mutase, epimerase, isomerase.
6. Ligases
• Enzymes that catalyze the joining together (ligation) of the
molecules in reactions coupled to the hydrolysis of ATP.
• exp. Piruvate carboxylase, etc.
7. Translocases
• Enzyme that catalyze the movement of ions or molecules
across membrane.
• Exp. Carnitine-acylcarnitine translocase, etc.
Ligand
= small molecule that is attached/bind to bigger
molecule.
S

E
S=SUBSTRAT
I=INHIBITOR
E
A=AKTIVATOR
A E=ENZIM

E
S
I LIGAND
I A
Figure. Diagramatic representation of protein life cycle
(Harper’s ilustrated Biochemistry, 31st ed, 2018)
FOUR ORDERS OF PROTEIN STRUCTURE
The modular nature of protein synthesis and folding are
embodied in the concept of orders of protein structure:
• Primary structure—the sequence of amino acids in a
polypeptide chain;
• Secondary structure—the folding of short (3-30
residue);
• Tertiary structure—the assembly of secondary
structural units into larger functional units such as the
mature polypeptide and its component domains;
• Quaternary structure—the number and types of
polypeptide units of oligomeric proteins and their
spatial arrangement.
 PROTEIN STRUCTURE
H O H O H O H
O
| || | || | || |
+H N – C – C – N – C–C–N–C–C–––N– C–C
3
| | | | | | |
O–
R1 H R2 H R3 H R
Free carboxyl end
Free amino end
Peptide bond

H
|
R – C – COOH • in aqueous solution, amino acids are in charged-form
| •  PROTEINS are also in charged-form
NH2
amino acid

R group of amino acids has important role in structure and function

of protein.
 exp. Catalytic function
Primary structure—the sequence of amino acids in a
polypeptide chain, from free amino end to free carboxyl end.

The sequence and the amount of amino acids are different

among various type of proteins.

Proteins are synthesized from 20 basic amino acids encoded by

nucleotide triplets (codons)

+H N
3 COO–
aa1 aa2 aa3 aa4 aa5 aa6
 H H H
| | |
+OH–
R – C – COOH R – C – COO– R – C – COO–
| | |
NH3+ NH3+ NH2
+H+
iep pH>iep
pH < iep
muatan=0

• pH of the aqueous environment affects the charge of amino

acids
• Molecules that contain an equal number of positively and
negatively charged groups bear no net charge  zwitterions.

The form of most amino acids

in blood and tissues.
Secondary structure—the folding of short (3-30
residue). Maintaned by hydrogen bond, disulfide
bond. Exp. Alfa-helix, beta-sheet, random coil.
H H O R
| | || |
–N–C–C– C–C–N–
| || | |
CH2 O H H
| : :
S Hydrogen : :
Disulfide bond bond : :
|
S : :
| H H O
CH2 | | ||
| –N–C–C
–N–C–C– |
| | || R
H H O
 Beta-sheet

Alfa-heliks
Tertiary structure—the assembly of secondary
structural units into larger functional units such as
the mature polypeptide and its component
domains
celah
aktif

• If the protein consist of one polypeptide chain  Monomer

• Exp. Myoglobin
• Tertiary structure is maintained by hydrogen bonds, van der
waals force.
•  DIMER Quaternary structure—the
number and types of
subunit polypeptide units of
oligomeric proteins and their
•  TETRAMER spatial arrangement.
subunit • Maintained by hydrogen
bonds, electrostatic bonds.
• Benefits : to make the
• OLIGOMER
molecule more stabile and
POLIMER
to gain particular function.
CELAH AKTIF
(ACTIVE SITE)
Monomer proteins do not
have quarternary structure.
The polypeptide chains that build one protein (dimer,
oligomer, polymer) :
- Same polypeptide chains
- Different polypeptide chains

• gene of chain   
Hb : 22 • Amino acids sequence of
LDH : M4 chain   

H4
ISOZYMES
M3H Catalyze the same reaction
M2H2
MH3
~ : DIMER OLIGOMER

: TETRAMER
POLIMER
4 POLYPEPTIDE CHAINS
~ consist of MANY SUBUNITS
4 SUBUNITS (MANY POLIPEPTIDE CHAINS)

