5

ENZYMES
M IMRAN
 OVERVIEW
Virtually all reactions in the
body are mediated by enzymes,
proteins catalysts that increase
the rate of reactions without
being changed in the overall
process.
 ENZYMES
definition

• Soluble , colloidal, thermolabile,

Highly pH sensitive, highly
specific, organic catalyst,
protein in nature, produced by
living organisms.
 ENZYMES
• all enzymes are proteins. With
the exception of a small group of
catalytic RNA molecules,
• Their catalytic activity depends
on the integrity of their native
protein conformation.
• If an enzyme is denatured or
dissociated into subunits,
catalytic activity is usually lost.
Enzyme structure
 like other proteins ,
have molecular weight
ranging from 12,000 th
over 1 million.
 They have a globular
shape
 A complex 3-D
structure

Human pancreatic amylase

© Dr. Anjuman Begum

© 2007 Paul Billiet ODWS

 SUBSTRATE

 The molecule the enzyme

acts upon to form product
is called the substrate.
Enzymes are specific to their
substrates
The specificity is determined by
the active site
The active site
 One part of an enzyme,
the active site, is
particularly important
 The shape and the
chemical environment
inside the active site
permits a chemical
reaction to proceed
© H.PELLETIER, M.R.SAWAYA
ProNuC Database more easily

© 2007 Paul Billiet ODWS

 CATALYST
By definition a catalyst is a
substance that enhances the
rate of a chemical reaction but
is not permanently altered by
reaction.
In addition, most of these
catalyst are non-specific; that
is, they accelerate a wide
variety of reactions
 NOMENCLATURE &
CLASSIFICATION OF ENZYMES
 Name of Enzymes
• End in –ase
• Identifies a reacting substance
sucrase – reacts sucrose
lipase - reacts lipid
• Describes function of enzyme
oxidase – catalyzes oxidation
hydrolase – catalyzes hydrolysis
• Common names of digestion enzymes still use –in
pepsin, trypsin
 NOMENCLATURE
In IUB system, each enzyme has a
unique name and code number that
reflect the type of reaction catalyzed
and the substrate involved.
Enzymes are grouped into six classes,
each with several subclasses and sub-
subclasses.
For example, the enzyme commonly
called “hexokinase” is designated
“ ATP:D-hexose-6-phosphotransferase
E.C.2.7.1.1.”
This identifies hexokinase as a member
of class 2 (transferses), subclass 7
(transfer of a phosphoryl group), sub-
subclass 1 (alcohol is the phosphoryl
acceptor).
Finally, the term “ hexose-6” indicates
that the alcohol phosphorylated is that
of carbon six of a hexose.
 Classification of Enzymes
Class Reactions catalyzed
1. Oxidoreductoases 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
 CLASSIFICATION OF ENZYMES
1. Oxidoreductases catalyze oxidation
and reduction.
2. Transferases catalyze transfers of
groups such as methyl or glycosyl
groups from a donor molecule to an
acceptor molecules.
3. Hydrolases catalyze the hydrolytic
cleavage of C-C, C-O, C-N, P-O, and
certain other bonds, including acid
anhydride bonds.
4. Lyases catalyze cleavage of
C-C, C-O, C-N, and other bonds
by elimination, leaving double bond.
5. Isomerases catalyze geometric or
structural changes within a single
molecule.
6. Ligases catalyze the joining together
of two molecules, coupled to the
hydrolysis of a pyrophophoryl group
in ATP or a similar nucleoside
triphosphate.
CO-ENZYMES
 Coenzyme are defined
as “heat-stable, low-molecular
weight organic compounds required
for the activity of enzymes.”
Cofactor An additional non-protein
molecule that is needed by some
enzymes to help the reaction
inorganic ions, such as Fe2+, Mg2+,
Mn2+, or Zn2+
Cosubstrate are the coenzyme linked
to enzyme by non-covalent forces.
 dissociate from enzyme in altered
state, example NAD+
prosthetic group
A coenzyme or metal that is very
tightly or even covalent bound to
the enzyme
 Can dissociate from enzyme in
unaltered state, example FAD
holoenzyme A complete, catalytically
active enzyme together with its
bound coenzyme and/or metal ion.
apoenzyme or apoprotein.
The protein part of such an enzyme i
 SIMPLE PROTEIN ENZYME

