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ENZYME TECHNOLOGY
Enzyme:-A protein with catalytic properties due to its power of
specific activation
 Introduction
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 A living system controls its activity through enzymes.

 An enzyme is a protein molecule that is a
biological catalyst.
 Enzymes are catalysts and increase the speed of a
chemical reaction without themselves undergoing
any permanent chemical change. They are neither
used up in the reaction nor do they appear as
reaction products.
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 Firefly bioluminescence
is produced by an
oxidation reaction
catalyzed by the
enzyme firefly luciferase
The oxidized substrate
(product of the reaction)
is in an electronically
excited state that emits
light as it returns to the
ground state.
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 Enzyme……..
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 The basic function of an enzyme is to increase the

rate of a reaction.
 In the laboratory, the average protein must be
boiled for about 24 hours in a 20% HCl solution to
achieve a complete breakdown. In the body, the
breakdown takes place in four hours or less under
conditions of mild physiological temperature and
pH.
 Specificity of Enzymes
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1. Absolute specificity - the enzyme will catalyze

only one reaction.
2. Group specificity - the enzyme will act only on
molecules that have specific functional groups, such
as amino, phosphate and methyl groups.
3. Linkage specificity - the enzyme will act on a
particular type of chemical bond regardless of the
rest of the molecular structure.
4. Stereochemical specificity - the enzyme will act on
a particular steric or optical isomer.
 Nature of Enzyme
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 All known enzymes are proteins. They are high molecular

weight compounds made up principally of chains of amino
acids linked together by peptide bonds.
 Enzyme structure
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 Enzymes are proteins

 They have a globular
shape
 A complex 3-D
structure

Human pancreatic amylase

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 Enzymes can be denatured and

precipitated with salts, solvents and
other reagents. They have molecular
weights ranging from 10,000 to
2,000,000.
 Many enzymes require the presence of
other compounds - cofactors - before
their catalytic activity can be exerted.
This entire active complex is referred to
as the holoenzyme; i.e., apoenzyme
(protein portion) plus the cofactor
(coenzyme, prosthetic group or metal-
ionactivator) is called the holoenzyme.
Nitrogenase enzyme with Fe, Mo and ADP cofactors
Jmol from a RCSB PDB file © 2007 Steve Cook
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS

IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

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 Apoenzyme + Cofactor = Holoenzyme

 The cofactor may be:

1. A coenzyme - a non-protein organic substance which is

dialyzable, thermostable and loosely attached to the
protein part.
2. A prosthetic group - an organic substance which is
dialyzable and thermostable which is firmly attached to
the protein or apoenzyme portion.
3. A metal-ion-activator - these include K+, Fe++,
Fe+++, Cu++, Co++, Zn++, Mn++, Mg++,
Ca++,and Mo+++.
 Enzyme Nomenclature
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 Enzymes are commonly named by adding a suffix "-

ase" to the root name of the substrate molecule it is
acting upon. For example, Lipase catalyzes the
hydrolysis of a lipid triglyceride. Sucrase catalyzes the
hydrolysis of sucrose into glucose and fructose.
 A few enzymes discovered before this naming system
was devised are known by common names. Examples
are pepsin, trypsin, and chymotrypsin which catalyzes
the hydrolysis of proteins.
 The latest systematic nomenclature system known as the
International Enzyme Commission (IEC) system is based
upon the type of reaction catalyzed.
 IEC Classification of Enzymes
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 Enzyme catalysis
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 The basic mechanism by which enzymes catalyze

chemical reactions begins with the binding of the
substrate (or substrates) to the active site on the
enzyme.
 The active site is the specific region of the enzyme
which combines with the substrate. The binding of the
substrate to the enzyme causes changes in the
distribution of electrons in the chemical bonds of the
substrate and ultimately causes the reactions that lead
to the formation of products.
 The products are released from the enzyme surface to
regenerate the enzyme for another reaction cycle.
 Lock and Key Theory
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 a Lock and Key

analogy first postulated
in 1894 by Emil Fischer.
 In this analogy, the lock
is the enzyme and the
key is the substrate.
Only the correctly sized
key (substrate) fits into
the key hole (active
site) of the lock
(enzyme).
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 Fit between the substrate and the active site of the

enzyme is exact
 Like a key fits into a lock very precisely
 The key is analogous to the enzyme and the substrate
analogous to the lock.
 Temporary structure called the enzyme-substrate
complex formed
 Products have a different shape from the substrate
 Once formed, they are released from the active site
 Leaving it free to become attached to another substrate
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 This explains enzyme specificity