~ PH/PH, t  DENATURATION

PROTEIN STRUCTURES ARE DAMAGED, EXCEPT PRIMARY
STRUCTURE
Peptide bond
~ PEPTIDE BOND
STRONG BOND
DISULFIDE BOND
~ PROTEIN :
- ENZYME  FUNCTIONAL
- COLLAGEN  STRUCTURAL
 ENZYME-SUBSTRATE INTERACTIONS
 LOCK AND KEY THEORY  FISHER

 INDUCED FIT THEORY (KOSHLAND)

BINDING OF S CONFORMATIONAL CHANGES

• COMPLEMENTARY BETWEEN “E” AND “S” IS FORMED AFTER THE

BINDING OF SUSSTRATE TO THE ENZYME.
~ REACTIVE / FUNCTIONAL GROUPS OF AMINO ACID  GROUP THAT
WILL PARTICIPATE DIRECTLY IN THE REACTIONS (“R” GROUP/SIDE
CHAIN).
H
SH R – C – COO– OH
| R | | R
CH2 NH3+ CH2
| |
H3+N – C – COO– H3 N – C – COO–
+

| |
H H
Cysteine (Cys) C Serin (Ser) S
SISTEIN
S
~
E

• FUNCTIONAL GROUPS THAT PARTICIPATE DIRECTLY IN CATALYTIC

REACTION  FUNCTIONAL GROUPS THAT RESIDE IN ACTIVE SITE.
• HEAVY METALS  BIND SH-GROUP  INACTIVATE ENZYMES
Hg++
 • Catalytic site
• ‘S’ binding site

Active Site
 A cleft or crevice in the enzyme, formed by one or more region
of the polypeptide chain.
 Active site is formed because of tertiary structure.
 Within the active site, cofactors and functional groups from the
polypeptide chain participate in transforming the bound
substrate molecule into product.
 The active site contains :
 The functional group that will bind substrate (substrate-binding
group)
 The functional group that participate in catalytic reactions
(catalytic group)
Enzymes employ multiple
mechanisms to fascilitate catalysis
 Catalysis by proximity
• When enzyme bind sunstrate in its active site 
creates region of high local substrate concentration in
which the substrate molecukes are oriented in a
position ideal for them to chemically interact.
 Acid-base catalysis
• In addition to contributing to the ability of the active
site to bind substrate, the ionizable functional groups
of aminoacyl side chain (and/or prosthetic group) may
serve as acid (proton donor) or bases (proton
acceptor).
Enzymes employ multiple
mechanisms to fascilitate catalysis
 Catalysis by strain
• For catalysis of lytic reactions, enzymes bind their
substrates in conformation that weaken the bond
targeted for cleavage through physical distortion and
electronic polarization.
 Covalent catalysis
• It involves the formation of between the enzyme and
one or more substrate.
• The modified enzyme becomes reactant  provide
new reaction pathway (lower activation energy) 
faster reaction
• The modification of enzyme is transient  back to
original form at the end of reaction.
 PROSTHETIC GROUP, COFACTOR &
COENZYME
ENZYME :
 SIMPLE  PROTEIN
 MORE COMPLEX  PROTEIN + SMALL MOLECULES/ METAL
IONS
 PARTICIPATE DIRECTLY IN SUBSTRATE BINDING OR IN CATALYTIC REACTION.
 EXTEND THE REPERTOIRE OF CATALYTIC CAPABILITIES BEYOND THOSE
AFFORDED BY FUNCTIONAL GROUPS PRESENT IN AMINOACYL SIDE CHAINS
OF PEPTIDES.
 PROSTHETIC GROUP, COFACTOR, COENZYME
 Many of them are derivatives of B vitamins. Nicotinamide (NAD, NADP),
riboflavin (FMN, FAD), panthotenic acid (CoA), thiamine (TPP).