+ +
SUCROSE INVERTASE GLUCOSE FRUCTOSE
(Protein)

 COMPLEX PROTEIN ENZYME

Apoenzyme
+
Coenzyme Holoenzyme

(Protein) (Non-Protein)
 COENZYMES

COENZYME
VITAMINS REACTION
FORM
Thiamine
Thiamine (B1) Decarboxylation
pyrophosphate

Riboflavin (B2) FAD and FMN Redox

Pyridoxine Pyridoxil Amino group

(B6) phosphate transfer

Nicotinic acid
NAD and NADP Redox
(B3)

Cont……
 COENZYME
1
VITAMINS REACTION
FORM

Pantothenic acid
Coenzyme A Acyl transfer
(B5)

Biotin Biocytin Carboxylation

Tetrahydrofolic One carbon

Folic acid (B9)
acid group transfer
Vitamin B12 Deoxyadenosylco Intermolecular
balamine rearrangement
 METALLOENZYMES

 The roughly one-third of all

enzymes that contain tightly
bound metal ions are termed
metalloenzymes.
 SOME INORGANIC ELEMENTS
THAT SERVE AS COFACTORS
Cu2+ Cytochrome oxidase
Fe2+ or
Catalase , peroxidase
Fe3+
K+ Pyruvate kinase
Mg 2+ Hexokinase
Mn 2+ Arginase
Se Glutathione peroxidase
Zn 2+ Carbonic anhydrase.
PROPERTIES OF ENZYMES

Lec 3
 1) ACTIVE SITES.
 It is special pockets or area in
Enzyme that contains amino acid
side chains that create a three-
dimensional complementary to the
substrate, forming an enzyme-
substrate (ES) complex.
 ES is converted to enzyme–product
(EP), which subsequently dissociate
to enzyme and products.

E + S ES E + P
 Active Site Is a Deep Buried Pocket

Why energy required to reach transition state

is lower in the active site?
It is a magic pocket
+ (1) Stabilizes transition
(2) Expels water
CoE (1) (2)
(3) Reactive groups
(4) - (4) Coenzyme helps
(3)
Juang RH (2004) BCbasics
 2) Catalytic efficiency
 Most enzyme-catalyzed reactions are
highly efficient, proceeding from 103
to 108 times faster than uncatalyzed
reactions.
 Typically each enzyme molecule is
capable of transforming 100 to 1000
substrate molecules into product
each second.
 The number of molecules of substrate
converted to products per enzyme
molecule per second is called the
turnover number.
 3)Specificity
Enzymes are highly specific,
interacting with one or a few
specific substrates and catalyzing
only one type of chemical
reaction.
 Specificity of Ser-Protease Family
Trypsin Chymotrypsin Elastase
cut at Lys, Arg cut at Trp, Phe, Tyr cut at Ala, Gly

O O
O O O O
–C–N–C–C–N–
–C–N–C–C–N– –C–N–C–C–N–
C
C
C CH3
C
Deep and negatively
charged pocket

C Shallow and
non-polar
NH3 pocket
+

Juang RH (2004) BCbasics

COO- Non-polar
pocket
C
Asp

Active Site
 4)Cofactors
 Some enzymes associated with a
nonprotein cofactor that is needed for
enzyme activity.
 Commonly encountered cofactors
include metal ions (for example, Zn2+ ,
Fe2+ ) and organic molecules, known as
coenzymes, that are often derivatives
of vitamins (for example NAD+, FAD,
coenzyme A.
 5) Regulation
Enzyme activity can be regulated
that is, enzyme can be activated
or inhibited so that the rate of
product formation responds to
needs of the cell.
• Detail ?
 6) Location within the cell
 Many enzymes are located in
specific organelles within the cell.
 Such compartmentalization serves
to isolate the reaction substrate or
product from other competing
reactions, to provide a favorable
environment for the reaction, and
to organize the thousands of
enzymes present in the cell into
purposeful pathways.
MECHANISM OF ENZYMES
ACTION
 HOW ENZYMES WORK
• The mechanism of enzyme action can be
viewed from two different perspectives.
1. catalysis in terms of energy changes that
occur during the reaction; enzyme
provide an alternate, energetically
favorable reaction pathway different from
the uncatalyzed reaction.
2. how the active site chemically facilitates
catalysis.
 ENERGY OF ACTIVATION

