TL3102 – REKAYASA PROSES BIOLOGI

ENZYMOLOGY

MARISA HANDAJANI

ENVIRONMENTAL ENGINEERING STUDY PROGRAM

FACULTY OF CIVIL AND ENVIRONMENTAL ENGINEERING
INSTITUT TEKNOLOGI BANDUNG
Metabolism
• All chemical reactions that occur in cells

• 2 major part:
• Energy-conserving reactions : release and conserve energy that provided by
organism’s energy source → CATABOLISM
• Energy-consuming reactions in order to build large-complex molecules from
small, simpler molecules → ANABOLISM
 ENERGY Cell works:
• Chemical work
• Transport work
• Mechanical work

Oxidation-reduction reactions
https://www.nature.com/scitable/topicpage/cell-metabolism-14026182/
Electron transport

e donor → e acceptor Energy is made available for work

(More negative) (More positive)
Enzyme
• Enzyme : biological catalyst
• Function : increase chemical reactions within living cells without
suffering any overall change
• Enzyme increase the chemical reaction by lowering the energy
activation of the reactions they catalyze

A+B→P
(substrate)
• Enzyme : specific in character
• Particular substrates
• Particular products

• Enzymes are proteins

Prosthetic
group

• Co-factor : non-protein component

• Co-enzyme : organic compound
• Metal ion
• Apoenzyme : Inactive protein component of enzyme
• Haloenzyme : Active enzyme including co-factor
• Prosthetic group : tightly bounded co-factor
• Naming : -ase (enzyme)
• Describe : substrate or reaction

• Isoenzyme : different enzymes that catalysing identical reactions

• All reactions catalyzed by enzymes are reversible to some degree

Mechanisme of Enzyme Reactions
• Enzyme increase the rate of reactions but do not alter the
equilibrium constants
Endergonic Reaction

A+B C+D • Enzyme will not shift the

enzyme equilibrium
• Enzyme simply speed up the
• Reactions require activation energy rate

• Enzyme accelerate the reactions by lowering the activation energy

• The formation of enzyme-substrate complex can lower the activation
energy
Mechanism
of Enzyme
Reactions
Interaction between Enzyme and Subtrate
• Enzyme brings substrates together at specific site on its surface to form an
enzyme-substrate complex
• Enzyme’s specific site is called active site or catalytic site
• Active sites contain amino acid side chains or other functional groups
which may serve as acids or bases or acceptors or donors of electron that
facilitating the reactions
• 2 ways of enzyme and substrate interaction:
1. Lock – key models
Enzyme may be rigidand shaped precisely fit to substrate so that the correct
substrate bind specifically and positioned properly for the reaction
2. Induce Fit models
Enzyme may change shape when it binds the substrate so that the active site
surround and precisely fits the substrate
 Substrate Enzyme
Interaction
• Lock and Key Model
• Induced Fit Model

https://alevelbiology.co.uk/notes/biological-catalysts-enzymes/
Effect of Environment to Enzyme Activity
• Substrate concentration
• At Low substrate concentration, enzyme makes products slowly since it seldom
contact with substrate.
• A substrate-saturated enzyme will operate at maximal velocity
• pH
• Enzyme function most rapidly at a specific optimum pH
• A great pH deviation, makes the enzyme function slower and may damage the
enzyme
• Temperature
• Enzyme function most rapidly at a specific optimum pH
• A high rise of temperature will disrupt the structure of enzyme and enzyme will lose
it activity.
Enzyme Inhibition
• Competitive Inhibition :
• directly competes with the substrate at an enzyme’s catalytic site and prevents the enzyme from forming
product
• Competitive inhibitors usually resemble normal substrates, but they cannot be converted to products.
• Uncompetitive Inhibition
• anti-competitive inhibition, takes place when an enzyme inhibitor binds only to the complex formed between
the enzyme and the substrate (the E-S complex).
• Typically occurs in reactions with two or more substrates or products. [1]
• While uncompetitive inhibition requires that an enzyme-substrate complex must be formed, non-competitive
inhibition can occur with or without the substrate present.
• Noncompetitive inhibitors :
• affect enzyme activity by binding to the enzyme at some location other than the active site.
• This alters the enzyme’s shape, rendering it inactive or less active.
• These inhibitors are called noncompetitive because they do not directly compete with the substrate. Heavy metal
poisons like mercury frequently are noncompetitive inhibitors of enzymes
Enzyme Inhibition Mechanism
 Metabolic Regulation
• Purpose :
• conserve raw materials and energy and
• maintain a balance among various cell components.
• Involves activating or inactivating pathways as needed.

• 3 ways of metabolic pathway regulation:

1. Metabolic channeling—this phenomenon influences pathway activity by localizing metabolites
and enzymes into different parts of a cell.
2. Regulation of the amount of synthesis of a particular enzyme—in other words, transcription and
translation can be regulated. These two processes function in synthesizing enzymes. Regulation at
this level is relatively slow, but it saves the cell considerable energy and raw material.
3. Direct stimulation or inhibition of the activity of critical enzymes—this type of regulation rapidly
alters pathway activity. It is often called posttranslational regulation because it occurs after the
enzyme has been synthesized.
Control Enzyme Activity
• Adjustment of the activity of regulatory enzymes and other proteins controls the
functioning of many metabolic pathways and cellular processes.
• This type of regulation is an example of posttranslational regulation because it
occurs after the protein is synthesized

• Posttranslational regulatory mechanisms

• Irreversible
• Reversible
• Allosteric regulation
• Covalent modification.
 Allosteric Regulation
• The activity of an
allosteric enzyme is altered by a small
molecule known as an effector or
modulator.
• The effector binds reversibly by noncovalent
forces to a regulatory site separate from the
catalytic site and causes
a change in the shape or conformation of
the enzyme
• A positive effector increases enzyme
activity, whereas a negative effector
decreases activity or inhibits the enzyme.
• These changes in activity
https://www.khanacademy.org/science/ap-biology/cellular- often result from alterations in the apparent
energetics/environmental-impacts-on-enzyme-function/a/enzyme- affinity of the enzyme
regulation for its substrate, but changes in maximum
velocity also can occur
Covalent Modification Regulation Regulatory enzymes also can
be switched on and off by
reversible covalent
modification through the
addition and removal of a
particular group, typically a
phosphoryl, methyl or adenyl
group.