THE BINDING OF ENZYME – SMALL MOLECULES/METAL IONS :

 STRONG/TIGHT (exp. COVALENT FORCES)
 WEAK/LOOSE
Prosthetic Group
o Prosthetic groups are incorporated into enzyme protein’s
structure tightly and stably by covalent or non-cavalent
forces.
o the examples of prosthetic groups :
o pyridoxal phosphate, FMN, FAD, TPP, lipoic acid, biotin.
o Transition metals : Fe, Co, Cu, Mg, Mn, Zn.
o Metal ions that participatein redox reactions  bound as
organometallic complexes / metalloenzymes.
o Exp. Prosthetic groups heme
The functions of prosthetic groups :
• Participate in catalytic reactions (redox reactions).
Metal ions as prosthetic gropus may also :
• fascilitate the binding and orientation of substrates.
• fascilitate formation of covalent bond with reaction
intermediate.
• Acting as Lewis acids or bases to render substrate
more electrophilic or nucleophilic  more reactive.
Cofactors
o Serve similar function as prosthetic groups.
o Cofactors binds weakly and transiently to their cognate
enzymes or substrate  dissociable complexes  so,
they must be present in the surrounding environment 
promote complex formation  catalysis to occur.
o Exp. Metal ions
o Enzyme that require metal ion cofactors  metal-
activated enzyme
 Coenzymes
 COENZYM + APOENZIM HOLOENZIM

Unspecific non-protein PROTEIN Catalitically active

organic molecules

 Serve as RECYCLABLE SHUTTLES  transport many substrates from one

point within the cell into another.
 This shuttle function as :
 to stabilize such species that is too reative in presence of water, etc :

Hydrogen atoms (FADH2), hydride ions (NADH).

 To increase number of points of contact between substrate and
enzyme  increase affinity & specifity with which small chemical
groups are bound to enzyme. Exp. Acetate (KoA), glucose (UDP), etc.
ISOZYME

• A GROUP OF ENZYMES THAT CATALYZES

THE SAME REACTIONS
• Example : Lactate dehydrogenase

• T.D. 4 RANTAI POLIPEPTIDA

• 2 JENIS RANTAI : M
H
 LDH isozymes :
I1=H4 I2=H3M I3=H2M2 I4=HM3 I5=M4

 M & H : amino acids sequences are different

 Isozymes distribution are varry among tissues
 To diagnose a disease
 Physical, chemical, immunology characteristics
 slightly different
 Enzyme concentration measurement in plasma
 Diagnosing disease

Plasma functional enzymes Non-plasma functional enzymes

(Enzymes that work in plasma ) (Enzymes that work in tissues)

exmple. : N : cell death  pasive diffusion of

Blood coagulation factors intracellular enzymes
(enzymes)
LYPOPROTEIN LYPASE
 : cell death 
 inflammation
Level/concentration  :  Ca
 Synthesis disturbances in liver  TRAUMA
 / (-) : genetic deffect  GENETIC DISEASE

 Deffiency of F VIII : HEMOPHYLIA (ex : DMD)

ENZYME :
KINETICS
FACTORS THAT INFLUENCE THE
VELOCITY OF ENZYME-CATALYZED
REACTIONS
1.Substrate concentration
2.Enzyme concentration
3.pH
4.Temperature
5.Modifier : activator and inhibitor

Velocity : rate of formation of product per unit of time

THE INFLUENCE OF ENZYME LEVEL ON VELOCITY OF
ENZYME-CATALYZED REACTION

Enzymatic reactions, like any other chemical reactions

comply with the rule : increasing concentration of
reactants (E &/ S) will increase the velocity of reaction.
Generally, in biological system, enzymes are found in
very low concentratiom  substrates concentration
are much higher.
Thus, the increase in enzyme concentration will not
cause “the saturation” of substrate to its enzyme.
Under natural condition, the increase in enzyme
concentration will directly proportional (not
hyperbolic) to the velocity of enzymatic reactions.
THE INFLUENCE OF ENZYME LEVEL ON THE VELOCITY OF
ENZYMATIC REACTION

Laju reaksi (Vo)