 This explains the loss of activity when enzymes
denature
 Induced Fit Theory:
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 The induced-fit theory assumes that

the substrate plays a role in
determining the final shape of the
enzyme and that the enzyme is
partially flexible. This explains why
certain compounds can bind to the
enzyme but do not react because the
enzyme has been distorted too much.
Other molecules may be too small to
induce the proper alignment and
therefore cannot react. Only the
proper substrate is capable of
inducing the proper alignment of the
active site.
 Induced Fit Theory:
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 Some proteins can change

their shape (conformation)
 When a substrate combines
with an enzyme, it induces a
change in the enzyme’s
conformation
 The active site is then moulded
into a precise conformation
 Making the chemical
environment suitable for the
reaction
 The bonds of the substrate
are stretched to make the
reaction easier (lowers
activation energy)
Hexokinase (a) without (b) with glucose
substrate
http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZY
MES/enzyme_mechanism.html
 Comparision of calytsed and
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uncatalysed reaction
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S
E
E
E

Enzyme- Enzyme may be

substrate used again
complex P

Reaction coordinate
 Making reactions go faster
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 Increasing the temperature make molecules

move faster
 Biological systems are very sensitive to
temperature changes.
 Enzymes can increase the rate of reactions
without increasing the temperature.
 They do this by lowering the activation energy.

 They create a new reaction pathway “a short

cut”
 ENZYME KINETICS
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 Developed by V.C.R. Henri in 1902 and by

L.Michalis and M.L.Menten in 1913 also
known as Michalis – Menten kinetics or
saturation kinetics
 An enzyme solution has a fixed number of
active sites to which substrate can bind. At
high substrate concentrations, all these sites
may be occupied by substrates or enzyme is
saturated.
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 The ES complex is established rather rapidly and the rate of

the reverse reaction of the second step is negligible.
 The assumption of an irreversible second reaction often
holds only when product accumulation is negligible at the
beginning of the reaction.
 Two major approaches
1. rapid –equilibrium
2. quasi-steady state
 Michaelies-Menten/rapid equilibrium
Approach(1913)
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 The balance of equilibrium between free

components and enzyme–substrate was
assumed to occur quickly, compared with the
formation of product ,so that k2 can be
neglected and ES depends on k1 & k-1.
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 Steady state Approximation(1925)
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 G.E.Briggs and J.B.S. Haldane proposed quasi

steady state assumpumtion
 the total enzyme concentration and the
concentration of the intermediate complex do not
change over time.
 [Eo] << [So]
 d[ES]/dt≈0, the concentration of the substrate-
bound enzyme ([ES]) changes much more slowly
than those of the product ([P]) and substrate ([S]).
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 Michaelis constant
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 The Michaelis constant is an approximation of the

affinity of the enzyme for the substrate based on the
rate constants within the reaction, and it is numerically
equivalent to the substrate concentration at which the
rate of conversion is half of vmax. A small KM indicates
high affinity, and a substrate with a smaller KM will
approach vmax more quickly. Very high [S] values are
required to approach vmax, which is reached only when
[S] is high enough to saturate the enzyme.
 KM is expressed in units of concentration, usually in
Molar units.
 Integral form of Michaelies-Menten
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 Evaluation of Kinetic parameters
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 Linear Burk Method or

Double Reciprocal plot
 Eadie-Hofstee
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 Hanes – Woolf Method
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 Determine the MM parameters for the reaction:
The rate of reaction is a function of urea concentration as shown in the following table:

[C], urea, 0.20 0.02 0.01 0.005 0.002

(kmol/m3)
-v urea, 1.08 0.55 0.38 0.2 0.09
(kmol/m3-s)

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 •The following data have been obtained for two different initial enzyme concentrations for an enzyme
catalysed reaction. Find the following (a) Km,(b)Vmax for both cases,(c) k2 using Hanes-Woolf method.

E0=0.021g/l
Rate=g 1.2 0.9 0.69 0.6 0.5 0.4 0.39 0.2
/l-min
[S],g/li 30 13 6.7 5.4 4.2 3.4 3 2.2
t
E0=0.00935 g/l
Rate=g 0.70 0.52 0.41 0.34 0.29 ---- ---- ----
/l-min

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 Allosteric Enzymes/Allosteric Inhibition
 Allosteric protein:-two or more
topological distinct binding sites
which interact functionally with each
other.
 Cooperatability:-modification of the
binding constant of the protein for a
small molecule by the prior binding
of another small molecule.
 +ve:-binding ability or affinity
increases
 -ve:- binding ability or affinity when 2,3-BPG binds to an allosteric site on
decreases hemoglobin, the affinity for oxygen of all
 Allosteric effectors(inhibitors & subunits decreases.
activators):- for speed up & to speed
down

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 Where n is cooperativity and n>1
,Indicates positive cooperativity.

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