STEADY STATE BKINETICS

(BRIGGS AND HALDANES)

An intermediate is in a steady state if its concentration does not change with time

Consider the equation for the reaction below:

…………………….. (1)
Where P is formed irreversibly from E + S via an intermediate ES. In equation (i), the rate constant 𝑘1 refers
to the conversion of E + S to ES and the rate constant 𝑘−1 and 𝑘2 relate the conversion of ES to E + S and E
+ P respectively.

Briggs and Halden in 1925 examined the Michaelis-Menten analysis and made an important development.
Instead of assuming that the first stage of the reaction was at equilibrium, they merely assumed, for all intents
and purposes, that the concentration of the enzyme-substrate complex scarcely change with time i.e., it was in
𝑑[𝐸𝑆]
a steady state. Written mathematically as =0
𝑑𝑡

The intermediate is in steady state if its concentration is constant. Mathematically, the change in the
𝑑[𝐸𝑆]
concentration of ES with time: is given by
𝑑𝑡

𝑑[𝐸𝑆]
= 𝑘1 [𝐸][𝑆] − (𝑘−1 + 𝑘2 )[𝐸𝑆] … … … … (2)
𝑑𝑡

Where the first term on the right hand side describes the rate at which ES is formed and the second term
𝑑[𝐸𝑆]
describes the rate at which ES breaks down. If the concentration of ES is constant then =
𝑑𝑡

0 𝑎𝑛𝑑 𝑘1 [𝐸][𝑆] = (𝑘−1 + 𝑘2 )[𝐸𝑆] … … … … … . (3)

Although equation (3) is exactly true for merely an instant during the reaction of equation (1), it can often be
a good approximation throughout the time course of the reaction. Specifically, since the reaction begins in the
absence of ES, the concentration of ES will be most nearly constant if it remains small throughout the time
course of the reaction. According to equation (3) the concentration of ES is small i.e. 𝐸𝑆 ≪ 𝐸 + 𝑆 𝑖𝑓 𝑘−1 +
𝑘2 ≫ 𝑘1 … … … … … . (4)

Where again the rate constants 𝑘−1 and 𝑘2 refers to the breakdown of ES, while 𝑘1 relates to the formation of
ES. The steady state assumption or approximation is therefore valid throughout the time course of the reaction
if the breakdown of the intermediate is much faster than its formation.

Page 1 of 16
However, introducing the concept of enzyme total [𝐸𝑡 ]

[𝐸𝑡 ] = [𝐸] + [𝐸𝑆]

[𝐸] = [𝐸𝑡 ] − [𝐸𝑆] … … … … … … … … . . (5)

Introducing and substituting equation (5) into equation (3)

𝑘1 ([𝐸𝑡 ] − [𝐸𝑆])[𝑆] = (𝑘−1 + 𝑘2 )[𝐸𝑆]

𝑘1 [𝐸𝑡 ][𝑆] − 𝑘1 [𝐸𝑆][𝑆] = 𝑘−1 [𝐸𝑆] + 𝐾2 [𝐸𝑆]

𝐾1 [𝐸𝑡 ][𝑆] = 𝐾1 [𝐸𝑆][𝑆] + 𝑘−1 [𝐸𝑆] + 𝐾2 [𝐸𝑆]

𝐾1 [𝐸𝑡 ][𝑆] = [𝐸𝑆](𝐾1 [𝑆] + 𝐾−1 + 𝐾2 )

𝑀𝑎𝑘𝑖𝑛𝑔 [𝐸𝑆 ]𝑡ℎ𝑒 𝑠𝑢𝑏𝑗𝑒𝑐𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑒𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛

𝐾1 [𝐸𝑡 ][𝑆]
[𝐸𝑆] =
𝐾1 [𝑆] + 𝐾−1 + 𝐾2

Simplifying the above expression by dividing both the numerator and the denominator by 𝐾1

𝐾1 [𝐸𝑡 ][𝑆]
𝐾1
[𝐸𝑆] =
𝐾1 [𝑆] 𝐾−1 + 𝐾2
𝐾1 + 𝐾1

[𝐸𝑡 ][𝑆]
[𝐸𝑆] = … … … … … … … … … … … … … … … … … … … … … … (6)
𝐾 + 𝐾2
[𝑆] + −1
𝐾1

𝑈𝑠𝑖𝑛𝑔 𝑡ℎ𝑒 𝑓𝑎𝑐𝑡 𝑡ℎ𝑎𝑡 𝑉0= 𝐾2 [𝐸𝑆] … … … … … … … … … … … … … … … … … (7)

Substituting equation (6) into (7)