[E]
THE INFLUENCE OF SUBSTRATE CONCENTRATION ON
THE VELOCITY OF ENZYMATIC REACTION
Substrate conc. in biological system are far much
greater than enzyme.
 the increase of substrate conc. will make
enzyme saturated with the subtrate, because all
enzymes has already bind substrate.
If all enzymes has already bind substrates, the increase
of substrate conc. Will fail to increase the velocity of
enzymatic reaction.
Thus, the increase of substrate concentration  will
increase the velocity of enzymatic reaction in
hyperbolic manner.
THE ENZYME SATURATED TO HIGH CONC. OF ITS
SUBSTRATE
THE INFLUENCE OF SUBSTRATE CONCENTRATION ON THE
VELOCITY OF ENZYMATIC REACTION (1)
Laju reaksi (Vo)

KM [S]
Kurva Michaelis-Menten
THE INFLUENCE OF SUBSTRATE CONCENTRATION ON THE
VELOCITY OF ENZYMATIC REACTION (2)
KM AND VMAX
Enzyme must bind substrate to form ES complex prior the
catalysis to transform substrate into product.

Pengikatan substat Katalisis

At any given instant, only substrate molecules that are

combined with the enzyme as ES complex can be transformed
into product. Thus, ES complexes >>  velocity >>.

Under saturating condition, Vi depends solely on-and thus is limited

by-the rapidity with which product dissociates from the enzyme
sothat it may combine with more substrate.
KM of the enzyme for the substrate is defined as the
concentration of substrate at which Vi equals ½
Vmax. Mathematically, it is described as:

Michaelis-Menten
equation

For enzymatic reaction:

k
KM = -1
+ k2
k1

The higher the k1 (means KM is smaller) → the more

enzymes are bound to substrate (ES), maka: (lihat slide
berikutnya)
 k-1 + k2
k1
KM determines affinity of enzyme to its substrate to form ES
complex.
The low KM determines enzyme has high affinity in binding its
substrate (more ES complex will be formed), on the contrary :
the high KM determines enzyme has low affinity in binding its
substrate.
KM can also be seen as the measurement of how many
substrate is required for effective catalysis to occur.
KM value is specific for each enzyme.

For example : enzyme A and enzyme B, both are able

to bind substrate C. Enzyme A has lower KM
compared to KM of enzyme B (which is higher), thus:
enzyme A will immediately bind substrate C
even under low substrate C concentration
(affinity of enzyme A to substrate C is higher ).
enzyme B will only bind substrate if the conc. Of
substrate C is higher.

Vmax is also specific for each enzyme, that

describes the maximum rate of substrate
transformation into product by the defined
enzyme.
 INHIBITOR
• One of characteristics which differrentiate enzyme
from general catalyst is that its vulnerability against
chemical compound’s negative impact known as
inhibitor.
• Inhibitor will decrease enzyme’s effectivity in
enhancing reaction rate.
• Physiologically, inhibitors play important role in
regulating metabolism within living system.
• Although, several inhibitors may be harmfull such as
toxin.
• Insecticide or pesticide  synthetic inhibitor
• Some inhibitors are used as therapeutic purposes such
as chemotherapy
INHIBITOR CLASSIFICATION

• Inhibitor will inhibit enzyme’s catalysis activity

• Based on its binding with enzyme, inhibitors are classified into :
1. REVERSIBLE INHIBITOR
THE BINDING OF INHIBITOR TO ENZYME IS REVESIBLE :
E + I EI
2. IRREVERSIBLE INHIBITOR
E + I EI

REVERSIBEL INHIBITORS : COMPETITIVE AND NON COMPETITIVE

IRREVERSIBEL INHIBITOR IS ALWAYS NONCOMPETITIVE

56
 BASED ON [SO] VS VO

• CLASSIFIED INTO :
• COMPETITIVE AND NON-COMPETITIVE
INHIBITOR
• COMPETITIVE :
• Competitive inhibitor “competes” with substrate for
binding at enzyme’s-substrate recognition site 
usually has analog structure
• An increase of substrate concentration can overcome
competitive inhibition.
• INHIBITOR NON KOMPETITIF:
• An increase of substrate concentration can not57
overcome non-competitive inhibition.
COMPETITIVE INHIBITOR
It is usually a close structural analog of the substrate
(classical)
Inhibitor competes with substrate to bind “active
site” of enzyme

