Enzymes

• all the reactions of the body are mediated by enzymes

• Enzymes are protein catalysts that increase the rate of
the reactions without being changed in the overall
process
• Catalysts for biological reactions
• Lower the activation energy
• Increase the rate of reaction
• Activity lost if denatured
• May be simple proteins
• May contain cofactors such as metal ions or organic
(vitamins)
 Enzymes
Nomenclature of enzymes
Enzyme has two names
a. Short Recommended name
b. Systemic name
Recommended name
• End in –ase,
• Identifies a reacting substance
sucrase – reacts sucrose
lipase - reacts lipid
• Describes function of enzyme
oxidase – catalyzes oxidation
hydrolase – catalyzes hydrolysis
lactate dehydrogenase, adenylate cyclase…
• Common names of digestion enzymes still use which don't provide
any hint as pepsin and trypsin
Systematic name
The international union of Biochemistry and Molecular Biology (IUBMB)
developed a system for nomenclature in which enzymes are divided into
6 groups and sub classes. These names are unambiguous and
informative but sometimes long and difficult to be of general use.

• Class Reactions catalyzed

1. Oxidoreductases oxidation-reduction
2. Transferases transfer group of atoms
3. Hydrolases hydrolysis
4. Lyases add/remove atoms to/from a double bond
5. Isomerases rearrange atoms
6. Ligases combine molecules using ATP
Enzyme Action:
Lock and Key Model
• An enzyme binds a substrate in a region
called the active site
• Active site is a special pocket or cleft
in the enzyme molecule
• The active site contains amino acids
side chains that form a three
dimensional surface complementary to
the substrate
• Only certain substrates can fit the
active site
• The active site binds to the substrate
and form enzyme-substrate complex
that will dissociate into the enzyme and
product.
• Amino acid R groups in the active site
help substrate bind
 Lock and Key
The active site of the unbound enzyme is complementary in shape to
that of the substrate
Enzyme Action: Induced Fit Model
• Enzyme structure flexible, not rigid
• Enzyme and active site adjust shape to bind substrate
• Increases range of substrate specificity
• Shape changes also improve catalysis during reaction

The enzyme changes

shape upon binding
substrate
The active site has a
shape complementary
to that of the
substrate only after
the binding
Cofactors
- Some enzymes associate with non-protein
cofactor that is needed for enzymatic activity
- These cofactor include metal ions (Zn, Fe) and
organic molecules called coenzymes such as
vitamins; NAD+, FAD, CoenzymeA..
- Holoenzyme refers to the enzyme with its
cofactor, Apoenzyme refers to the protein
portion of the holoenzyme and it dose not
show biological activity.
- Prosthetic group is a tightly bound enzyme
that dose not dissociate from the enzyme
Location of enzymes
Many enzymes are located into specific
organelles in the cell serve to isolate the
reaction substrate or product from each other
and to provide a special environment for a
reaction and to organize these reactions
Turnover number and catalytic efficiency
- Enzyme-catalyzed reactions are highly efficient, proceeding from 10 3 – 108
times faster than uncatalyzed reactions.
- The number of substrate molecules converted into product per enzyme per
second is called Turnover number, each enzyme molecule is capable of
transforming 100-1000 of substrate molecules into product per second
- Enzymes are highly specific, interacting with one or a few specific substrate

Enzymes could be enantiomers specific

 Enzymes could be enantiomer specific

Enantiomer are isomers that are mirror images of each other and that
differ in shape due to the presence of an asymmetric carbon
 How Enzymes work
The mechanism of action of enzymes can be explained by two different
modes
A. Energy changes during the enzyme-catalyzed reaction, enzyme provide
an energetically reaction pathway different from uncatalyzed reaction
B. The active site chemically facilitates catalysis
Energy Changes during the reaction
- the reactant and the product are separated by energy gap or energy
barrier that called free energy of activation and it equals to the energy
difference between the energy of reactant and the energy of high-
energy intermediate (Transition state)
A  T*  B
- The peak of energy represents the Transition state in which the high-
energy intermediate is formed during the conversion of reactant into
product.
- because of large activation energy the rate of uncatalyzed reaction is
slow
Rate of Reaction
-The substrate molecules should have sufficient energy to
overcome energy barrier to be converted into products.
- only few molecules have energy to pass the energy gap 
rate of reaction is determined by number of molecules that
is converted into the product.
- the lower the free energy of activation, the more molecules
have sufficient energy to pass over the transition state
the faster the rate of the reaction
 Factors Affecting Enzyme Action

• Temperature

• pH

• Cofactors & Coenzymes

• Inhibitors

From the Virtual Cell Biology Classroom on ScienceProfOnline.com Image: Animation of Enzyme, Wiki
Factors Affecting Enzyme Action
Different enzymes show different response to changes in substrate
concentration, temperature, and pH
Reaction Rate (velocity of a reaction):
• Reaction rate is the number of substrate molecules converted to product per
unit time and is usually expressed as µmoles product formed per minute
• The rate of reaction (enzyme catalyzed) increases with substrate
concentration until a maximal velocity (Vmax), the plateau reflects the
saturation with a substrate of all available binding sites on the enzyme