The enzyme with an attached

group
can be either activated or
inhibited.

Systems of covalently
modified enzymes are able to
respond to more stimuli in
varied
and sophisticated ways.
Feedback Inhibition / End Product Inhibition

Feedback inhibition ensures balanced production of a pathway end product. If the end product becomes too
concentrated, it inhibits the regulatory enzyme and slows its own
synthesis. As the end product concentration decreases, pathway activity again increases and more product is formed.
Enzyme Kinetics (Michaelis-Menten Kinetics)
Basic assumption :
Enzyme catalysis occurs through a series of elementary reactions involving the
enzyme-substrate complex
k1 S : Substrate
S+E ES
k1
E : Free enzyme
k2 k-1
k-1
ES E+P ES : Enzyme-substrate
complex
The reaction is assumed in equilibrium : P : Product
k1 CE CS = k-1 CES
CES = CE CS / K*m
K*m= k-1 /k1
K*m : dissociation constant → reciprocal of the equilibrium constant
Mass Balance on Enzyme
CE + CES = CE0

Reaction rate (rp) = k2 CES

CES is not readily measureable → replaced by concentration of readily measurable
species such as
substrate and initial enzyme
CES = CE CS / K*m
Substitusi CE = CE0 - CES
CES = (CE0- CES) CS / K*m
CES = CE0CS /(K*m+ CS)
rp = k2 CE0CS /(K*m+ CS)
rp = VmCS /(K*m+ CS) where Vm = k2 CE0
 rP = - rS = VmCS /(Km+ CS)
Km= (k-1 + k2)/k1
Vm and Km can be
Km= Michaelis-Menten constant estimated by relating the
data of v and Cs for
v = - rS = VmCS /(Km+ CS) constant CE0 with linear
transfromation
V = velocity
At low concentration of substrate:
the reaction rate is approximately
first order

At high concentration of substrate,

the reaction rate is approximately
zero order
Enzyme Inhibition Kinetics
• Competitive Inhibition k1 k2
S+E ES E+P
k-1
k3
I+E EI
k-3

rP = VmCS /[Km(1+CI/KI)+ CS)]

KI= k-3 /k3
K’m= Km(1+CI/KI)

Effect of competitive inhibitor is to increase the Michaelis-Menten constant.

At high substrate concentration, a rate is equal to the rate that attained in the absence of
the inhibitor.
Enzyme Inhibition Kinetics
• Uncompetitive Inhibition k1 k2
S+E ES E+P
k-1
k3
I + ES ESI
k-3

rP = VmCS /[Km+ Cs(1+CI/KI)+ CS)]

V’m = Vm /(1+CI/KI)
K’’m= Km(1+CI/KI)

Effect of uncompetitive inhibitor is to reduce the Michaelis-Menten constant.

A high substrate concentration will not overcome the effect of inhibitor, because the
inhibitor binds with the enzyme-substrate complex rather than with the free enzyme.
Enzyme Inhibition Kinetics
• Noncompetitive Inhibition
k1 k2
S+E ES E+P
k-1
k3
I+E EI
k-3

k4
I + ES ESI
k-4

rP = VmCS /(Km+Cs)(1+CI/KI)
V’m = Vm /(1+CI/KI)
Effect of noncompetitive inhibitor is to reduce the Vm without affecting Km
A high substrate concentration will not overcome the effect of inhibitor, because the
inhibitor binds with the enzyme-substrate complex rather than with the free enzyme.
Enzyme Inhibition Kinetics
• Substrate Inhibition k1 k2
S+E k-1 ES E+P
k3
k-3
S + ES SES

Ks and K’s are the

rp = k2 CE0CS /(Ks+ CS+CS2/K’s) dissociation constant for ES
Vm = k2 CE0 /(1 + 2(Ks/K’s)0,5 and SES

When substrate concentration are very high, some substrates will bind with enzyme-
substrate complex as well as with free enzyme
The formation of enzyme-substrate-substrate complex will inhibit the reaction to yield the
product
Enzyme Inhibition Kinetics
• Product Inhibition k1 k2
S+E ES E+P
k-1

ES + P k3 ESP
k-3
rP = VmCS /(Km+Cs)(1+CP/KP)
KP= k-3 /k3

The Product of enzymatically catalyzed reaction may act to inhibit the reaction forming it, because
the product is bind with enzyme-substrate complex and forming an unreactive enzymee-substrate-
product complex
This inhibition tend to behave like uncompetitive inhibition.
Any Question???
References
• Willey, Joanne M. at al (2008) Prescott, Harley, and Klein’s Microbiology, McGraw Hill, 7th ed.
• Hogg, Stuart (2013), Essential Microbiology, John Wiley&sons, 1st ed.
• Palmer and Bonner (2007) ENZYMES:Biochemistry, Biotechnology and Clinical Chemistry, WP, 2nd ed
• Grady and Lim (1980), Biological Wastewater Treatment, Marcel Dekker inc.