E
COMPETITIVE INHIBITOR STRUCTURE

Classical COMPETITIVE Inhibitor

 STRUKTUR I MIRIP S I ANALOG S.
 I BEREBUT DANGAN S UNTUK MENEMPATI
CELAH AKTIF ENZIM
II

59
• NON-CLASSICAL

• STERICAL HINDRANCE INHIBITOR

INHIBITOR HALANGAN STERIK

E
60
COMPETITIVE INHIBITOR

- Vmax is not changed

- KM ↑
NON-COMPETITIVE INHIBITOR
Non-competitive Inhibitors’s structure is not analog
to substrate.
Inhibitor does not bind enzyme’s active site.
The binding of inhibitor at the enzyme does not
interfere the binding of substrate at the same
enzyme.
Complex E-I-S can not generate product.
NON-COMPETITIVE INHIBITOR

- Vmax ↓
- KM is not changed
Competitive
inhibition

Uncompetitive
inhibition

Nonkompetitif
inhibition
THE INFLUENCE OF PH ON ENZYMATIC
REACTION
Amino acid formula R – CH – NH2
COOH
 In aqueous solution, amino acids always in a charged form.
R – CH – NH3+ R – CH – NH3+ R – CH – NH2
COOH H+ COO- OH- COO-
pH < iep pH = iep ph> iep

 AT ISOELECTRIC POINT, AN AMINO ACID BEARS NO NET CHARGE.

 ZWITTERIONS  THE FORM OF MOLECULE THAT HAS AN EQUAL NUMBER
OF POSITIVE AND NEGATIVE CHARGES AND THUS IS ELECTRICALLY
NEUTRAL.
 NET CHARGE = 0
 BRONDSTET : ACID : H+ DONOR 65
BASE : H+ ACCEPTOR
 • Optimum pH of the
pH I enzyme is the pH
pH II where enzyme’s
activity is at the
P
pH III
highest  pH I
pH IV • At optimum pH, P/t
o
is always higher
compared to other
o t
pH.

66
 • OPTIMUM pH of
100 enzymes is 5 – 9
(bell-shaped)
Akrivitas E (%)

• exception:
• PEPSIN : optimum
pH is 1-2

pH < pH opt pH>

67
A plot of pH versus enzyme’s activity
reveals the bell-shaped curve.

 Protein will denaturate if the pH is too low or too high.

 The influence of pH on enzyme’s and substrate’s charge :
E- dan SH+  ESH
If pH >  SH+  S + H+
S fails to bind E-
If pH <  E- will react with H+  EH
EH cannot bind SH+
 pH inluences conformation/structure of Enzyme.

68
THE INFLUENCE OF TEMPERATURE ON
ENZYMATIC REACTION

• TEMPERATURE
• KINETIC ENERGY REACTION OCCURS FASTER

• ENZYME IS PROTEIN DENATURATION

50o C
60o C
P
40o C
80o C
69
o
o t1 t t2
• “OPTIMUM TEMPERATURE” DEPENDS ON OBSERVATION TIME
(SEE THE PREVIUOS PLOT)
• AT t1  60oC
• AT t2  50oC
• GENERALLY, ENZYME IS :
• STABIL UNDER LOW TEMPERATURE
• VULNERABLE TO HEAT (70oC WILL DENATURATE ENZYME IMMEDIATELY)

70
 THE INFLUENCE OF ACTIVATOR ON ENZYMATIC
REACTIONS
A = ACTIVATOR
I = INHIBITOR
S = SUBSTRATE
E = ENZYME
• ENYMATIC REACTION’S
ACTIVATOR : Any substances S
that enhance enzymatic reaction.
• Examples : metal ions,
cofactors.
• Not all activators are cofactors.
E
I

A
 ENZYMES WITH M-M KINETICS

[So]1 < [So]2 < [So]3 < [So]4 < [So]5 < [So]6 < [So]7 < [So]8
vomax MONOMERIC
ENZYMES
vo
½ vomax

O
O
KM [So] 72
[So]6 [So]7
[So]1 < [So]2 < [So]3 < [So]4 < [So]5 < [So]6 < [So]7 < [So]8

vomax OLIGOMERIC
ENZYMES

vo
½ vomax

O
O
KM 73
[So] [So] [So]
• OLIGOMERIC ENZYME WITH M-M KINETIC
LACTATE DEHYDROGENASE (LDH)

• The binding of a substrate to one subunit does

not influence the binding of substrates to
other subunits of the same enzyme molecule.

