Catalytic Mechanism of Enzyme Reaction
Remarkable properties of Enzymes as catalysts
1. Catalytic power
2. Specificity
3. Regulation
Ad1. Catalytic Power
- enzymes increase the rate of reaction by as
much as 1012 –fold
- not many examples  to compare between the
rates of an enzyme-catalyzed reaction and the
reaction occurring under similar conditions
(temperature, pH etc) but in the absence of
enzyme  as it is too low to be measured
- Comparison between enzymatic and nonenzymatic catalysts  also difficult. As
enzymatic catalysts occur :
o Much higher rates
o Lower temperature
It is well illustrated by the process of nitrogen
fixation (N2  ammonia)  catalyzes by
nitrogenase complex (300 K ,pH neutral)
Industrial synthesis of ammonia from nitrogen
and hydrogen  temp 700-900 K, pressure 100900 atm, with iron catalyst
Ad2. Specificity
- enzyme  highly specific both in :
o nature of substrate
o the reaction they catalyze
- the range of specificity varies between enzymes
o low specificity  the specificity base on
bond specificity
ex. Peptidase, phoshatase, esterase 
utilize a wide range of substrates which
contain the required chemical bond
mostly for degradative enzymes but not
biosynthetic enzymes.
o intermediate specificity  group specificity
ex. Hexokinase  catalyse the
phosphorylation of a variety of sugars
provided they are aldohexose
o absolute / near absolute specificity.  only
catalyse a reaction with a single substrate at
an appreciable rate
Ad3. Regulation
- The catalytic activity  regulated by small ions
or other molecules
- Ex.
The breakdown of glycogen in skeletal muscle
Carbohydrate reserved  degraded to generate
ATP  required for muscle contraction
The muscle contraction is triggered by release
of Ca2+ from the sarcoplasmic reticulum 
also ensure the continuation of ATP
production
Feedback inhibition phenomena  common in
many biosynthetic pathway
Ex. Biosynthetic pathway leading to the
synthesis of pyrimidine nucleotide  the end
product UTP and CTP  block the first
enzyme
 they are able to to limit the flow of
metabolites into the pathway and regulate
their own biosynthesis
HOW ENZYME WORKING
 A catalyst works simply by lowering the energy
barrier of a reaction, ΔGº±
 Catalysts provide an alternative way
 the catalyst increases the fraction of molecules
that have enough energy to attain the transition
state, thus making the reaction go faster in both
directions.
 The position of the equilibrium (the amount of
product versus reactant) is unchanged by a
catalyst.
 Catalysts lower the energy barrier in two ways:
o The catalyst binds a substrate in an
intermediate conformation that resembles
the transition state, but has a lower
energy.  lead to multiple intermediate
states that bypass the transition state. An
intermediate state is a metastable state of
a molecule.
o In a non-catalyzed reaction the entropy
may be highly negative due to the highly
specific orientation required in order for a
reaction to occur. Catalysts can lower the
negative entropy by binding reacting
molecules only in the proper mutual
orientation, thus increasing their reactivity
E + S ↔ ES ↔ EP ↔ E + P
Collision Theory
Molecular velocity determines the binding of enzyme
and substrate, thereby determine ES formation and
Enzyme catalytic velocity
Transient ES complex undergo INTRA-Molecular
straining  decreasing initial energy requirement for
catalytic reaction to produce reaction product
The highest point of the energy profile is
designated the transition state of the reaction
In all cases,  the catalyst does not cause a shift in
the equilibrium between reactant (s) and product (s)
 only increases the rate at which that equilibrium
is attained.
