• Enzyme kinetics is the study of the chemical reactions that are

catalyzed by enzymes. Enzymes are usually protein molecules that

manipulate other molecules, the enzyme’s substrates. These target
molecules bind to an enzyme’s active site and are transformed into
products through a series of steps known as the enzyme mechanism.
These mechanisms can be divided into:

Single
substrate Multiple-
mechanism substrate
mechanism.

• An essential feature of enzyme-catalyzed reactions is saturation: at increasing

concentrations of substrates the rate increases and approaches a limit where
there is no dependence of rate on concentration.(Vmax)
Involve more than one substrate often resulting in multiple products.
Known as bi bi reactions ( two substrates two products one enzyme)
The following equation represents a bi bi reaction in which A and B are
the substrates, E is the enzyme and P, Q are the products (considering
the forward direction).
A+B P+ Q
enzyme

Overall reaction does not give a good representation of what actually

happens in reality.
The Multi-Substrate reactions in fact, follow a complex create
equations that describe how the substrates bind and in what sequence.
More information about the enzyme-reagent interaction is obtained
when looking at the enzyme mechanism which consists of microscopic
reactions
Mechanisms for multi-reactant enzymatic reactions have been classified in
some general groups.
Often a division is made between sequential mechanisms and non-
sequential mechanisms. In the sequential type, both substrates must
bind to the enzyme before any product is released.

The sequential mechanism can be subdivided in: random-order

mechanisms, in which the order of binding the substrates is not
important, and compulsory-order mechanisms, when the order of
binding is important. For bi bi reactions the sequential mechanisms are
also called ternary-complex mechanisms because of the ternary
EAB/EPQ intermediate.

Non-sequential mechanisms on the other hand, do not require all the

substrates to bind before a product is released and thus a substituted-
enzyme intermediate, E* will be formed. Such mechanisms are generally
referred to as ping-pong or substituted-enzyme mechanisms.
Ping-pong mechanism, also called a double-displacement reaction, is characterized
by the change of the enzyme into an intermediate form when the first substrate
to product reaction occurs. It is important to note the term intermediate
indicating that this form is only temporary. At the end of the reaction the
enzyme must be found in its original form.

The Ping Pong mechanism can be shown by following expression

Enzymes with a ping-pong mechanism can exist in two states, E and a chemically
modified form of the enzyme E*; this modified enzyme is known as an
intermediate. In such mechanisms, substrate A binds, changes the enzyme to E*
by, for example, transferring a chemical group to the active site, and is then
released. Only after the first substrate is released can substrate B bind and
react with the modified enzyme, regenerating the unmodified E form and
product Q is released.

For this mechanism, Lineweaver-Burk plots at fixed A and different varying values
of B give a series of parallel lines.
More specifically, chymotrypsin operates through a particular type of ping-pong
mechanism called covalent hydrolysis. This means that the enzyme first forms a
covalent bond with the target substrate, displacing the more stable moiety into
solution. This enzyme-substrate complex is called the enzyme intermediate. The
intermediate then reacts with water, which displaces the remaining part of the
initial substrate and reforms the initial enzyme
Other enzymes that work through ping pong mechanism include
some oxidoreductases such as peroxidase, and some
transferases such as acylnuraminate etc.
In these enzymes, both substrates bind to the enzyme at the same time to produce
an EAB ternary complex. The form of an enzyme that exists in solution in the
absence of any substrate or other small molecule that can bind to it is called the
free enzyme. An intermediate derived from the free enzyme by binding of a
substrate molecule is called an enzyme-substrate complex, and terms such as
enzyme-product complex, enzyme-inhibitor complex. A complex derived from the
free enzyme and one other molecule is called a binary complex; one derived from
the free enzyme and two other molecules is called a ternary complex

These mechanisms can be divided into further two types:

(i)Random order mechanism
(ii) Compulsory order mechanism
 Ordered Sequential Reactions

In ordered sequential reactions, all the substrates are first bound to the
enzyme in a defined order or sequence. The products, too, are released after
catalysis in a defined order or sequence.
Example:
An example is the lactate dehydrogenase enzyme, which is a protein that
catalyzes glucose metabolism. In this ordered mechanism, the coenzyme, NADH,
always binds first, with pyruvate binding afterward. During the reaction, the
pyruvate is reduced to lactate while NADH is oxidized to NAD+ by the enzyme.
Lactate is then released first, followed by the release of NAD+.
This is a characteristic of a ternary complex, which consists of three molecules
that are bound together. Before catalysis, the substrates and coenzyme are
bound to the enzyme. After catalysis, the complex consists of the enzyme and
products, NAD+ and lactate.
 Random Order Mechanism:

In random sequential reactions, the substrates and products are bound and then
released in no preferred order, or "random" order. This mechanism can generally
be expressed by following equation:
Where
A, B = Substrates
E = Enzyme
P, Q = Products
Example:
An example is the creatine kinase enzyme, which catalyzes the substrates,
creatine and ATP, to form the products, phosphocreatine and ADP. In this case,
either substrates may bind first and either product can be rel eased.
A ternary complex is still observed for random sequential reactions. Before
catalysis, the complex includes the enzyme, ATP and creatine. After catalysis,
the complex consists of the enzyme, ADP, and phosphocreatine.
We first find out the values of the constants

These are the values of the reactants over the values of product.