74
 HOMOTROPIC KINETICS

OLIGOMERIC ENZYMES
“HIDDEN” ACTIVE SITE

• If the binding of a substrate molecule to one

subunit influences the binding of next (same kind
of) substrates to other subunits of the same
enzyme molecule.
• Positive co-operativity (“easier”)  allosteric
enzyme
• Negative co-operativity (“more difficult”)
75
 ALLOSTERIC ENZYMES
vomax

SIGMOID
[So]0.5 is on a vo
steep area ½ vomax
Mostly are
Regulatory
enzymes

o
o [So]
[So]0.5 76
• Regulatory enzymes are (mostly) an allosteric enzymes
A B C D E P
E1 E2 E3 E4

E1 is Regulatory Enzyme

• Enzyme with homotropic kinetics-positive cooperativity

s

77
 HETEROTROPIC KINETICS
• Allosteric enzymes are influenced by allosteric
activator/inhibitor  Heterotropic Kinetics
• SIGMOID-shaped Curve :
• A  shifts the curve to the left
• I  shifts the curve to the right  more
SIGMOID

78
 S

EFFECTOR SITE:
TEMPAT A
TEMPAT I
79
 vomax

CONTROL : ALLOSTERIC +ATP

ENZYME (ASPARTATE CONTROL
TRANSCARBAMOYLASE) –
WITHOUT MODULATOR vo +CTP

ATP : ALLOSTERIC ½ vomax

ACTIVATOR

CTP: ALLOSTERIC
INHIBITOR

PRODUCT : N- CARBAMOYL
–L-ASPARTATE
o
o [So] 80
[So]0.5
 REGULATION OF ENZYMATIC REACTION

• An enzymatic reaction is a part of metabolic pathway.

•A B C D E P
E1 E2 E3 E4

• E1 catalyzes one way reaction  Regulatory Enzyme

regulating the rate of reaction
Metabolic pathway regulation by Enzyme

• Many metabolic pathways take place in the cells of organism.

• Every metabolic pathway consist of series of chemical
reactions in which each reaction is catalyzed by specific
enzyme.
• Each metabolic pathway must be controllable so that the
reactions run at a rate that is in accordance with the needs of
the cells/organism/living creature.
• The metabolic pathway rate is regulated by regulatory enzyme.
• The Regulatory enzyme (in general) is an enzyme that has
lowest catalytic rate in a metabolic pathway.
• The final products of metabolic pathway are mostly
allosteric inhibitor of the regulatory enzyme of the pathway.
• Accumulation of product will inhibit regulatory enzyme’s
activity (feedback inhibition)  product accumulation
will be avoided.
(-)

E2 E3 E4 E5
E1 A B C D P
S1 S
e1
a e2 b e3 c e4 d e5 p
E1 is regulatory enzyme of S methabolic pathway  P
P is allosteric inhibitor of E1
• In general, the regulation of enzymatic reaction can be achieved
through several mechanisms :
• The changes in Substrate concentration
• the changes in substrate concentration generate corresponding
changes in metabolic flux
• The changes in Enzyme concentration
• The changes in enzyme’s synthesis rate or enzyme’s
degradation rate (slow response, hours/days - weeks)
• Isolation of enzymes in intracellular compartment
• Hydrolytic enzymes are placed inside the lysosome  to avoid
enzymes hydrolize important components of the cell.
• Modulation of Enzyme’s catalytic activity
• Immediate response (seconds – minutes)
• Consist of : (1) allosteric regulation, (2) reversible covalent
modification, (3) proteolytic activation, (4) regulation by
regulator protein.
Regulation of Enzyme’s synthesis
MODEL a
OP E1 E2 E3
REGULATOR