𝐾2 [𝐸𝑡 ][𝑆]
𝑉0 = … … … … … … … … … … … … … … … … … … … … … … … (8)
𝐾 + 𝐾2
[𝑆] + −1
𝐾1

Equation (8) has the same form as the Michaelis Menten’s equation, provided
𝐾−1 +𝐾2
𝐾𝑚 𝑖𝑠 𝑖𝑑𝑒𝑛𝑡𝑖𝑓𝑖𝑒𝑑 𝑤𝑖𝑡ℎ 𝑎𝑛𝑑 𝑉𝑚𝑎𝑥 𝑖𝑠 𝑖𝑑𝑒𝑛𝑡𝑖𝑓𝑖𝑒𝑑 𝑎𝑠 𝐾2 [𝐸𝑡 ]
𝐾1

𝑉𝑚𝑎𝑥 [𝑆]
∴ 𝑉0 = 𝑡ℎ𝑖𝑠 𝑖𝑠 𝑀𝑖𝑐ℎ𝑎𝑒𝑙𝑖𝑠 𝑀𝑒𝑛𝑡𝑒𝑛′ 𝑠𝑒𝑞𝑢𝑎𝑡𝑖𝑜𝑛
𝐾𝑚 + [𝑆]

Several features of equation (8) are worth noting and they include:

a) Because K2 describe the number of molecules of substrate converted to product per second per
molecule of enzyme, it is called the turnover number of the enzyme. Generally, in more complex
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 enzyme mechanisms, the expression for 𝑉𝑚𝑎𝑥 is complicated by 𝐾2 being replaced by an expression
that is a ratio of sum of products of unitary rate constants, this grouped expression is then called 𝐾𝑐𝑎𝑡 .

b) If an enzyme is not pure, it may not be possible to determine accurately the concentration of the active
form [𝐸𝑡 ]. Nevertheless, Vmax can still be obtained by steady-state kinetics analysis. So to standardize
experimental results, will refer to one enzyme unit (or katal) as the amount of enzyme solution required
to transform 1µmol of substrate into product(s) in 1minute, under standard condition of pH, in ionic
strength and temperature.

c) When [𝑆0 ] is very large compared with 𝐾𝑚 , virtually all 𝐸 is in form of [𝐸𝑆] so the enzyme is said to
be saturated, i.e. it is then operating at its maximum velocity.

DERIVATION OF COMPLICATED STEADY STATE EQUATION

In principle, the steady-state expression of any enzyme with any number of reactants can be derived using the
method of Briggs and Haldane. In practice, the procedure is very laborious, so use if made of and algorithmic
method, introduced by King Altman in 1956, it is applicable to the followings:

1. Non enzymatic reaction (each reactant concentration must be ≫ [𝐸𝑡 ]

2. Mixture of enzymes

3. Reactions with non-enzymatic steps.

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 THE BINDING OF LIGAND TO PROTEIN
Anything which binds to an enzyme or other protein is ligand, regardless of whether or not it is a substrate and
undergoes a subsequent reaction.

The binding of a ligand to a protein having a single ligand-binding site

Consider the binding of a ligand of ligand (S) to a protein (E) according to reaction

E + S ES

[𝐸𝑆]
The binding constant 𝐾𝑏 = [𝐸] [𝑆] ……………………………………………………………… (1)

1
Note that 𝐾𝑏 = 𝐾𝑠 i.e dissociation constant

From equation (1), [𝐸𝑆] = 𝐾𝑏[𝐸][𝑆] …………………………………………………………... (2)

The fractional saturation Ȳ of the protein is given by:

[𝐸𝑆]
𝑌̅ = [𝐸 ] Note that [𝐸0 ] = [𝐸𝑆] + [𝐸]
0

[𝐸𝑆]
Therefore 𝑌̅ = [𝐸𝑆]+[𝐸] …………………………………………………………………………… (3)

Substitute equation (2) into equation (3)

𝐾𝑏 [𝐸][𝑆] 𝐾𝑏 [𝐸][𝑆]
𝑌̅ = =
𝐾𝑏 [𝐸][𝑆] + [𝐸] [𝐸](𝐾𝑏 [𝑆] + 1)

𝐾𝑏 [𝑆]
𝑌̅ =
𝐾𝑏 [𝑆] + 1

A graph of Ȳ against [S] at constant [E0] will be hyperbolic

ȳ

[S]
Graph of fractional saturation [Ȳ] against ligand concentration [S] at a fixed concentration of a protein having
a single binding site for S.

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If the binding of S to E is the first step in a process whereby a product P is formed. If the reaction proceeds
under a steady-state condition, where [S0], >> [E0] and [S] ≅ [S0] then [ES] does not vary with time and in
most straight forward system V0 is proportional to [ES]

𝑖. 𝑒. 𝑉0 = 𝐾2 [𝐸𝑆]𝑎𝑛𝑑 𝑉𝑚𝑎𝑥 = 𝐾2 [𝐸0 ]

𝑉0 [𝐸𝑆]
= = 𝑌̅
𝑉𝑚𝑎𝑥 [𝐸0 ]

So graph of V0 against [S] will be the same shape as that of Ȳ against [S] i.e. hyperbolic. This hyperbolic
relationship between 𝑉0 and [S0] under steady-state conditions is of course predicted by the Michaelis-Menton
equation. If on the hand the reaction proceeds in a way which is not consistent with all of the assumptions
made in the derivation of Michaeleis–Mentens equations, then the kinetic characteristics of the reaction will
not usually run parallel to the binding characteristics.