As we represented the total value of enzyme through E0, So it equals to

1
E0 = E+ EA+ EB+ EAB
Here
E = It represents the value of the enzyme present in free form
EA = It represents the value of the enzyme that bound with substrate A
EB = It represents the value of the enzyme that bound with substrate B
EAB = Enzyme in bound state with both substrate A and substrate B
Now we found out the value of enzymes in different states given as above
Follow the basic mechanism as given above each value of enzyme is equal to
the value of the constant multiply with the product formation divided by
the reactant. So these values came out………
The value of EAB find out by the help of basic Michaelis–Menten equation.
Putting the values of the EA, EB and E in equation (1), we get
Eo= E + EA + EB + EAB
2

On the right hand side of the equation EAB is common in all factors so
we take is as common

As we know that E0 Kcat = Vmax and (EAB) Kcat=V0

Multiply both sides of the equation by Kcat,
4
So put these values of Vmax and Vo in equation 4 E0 Kcat = Vmax
(EAB) Kcat=V0

Divide the right factors on left hand side in order to find out the value
of V0

Now take the L.C.M of the denominator of the right hand side and the
denominator of the denominator with numerator so AB woud be written
with Vmax, equation number 6 would be like this

6
Kib does not appear in the final equation. Assuming rapid equilibrium,
KiaKb=KibKa.
The following equation can be derived from Ping-Pong bi-bi mechanism

For simplicity, all of the enzyme kinetic equations have been derived
assuming no products are present.

By inspection, there would appear to be two types of "effective"

dissociation constants for reactant A. One describes the binding of A to E
(Kia) and the other the binding of A to EB (Ka). Using mass balance for E
and relationship that v0=kcat [EAB], the following initial rate equation can
be derived.Here Kia is the apparent dissociation constant for A.
The pre-steady-state in enzyme-catalyzed reactions occupies very short
period of time usually fractions of a second and very low product
concentrations. For reactions that are not too fast the stopped-flow
techniques can be used to measure easily the rate of reaction as
compare to fast orders reaction.
For reactions that have to be studied over periods of less than 1 minute
relaxation techniques are used. In these techniques the system is
disturbed, usually but not necessarily from a state of equilibrium, after
which it relaxes to equilibrium or a new steady state. In
the temperature-jump (T jump) technique the temperature is increased
rapidly and the system relaxes to a new state of equilibrium or a new
steady state at the final temperature. In the pressure-jump technique
the pressure is rapidly changed.
 What is Steady State?

A condition of a physical system or device that does not change over time, or in
which any one change is continually balanced by another, such as the stable
condition of a system in equilibrium.

The general rate equation of Alberty (1953)

• Many two-substrate reactions obey the MM equation with respect to one
substate at constant concentration

VmaxAX B 
v B
K m AX   K mAX B   AX B   K sAX K mB
Vmax : max vo when both AX and B are saturating
K mAX : [AX] which gives 1/2Vmax when B is saturating
K mB : [B] which gives 1/2Vmax when AX is saturating
K SAX : dissociation constant for E + AX EAX
 Vm axAX B 
v
K mB AX   K mAX B   AX B   K sAX K mB

Vm axAX 
At very large [B]: v 
AX   KmAX
Vm axK1 AX 
At constant but non saturating [B]: v 
AX   K 2

K1  B
B  K sAX KmB  KmAX B
K m  B  K2 
KmB  B

It works well for reactions using 1 or 2 substrate and producing 1 or

2 products but for more complex reactions, other approaches are
used
 [E ] AX B AXB
 0   
v [ AX ] [ B ] [ AX ][ B ]

 terms: kinetic coefficients found from primary and secondary

plots
• Primary plots of [E]/v versus 1/[AX] at constant [B] are
drawn for series of different [B]
• Secondary plots:
• Slope vs 1/[B]  intercept: AX, slope: AXB
• Intercepts vs 1/[B]  intercept: 0, slope: B
 Vm axAX B 
v B
K m AX   K mAX B   AX B   K sAX K mB
 Compulsory Order Mechanism
• In multi reactant enzyme catalyzed reactions, if the
reactants must bind to the enzyme active site in a
specific order the reaction type is called a compulsory
order reaction or compulsory order mechanism.