Maximum activity
 Increasing substrate
concentration increases the Reaction Rate
rate of reaction (enzyme
concentration is constant)
 Maximum activity occurs when
all of enzyme combines with
substrate

substrate concentration
Factors Affecting Enzyme Action: Temperature
• Little activity at low temperature
• Rate increases with temperature (the velocity increased with Tem
until a peak due to the increased number of molecules having
sufficient energy to pass over the energy barrier and form
product)
• Most active at optimum temperatures (usually 37°C in humans)
• Activity lost with denaturation at high temperatures decrease
the velocity
Factors Affecting Enzyme Action: pH
• pH affect the ionization of the amino acids in the active site.
• R groups of amino acids in the active site should have proper charge to bind
with the substrate (the ionization or unionization is affected by the pH)
• Maximum activity at optimum pH
• Tertiary structure of enzyme is correct
• Most lose activity in low or high pH, extremes of pH can also lead to
denaturation of the enzyme because the structure of active protein depends on
the ionic character of the amino acid

• Narrow range of activity,

and the pH optimum varies
Reaction

for different enzymes

Rate

Optimum pH

11 9 7 5 3

pH
Each enzyme has
its optimum pH
Michaelis-Menten Assumption of An Intermediate Complex
Michalis and Menten proposed model that accounts for most features of enzyme-
catalyzed reactions. In this model the enzyme reversibly binds its substrate to
form ES complex that subsequently breaks down to product
k1 k2
E+S ES E+P
Where k-1
E: enzyme
S: substrate
ES: enzyme-substrate complex
P: product
k1 k-1 k2 are rate constants

Michaelis-Menten Equation

The Michaelis-Menten Equation describes how reaction velocity varies with

substrate concentration
V [S]
Rate v =
km + [S]
Assumptions Required for Michaelis-Menten Equation
• E and S combine to form ES complex
• the concentration of S is much greater than concentration of enzyme the
amount of the substrate bound by the enzyme at any one time is small.
• E + S = ES rapidly reaches equilibrium: steady state assumption, concentration
of ES is constant, dose not change with time the rate of formation of ES is
equal to that of the breakdown of ES (to E and P)
• Reaction from P is irreversible
• only the initial reaction velocities are used in the analysis of enzyme reactions-
the rate of reaction at zero time, at that time the concentration of product is
very small  the rate of the back reaction from P to S can be ignored

Michaelis-Menten Equation
Vo: initial reaction velocity
Vmax: maximal velocity V max[S]
Km: michaelis-menten constant=
(k-1+K2)/K1
vo =
[S]: concentration of substrate km + [S]
 Vmax Maximum activity

Most of enzymes show hyperbolic

dependence of velocity on

Reaction Rate
substrate concentration Vmax/2

Km

substrate concentration
 Conclusions about Michaelis-Menten kinetics

Characteristics of Km:
 The Km constant is a characteristics
of an enzyme and particular substrate
 Km reflects the affinity of the
enzyme for a particular substrate
 Km : equal to the concentration
substrate at which the reaction
velocity is equal to ½ Vmax.
 Km dose not vary with concentration
of the enzyme
 low Km  high affinity; low [S] is
needed to half-saturate the enzyme
 large Km  low affinity; high [S] is
needed to half-saturate the enzyme
Relationship between the velocity to
enzyme concentration: the rate of
reaction is directly proportional to the
enzyme concentration at all substrate
conc. if the enzyme concentration is
halved  the Vo is reduced to half

Order of reaction:
 When [S] << Km, V (velocity of
reaction) is roughly proportional to
[S] the rate of the reaction is first
order reaction

 When [S] >> Km, the V is constant and

equal Vmax. The rate of reaction is
independent on the [S] the reaction
rate is Zero order
 Double reciprocal of Michaelis-Menten equation

When V is plotted versus the [S], it is not always possible to determine

the Vmax or Km from the graph because of gradual upward slope of the
hyperbolic curve

vo =Vmax [S] / (Km + [S]) (Michaelis-Menten Equation)

Equation of straight line: y= ax + b (a = slope, b = y intercept

Linear Transform: take reciprocal of each side of equation

to obtain straight line
1/vo = Km/Vmax • 1/[S] + 1/V
y = a x + b
 Double Reciprocal Plot
1/vo = Km/Vmax • 1/[S] + 1/Vmax

straight line allow us to

determine the Vmax and Km
simply
The x intercept is -1/km
The y intercept is 1/Vmax
 Enzyme Inhibition
• Enzyme inhibitor: any substance that can decrease the velocity of an
enzyme-catalyzed reaction and cause a loss of catalytic activity
• Inhibitors can be reversible or irreversible.
- Reversible inhibitors bind to enzymes through a non-covalent bonds.
Dilution the EI (enzyme-inhibitor) complex dissociate and recover the
enzyme activity, may be competitive or noncompetitive
- Irreversible inhibition occur when the inhibited enzyme can not
recover its activity by dilution. The inhibitors form a covalent bonds
with the active site enzyme or destruction of the protein structure of
the enzyme