mRNA

REPRESOR

OP E1 E2 E3
REGULATOR

mRNA mRNA

REPRESOR INDUCER E1 E2 E3
• OPERON: A group of structural genes containing genetic
codes of a particular metabolic pathway’s enzymes.
• OPERATOR ; a DNA segment which has function as
ON/OFF button for OPERON transcription.
• REGULATORIC GENE: Gene that contains genetic codes of
Repressor protein.
 MODEL a: INDUCIBLE ENZYME
• Used in CATABOLIC pathway
• When REPRESOR locks OPERON (inactivate OPERON)  No
enzyme synthesis
• If INDUCER is available  INDUCER binds REPRESOR  OPERON
is unlocked  OPERON is activated  ENZYME synthesis.
• INDUCER is usually substrate of a metabolic pathway, for example
substrate A
A B C D E P
• This kind of enzyme is known as INDUCIBLE enzyme
 MODEL a

E.COLI was cultured in a medium containing glucose 

proliferate

E.COLI was cultured in a medium containing lactose (not

glucose)  failed to proliferate, at first  after some time 
proliferate
Lactose : inducer  induce synthesis of the enzyme that
hydrolyzes lactose into glucose and galactose.
The enzyme is known as inducible enzyme
 MODEL b : REPRESSIBLE ENZYME

REGULATOR OP E1 E2 E3

mRNA mRNA

REPRESSOR
E1 E2 E3

REGULATOR OP E1 E2 E3

mRNA

REPRESSOR = COREPRESSOR
• REGULATORORY GENE  mRNA  REPRESSOR
• REPRESOR DOES NOT BIND OPERATOR  ENZYME
SYNTHESIS (OPERON IS ACTIVATED)
• IF COREPRESSOR IS AVAILABLE  BINDS TO REPRESSOR
 LOCKS THE OPERATOR  INHIBITS ENZYME SYNTHESIS
• COREPRESSOR IS USUALLY PRODUCT OF A METABOLIC
PATHWAY.
• COMMONLY USED IN BIOSYNTHETIC PATHWAY
 E. COLI SYNTHESIZED
ALL AMINO ACIDS
REQUIRED INCLUDING
LEUSIN
MEDIUM THAT DID NOT
CONTAINS LEUSIN

E. COLI DID NOT

SYNTHESIZE LEUSIN
MEDIA CONTAINED LEUSIN

E. COLI SYNTHESIZED
LEUSIN
MEDIUM THAT DID NOT
CONTAINS LEUSIN
• This phenomenon is known as REPRESSION
• LEUSIN is COREPRESSOR
• ENZYMES that catalyze SYNTHESIS OF LEUSIN is known
as REPRESSIBLE ENZYMES
• DEREPRESSION: IF E. COLI IS CULTURED BACK IN
MEDIUM THAT DOES NOT CONTAIN LEUSIN 
ENZYMES that catalyze SYNTHESIS OF LEUSIN IS
AVAILABLE  LEUSIN IS SYNTHESIZED
• CONSTITUTIVE ENZYMES :
• Enzymes that are synthesized in all circumstances
• INDUCIBLE ENZYMES:
• Enzymes which its synthesis is inducible by inducer
• ENZIM REPRESIBEL:
• Enzymes which its synthesis is repressed by
corepressor
 REGULATION OF ENZYME’S CATALYTIC
EFFICIENCY

• ALLOSTERIC MODULATION
• Allosteric : occupy other place
• ALLOSTERIC ENZYME : Enzyme where its active site is
modulated by effector that occupies allosteric site
• Allosteric regulator will induce conformational changes
of allosteric enzyme’s active site
• COVALENT MODIFICATION
• Mostly, by enzyme phosphorylation-
dephosphorylation.
• Example :
1. GLYCOGEN PHOSFORYLASE :
• PHOSPHO-ENZYME : ACTIVE FORM
• DEPHOSPHO-ENZYME : INACTIVE FORM
2. GLYCOGEN SHYNTETASE :
• DEPHOSPHO-ENZYME : ACTIVE FORM
• PROENZYME – ENZYME MECHANISM
• EXAMPLE : PEPSINOGEN (PROENZYME)  PEPSIN (ACTIVE
ENZYME)
• BLOOD COAGULATION FACTORS/ENZYMES  CASCADE