 Decomposition of H2O2 has an

activation energy of 78 k.J/mol.
 In presence of colloidal platinum
the activation energy is 49 kJ/mol.
 In the presence of enzyme
catalase it is less than 8 kJ/mol.
active site chemically facilitates
catalysis
1) general acid and general base
catalysis-- good proton donors
& acceptors positioned just right.
2) covalent catalysis- unstable intermediate
3) metal ion catalysis
- electron donor or acceptor
4) electronic effects- “orbital steering”
steering aromatic groups
5) proximity and orientation
- close to reactive group
and aligned versus random
in solution chemistry.

6) conformational strain distortion

and induced fit
old idea, lock-and-key=
substrate fits active site
induced fit-
fit enzyme changes its
conformation to accept the
transition state of substrate/product well.
Enzyme conformational
change works to distort
and strain substrate forcing
it into transition state.
 MECHANISM OF ACTION OF ENZYMES
Active site

Lock-and-key model of the interaction of substrate and

enzyme. The active site of the enzyme by itself is
complementary in shape to that of the substrate.
E+S ES EP E+P
 Enzyme Action:
Lock and Key Model
• An enzyme binds a substrate in a region called the active
site
• Only certain substrates can fit the active site
• Amino acid R groups in the active site help substrate
bind
• Enzyme-substrate complex forms
• Substrate reacts to form product
• Product is released
 Lock and Key Model

P
+ S + S
P

E + S ES complex E + P
Induced-fit model of interaction of substrate and enzymes.
The enzyme changes shape upon binding substrate. The
active site has a shape complementary to that of the
substrate only after the substrate is bound.
Factors regulating the Enzyme
activity
1. Allosteric regulation
2. Covalent modification
3. Trimming
4. Hormones through signal
transduction
 ALLOSTERIC SITE
In some enzyme there is another
region of the molecule, the
allosteric site, that is not at the
active site or substrate binding site,
but is some where else on the
molecule.
The allosteric site is the site where
small molecule bind and effect a
change in the active site or the
substrate binding site.
The binding of a specific small
organic molecule at the allosteric site
causes a change in the conformation
of the enzyme, and that
conformational change may cause
the active site to become either more
or lass active.
It may cause the binding site to have
a greater affinity of substrate.
Such interaction are involved in the
regulation of the activity of enzymes
Allosteric enzymes
 Are usually composed of
multiple subunits.
 Catalyze a rate-limiting reaction.
 Bind substrate cooperatively.
 Show a sigmoid curve when v° is
plotted against [S].
 A. Allosteric binding sites
 Allosteric enzymes are regulated by
molecules called effectors (also
modifiers) that bind noncovalently
at a site other than the active site.
 These enzymes are composed of
multiple subunits, and the
regulatory site that binds the
effector may be located on a
subunit that is not itself catalytic.
 The presence of an allosteric
effector can alter the affinity of
the enzyme for its substrate, or
modify the maximal catalytic
activity of the enzyme, or both.
 Effectors that inhibit enzyme
activity are termed negative
effectors, whereas those that
increase enzyme activity are
called positive effectors.
 Allosteric enzymes usually
contain multiple subunits, and
frequently catalyze the
committed step early in a
pathway.
1. Homotropic effectors:
 When the substrate itself serves
as an effector
 Most often, an allosteric substrate
functions as a positive effector.
 substrate- binding sites—that is,
their binding sites exhibit
cooperativity.
 These enzymes show a
sigmoidal curve when reaction
velocity (v0) is plotted against
substrate concentration [S].
 This contrasts with the hyperbolic
curve characteristic of enzymes
following Michaelis-Menten
kinetics.
 Positive and negative
effectors of allosteric enzymes
can affect either the Vmax or
the Km, or both.
 2. Heterotropic effectors: The
effector may be different from
the substrate, in which case the
effect is said to be
heterotropic.
 For example, feedback
inhibition the enzyme that
converts A to B has an allosteric
site that binds the end-product,
E.
 If the concentration of E
increases (for example,
because it is not used as
rapidly as it is synthesized),
the initial enzyme in the
pathway is inhibited.
 Mechanism and Example of Allosteric Effect
Kinetics Models Cooperation
Allosteric site
R = Relax R
(active)
Homotropic
vo (+) S
S (+)
S
R Concerted
Allosteric site
[S]
R
(+) A T Heterotropic
vo (+)
S S (+)
R Sequential
X [S]
T
T = Tense I
vo
T Heterotropic
(inactive) (-)
(-) X X (-)
T Concerted
[S]
Juang RH (2004) BCbasics
Enzyme Activity is Often
Regulated