Various factors leading to the rate enhancements 
a. proximity and orientation effects
b. acid-base catalyst
c. covalent catalyst
d. strain or distortion
e. changes in environment
ad.a proximity and orientation effects
commonly  an enzyme could increase the rate of
reaction involving more than one substrate by 
binding the substrates at adjacent sites and
therefore cringing them into close proximity with
each other
so reaction occur more readily
Orientation with respect to each other  influence
the rate of reaction
Enzymes  make sure that the reactants are in
the correct orientation as they approach each other
Example
In this examples the restrictions placed on rotation
of single bonds by bridge structure  ensures that
the preferred orientation of the reacting groups
closely resembles that of the transition state of the
reaction
Less rotational entropy (degrees of freedom) occurs
as the reaction proceed towards the transition state
The smaller negative entropy of activation  lead to
an increase in the rate of reaction
Adb. Acid-Base catalyst
Many reactions of the type catalyzed by enzymes are
known to be catalyzed by acids and/or base
Since enzymes contain a number of amino acid side
chains which are capable of acting as proton donors
or acceptors  acid-base catalysis  important
Adc. Covalent catalysis (intermediate formation)
Reactions can be speeded up by the formation of
intermediates
Many of the examples of covalent catalysis in
enzyme-catalyzed reactions involve attack by a
nucleophilic side chain at electron-deficient centre in
the substrate  nucleophilic catalysis
Ad.d Strain or distortion
A substrate may be distorted on binding to the
appropriate enzyme  speed up the reaction if the
distortion lowered the free energy of activation by
making the geometry and electronic structure of the
substrate more closely resemble the transition state
Strain also give a stabilization of the transition
state of the reaction
Enzymes  make favorable contact with the
transition state of the substrate
Ad5. changes in environment
The rates of many organic reactions are highly
sensitive to the nature of of the solvents in which
they occur
ENZYME MODEL
 The induced fit model of enzyme action is a
modification of the lock-and-key model originally
proposed by Emil Fischer in 1894
 The lock-and-key model proposes that an
Enzyme/substrate pair is like a lock and key.
Though it explains the specificity of enzyme
/substrate pairs, it does little to explain
catalysis, because a lock does not change a key
the way an enzyme changes a substrate.
 In 1958, Daniel Koshland proposed the induced
fit model to explain enzymatic catalysis 
proposed that distortion of the enzyme and the
substrate is an important event in catalysis.
Enzymes do more than simply bind and position
substrates, however. Enzymes
1. Bind substrate(s);
2. Lower the energy of the transition state; and
3. Directly promote the catalytic event  occur as
a result of specific amino acid side chains that
physically interact with the substrate and end
up promoting the reaction.
When the catalytic process is complete, the enzyme
must be able to release the product or products and
return to its original state for another round of
catalysis.
Triose Phosphate Isomerase Catalysis
Triose phosphate isomerase catalyzes the following
reaction:
Glyceraldehyde-3-Phosphate (G3P) <=> cisenediol intermediate <=> Dihydroxyacetone
Phosphate (DHAP)
The active enzyme is a dimer of two identical
subunits.
The active site (the place on the enzyme where
catalysis occurs) can accommodate either G3P or
DHAP
At the active site, a glutamate residue (Glu 165) and
a histidine (His 95) are essential for function of the
enzyme. The reaction steps :
E + G3P <=> E-G3P (Binding of G3P)
E-G3P <=> E-ed (Conversion to enediol)
E-ed <=> E-DHAP (Conversion to DHAP)
E-DHAP <=> E + DHAP (Release of DHAP)
Like other enzymes, triose phosphate isomerase
lowers the energy barriers of the transition
States
Both triose phosphate isomerase and serine
proteases have a histidine and an acidic residue
in their active site. Histidine is very common in
active sites, because it readily accepts or donates
protons at physiological pH.
Multisubstrate Reactions
 Most biochemical reactions involve two or more
substrates, often resulting in multiple products.
 An example is proteolysis, which involves two
substrates (the polypeptide and water) and two
products (the two fragments of the cleaved
polypeptide chain).
 When an enzyme binds two or more substrates
and releases multiple products, the order of
the steps becomes an important feature of the
enzyme mechanism. Several classes of
mechanisms include the following:
o Random substrate binding
o Ordered substrate binding
o The "ping-pong" mechanism
Ad. Random substrate binding
In this mechanism, either substrate can be
bound first, though in many cases one substrate
will be favored for initial binding, and its
binding may promote the binding of the other.
Ad. Ordered substrate binding
This mechanism occurs when one substrate
must bind before a second one can bind
significantly.
 often observed in oxidations of substrates by
the coenzyme nicotinamide adenine dinucleotide
(NAD+).
Ad. The ping-pong mechanism
This occurs when a catalytic sequence of events
occurs, such as one substrate is bound, one
product is released, a second substrate is bound,
and a second product is released.