PRO-E1 E1

PRO-E2 E2

PRO-E3 E3

PRO-E4 E4

ETC
 PROENZYME=ZYMOGEN
• ENZYME THAT IS SECRETED IN AN INACTIVE FORM
• PURPOSE :
• TO PTOTECT BODY ORGANS
• TO PROVIDE “HALF-FINISHED” MATERIALS
• EXAMPLE : PEPSINOGEN
• TO ACTIVATE PROENZYME, PROTEOLYTIC ENZYME OR H+ IS UTILIZED

PEPSINOGEN PEPSIN
H+ / PEPSIN
H+ / proteolytic enzyme

PEPSINOGEN

PEPSIN

97
 THE REGULATION OF METABOLIC PATHWAY
X
-

A B C D P
E1 E2 E3 E4
+ -
+
Y
• X, Y : SUBSTANCES OUTSIDE METABOLIC PATHWAY, example.
Hormone, etc  covalent modification
• A, P : MEMBERS OF METABOLIC PATHWAY  regulating the enzyme
synthesis (induction/repression) or allosteric regulation
• A activates E2 (A = substrate)
• positive feedforward regulation

• P inhibits E2 (T = product)
• negative feedback regulation
REGULATION THROUGH ENZYME SYNTHESIS
Influences the amount of enzyme

A B C D P

If repression involves >1 or all enzymes in

one metabolic pathway  coordinated
Repression
In general, Repression is utilized in
biosynthetic/anabolic pathway
 R S T U V

 if induction involves >1 or all enzymes in one

metabolic pathway  coordinated induction

In general, induction is utilized in

catabolic/degradation pathway
 ALLOSTERIC REGULATION
influences enzyme’s catalytic efficiency

• ALLOSTERIC ACTIVATOR : It accelerates/activates

enzyme

• ALLOSTERIC INHIBITOR : It inhibits enzyme

 REGULATION OF TWO-WAY METABOLIC PATHWAY

GLUCOSE
HEXOKINASE ATP Pi GLUCOSE -
6P- ASE
ADP H2O
GLUCOSE-6P

GLYCOLYSIS

PIRUVATE/ LACTATE
• GLYCOLYSIS :
• GLUCOSE  PIRUVATE / LACTATE
• GLUCONEOGENESIS
• LACTATE  GLUCOSE
• IRREVERSIBLE REACTION  ONE-WAY
• REVERSIBLE REACTION  TWO-WAY
• IN GLUCONEOGENESIS, MOST REACTIONS ARE THE
REVERSAL OF GLYCOLYSIS REACTIONS.
• REACTIONS THAT IS CATALYZED BY REGULATORY ENZYMES
IRREVERSIBLE)  IS OVERCAME BY UTILLIZING OTHER
ENZYMES.
• HOW TO REGULATE? BOTH PATHWAYS ARE REGULATED
RECIPROCALLY.
• WHE BLOOD GLUCOSE IS HIGH  GLYCOLYSIS IS ON,
GLUCONEOGENESIS IS OFF.
• WHEN BLOOD GLUCOSE IS LOW  GLUCONEOGENESIS IS
ON, GLYCOLYSIS IS OFF.
 Another example :
GLYCOGEN
IN LIVER
GLYCOGENESIS:
GLUCOSE -1P GLUCOSE  GLYCOGEN
GLYCOGENOLYSIS
GLUCOSE -6P GLYCOGEN  GLUCOSE
When glycogenesis is on 
GLUCOKINASE GLUCOSE 6- glycogenolysis is off
PHOSPHATASE
Both pathways are regulated
reciprocally
GLUCOSE
 GLUCOSE
HEXOKINASE/ ATP Pi GLUCOSE -
6P- ASE
GLUCOKINASE ADP H2O
GLUCOSE -6P
REGULATING BOTH PATHWAY RECIPROCALLY  TO
PREVENT THE WASTE OF ATP (FUTILE CYCLE)
_
e2
+
A B C D E F
_
e5
+