Feedback inhibition - a
common form of enzyme
regulation in which the
product inhibits the enzyme .
 Feedback inhibition provides the
cell with a product it needs by
regulating the flow of substrate
molecules through the pathway
that synthesizes that product.
 Heterotropic effectors for
example, the glycolytic enzyme
phosphofructokinase is
allosterically inhibited by citrate,
which is not a substrate for the
enzyme.
ENZYME KINETICS
 An Example for Enzyme Kinetics (Invertase)
1) Use predefined amount of Enzyme →E
2) Add substrate in various concentrations → S (x 軸 )
3) Measure Product in fixed Time (P/t) → vo (y 軸 )
4) (x, y) plot get hyperbolic curve, estimate → Vmax
5) When y = 1/2 Vmax calculate x ([S]) → Km

1 Vmax
vo vo
1/2

Juang RH (2004) BCbasics

-1
Km 1
Vmax
Double reciprocal 1/S Km Direct plot S
 MICHAELIS-MENTON EQUATION
A. Reaction model
Michaelis and Mention proposed a simple
model that accounts for most of the
features of enzymes-catalyzed reactions .
In this model the enzyme reversibly
combines with its substrate to form an ES
complex that subsequently breaks down to
product, regenerating the free enzymes.
k1 k2
E+S ES E+P
k-1
 MICHAELIS –MENTEN EQUATION

The Michaelis-Menten equation describe how

reaction velocity varies with substrate
concentration :
Vmax [S]
v° =
Km + [S]
v° = initial reaction velocity
Vmax = maximum velocity
Km = Michaelis constant = (k-1 + k2) /k1
[S] = substrate concentration
1. Relative concentration of E and S

The concentration of substrate,

[S], is much greater than the
concentration of enzyme, [E], so
that the amount of substrate
bound by the enzyme at any one
time is small.
 2. Steady-state assumption :
[ES] does not change with time (the
study-state assumption) – that is,
the rate of formation of ES is equal
to that of the breakdown of ES (to E
+ S and to E + P).
In general, an intermediate in a
series of reaction is said to be in
steady-state when its rate of
synthesis is equal to its rate of
degradation.
Constant ES Concentration at Steady State

S
P
Concentration

ES
E

Reaction Time
Juang RH (2004) BCbasics
 3. Initial velocity 6
Only initial reaction velocities are
used in the analysis of enzyme
reactions – that is, the rate of the
reaction is measured as soon as
enzyme and substrate are mixed.
At that time the concentration of
products is very small and ,
therefore, the rate of the back
reaction from P to S can be
ignored.
 IMPORTANT CONCLUSIONS ABOUT
MICHAELIS-MENTEN KINETICS
Characteristics of Km; The Michaelis
constant is characteristic of an enzyme
and a particular substrate, and reflects
the affinity of the enzymes for the
substrate.
Km is numerically equal to the substrate
concentration at which the reaction
velocity is equal to ½ Vmax.
Km does not vary with the concentration
of enzyme.
 SMALL Km
A numerically small (low) Km
reflect a high affinity of the
enzyme for the substrate because
a low concentration of substrate is
needed to half-substrate the
enzyme –that is, reach a velocity
that is ½ Vmax.
 LARGE Km

A numerically large (high) Km

reflects a low affinity of enzyme
for substrate because a high
concentration of substrate is
needed to half saturate the
enzyme.
 Lineweaver-burke plot
When the reaction velocity, is
plotted against the substrate
concentration [S] it is not always
possible to determine when Vmax has
been achieved.
However, if 1/v° is plotted vs. 1/[S],
is a straight line is obtained.
This plot is called the Lineweaver-
Burks plot, and can be used to
calculate Km and Vmax as well as to
determine the mechanism of action
of enzyme inhibitors.
 This equation describing the
Lineweaver-Burk plot is

1 = Km 1
+
V0 V max [S] V max