Irreversible Inhibitors
Competitive inhibitors
This type of inhibition occurs when the inhibitors bind reversibly to the
same site that the substrate normally occupy  compete the substrate
for that site
A competitive inhibitor
• Has a structure similar to substrate
• Occupies active site
• Competes with substrate for active site
• Has effect reversed by increasing substrate concentration
Competitive inhibitors
Vmax: the effect of the competitive inhibitors is reversed by increasing the [S].
At sufficient high substrate concentration, the reaction velocity reaches the Vmax
observed in the absence of the inhibitors. The y-intersect is unchanged
Km: a competitive inhibitors increases the Km for a given substrate in the
presence of this inhibitors , more substrate is needed to reach the Vmax. The x
intersect is changed indicating that the Km is increased
Noncompetitive Inhibition
• Non competitive inhibition occurs when the inhibitor at a site distinct from
the substrate site; the inhibitor bind to site other than active site
• A noncompetitive inhibitor does not have a structure like substrate.
• Alter the shape of enzyme and active site Substrate cannot fit altered
active site
• It binds either to the enzyme or to the ES complex No reaction occurs
• Effect is not reversed by adding substrate
• It may bind to free E or to ES. Once bound it will prevent P formation.
• And the affinity of the I to both E and Es is the same.
• Non-competitive inhibitors decrease the Vmax, and can not be
overcome by increasing the substrate concentration
• Non-competitive inhibitors don’t affect the Km, the enzyme
show the same Km in the absence or the presence of the
inhibitors
Uncompetitive
Inhibitors
Inhibitor binds at an
allosteric site, but only
to the ES complex and it
lower both Vmax and Km
Vo
 Enzyme Inhibition (Mechanism)

I Competitive I Non-competitive I Uncompetitive

Substrate E
Cartoon Guide

S S E I
S X
E S I I
I
Compete for S I
Inhibitor active site Different site

E + S←
→ ES → E + P E + S←→ ES → E + P E + S←
→ ES → E + P
Equation and Description

+ + + +
I I I I
↓↑ ↓↑ ↓ ↑ ↓↑
EI EI + S →EIS EIS
[I] binds to free [E] only, [I] binds to free [E] or [ES] [I] binds to [ES] complex
and competes with [S];
complex; Increasing [S] can only, increasing [S] favors
increasing [S] overcomes
not overcome [I] inhibition. the inhibition by [I].
Inhibition by [I].

Juang RH (2004) BCbasics

 Enzyme Inhibition (Plots)

I Competitive I Non-competitive I Uncompetitive

Vmax Vmax Vmax
vo vo
Direct Plots

Vmax’ Vmax’
I I I

Km Km’ [S], mM Km = Km’ [S], mM Km’ Km [S], mM

Vmax unchanged Vmax decreased
Both Vmax & Km decreased
Km increased Km unchanged
Double Reciprocal

1/vo I 1/vo I 1/vo

I
Two parallel
Intersect lines
at Y axis 1/ Vmax 1/ Vmax
Intersect 1/ Vmax
at X axis

1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S]

Juang RH (2004) BCbasics

 Allosteric Enzymes and Allosteric Regulation
Allosteric enzymes: are regulated by molecules
called effectors (modulators) that bind non-
covalently at site other than the active site.
- Allosteric enzymes show Sigmoidal curve
and don't follow Michalaelis-menten
equation
- Allosteric modulators affect the enzyme
affinity to its substrate and /or modify the
maximal catalytic activity of the enzyme
- Enzyme regulation is essential to coordinate
the numerous metabolic processes
- Most of enzymes respond to changes of
substrate concentration  an increase in
the substrate conc. lead to increase in the
rate of enzyme action  return the
substrate conc. to normal level
- Some enzymes respond to allosteric
effectors or covalent modification that
affect the velocity of enzyme
Allosteric regulators (effectors)
-Effectors that inhibit enzyme activity are termed negative effectors
while those increase the enzyme activity are called positive effectors
- Homotropic effectors:
• The substrate itself acts as effector, usually allosteric substrate are
positive effector.
• The presence of substrate molecule at allosteric site will increase the
catalytic activity of the substrate-binding site  sites cooperativity
 Sigmoidal curve of V vs [S] not hyperbolic
- Heterotropic effectors
The effectors are different from substrate
Feed back inhibition
A B C DE
This enzyme has allosteric site that can bind to E
The final product may have a feedback inhibition of on the enzyme that
convert AB
Feed back inhibition serves to coordinate the flow of substrate
molecules through a series of reactions with the need of the cell.
Enzymatic Regulation
 Covalent modification-Protein phosphorylation

- Many enzymes may be

regulated by covalent
modification; addition or
removal of phosphate
group
- Phsphorylation occurs at
the –OH of serine,
threonine or tyrosine
residues
- Phosphorylation process active Inactive or

could be Activation or
Deactivation process 
depending on the
enzyme itself
The End