Cooperativity
If more than one ligand-binding site is present on a protein there is a possibility of interaction between the
binding sites during the binding process. This is termed cooperativity. Cooperativity can either be positive or
negative

Positive cooperativity: is said to occur when the binding of some molecule of a substrate of ligand increases
the affinity of the protein for another molecule of the same or different substrate or ligand

Negative cooperativity: occurs when the binding of one molecule of a substrate of ligand decreases the
affinity of the protein for other molecule of the same or different substrate or ligand.

Homotropic cooperativity; occurs when the binding of one molecule of a substrate or ligand affects the
binding to the protein of subsequent molecules of the same substrate or ligand (i.e. the binding of one molecule
of A affects the binding of further molecules of A).

Heterotropic cooperativity: occurs when binding of one molecule of a substrate or ligand affects the binding
to the protein of molecule of a different substrate or ligand (i.e. the binding of one molecule of A affects the
binding of B)

Cooperativity effects may be positive and homotropic, positive and heterotropic, negative and homotropic or
negative and heterotropic. Allosteric inhibition is an example of heterotropic cooperativity and allosteric
activation is an example of positive heterotropic cooperativity.

Positive Homotropic Cooperativity and Hill Equation

Consider the simplest case of positive homotropic cooperativity in a dimeric protein: There are two identical
ligand binding sites and when the ligand binds to one, increases the affinity of the protein for the ligand on the
other site. So the reaction sequence is

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 𝑠𝑙𝑜𝑤
𝑀2 + 𝑆 → 𝑀2 𝑆

𝑟𝑎𝑝𝑖𝑑
𝑀2 𝑆 + 𝑆 → 𝑀2 𝑆2

(Where M is the monomeric subunit, termed a protomer, and M2 is the diametric protein)

If the increase in affinity is sufficiently large M2S will react with S almost immediately it is formed; under the
𝑀 𝑆2
condition [𝑀2 𝑆2 ] ≫ [𝑀2 𝑆] 𝑎𝑛𝑑 𝑌̅ = [(𝑀2 )0]
2

Where [(M2)0] is the total concentration of the dimer present, also a graph of Ȳ against [S] will be sigmoidal
(S-shape) rather than hyperbolic.

For complete cooperativity, where each protein molecule must be either free of ligand or completely saturated
the reaction may be written as:

M2 + 2S M2S2

The binding constant for this reaction is expressed as:

[𝑀2 𝑆2 ]
𝐾𝑏 =
[𝑀2 ][𝑆]2

[𝑀2 𝑆2 ] = 𝐾𝑏 [𝑀2 ][𝑆]2 … … … … … … … … … … … … … … … … … … … … … … … … (1)

𝐹𝑖𝑙𝑙𝑒𝑑 𝑠𝑖𝑡𝑒𝑠
𝐹𝑟𝑎𝑐𝑡𝑖𝑜𝑛𝑎𝑙 𝑠𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑌̅ =
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑖𝑡𝑒𝑠

[𝑀2 𝑆2 ]
𝑌̅ = … … … … … … … … … … … … … … … … … … … … … … … … . (2)
[𝑀2 ] + [𝑀2 𝑆2 ]

Substitute equation (1) into (2)

𝐾𝑏 [𝑀2 ][𝑆] 2
𝑌̅ = [𝑀 ]+𝐾 [𝑀 ][𝑆]2
2 𝑏 2

𝐾𝑏 [𝑀2 ][𝑆] 2
𝑌̅ = [𝑀 ](1+𝐾 [𝑆]2
2 𝑏 )

𝐾𝑏 [𝑆]2
𝑌̅ =
1 + 𝐾𝑏 [𝑆]2

𝐾𝑏 [𝑆]2 = 𝑌̅ + 𝑌̅𝐾𝑏 [𝑆]2

Page 6 of 16
Collect like terms

𝐾𝑏 [𝑆]2 − 𝑌̅𝐾𝑏 [𝑆]2 = 𝑌̅

𝐾𝑏 [𝑆]2 (1 − 𝑌̅) = 𝑌̅

Dividing both side of the expression by (1 − 𝑌̅)

𝐾𝑏 [𝑆]2 (1 − 𝑌̅ ) 𝑌̅
=
(1 − 𝑌̅) 1 − 𝑌̅

𝑌̅
𝐾𝑏 [𝑆]2 =( )
1 − 𝑌̅

Taking log of both sides

𝑌̅
𝑙𝑜𝑔𝐾𝑏 + 2𝑙𝑜𝑔[𝑆] = 𝑙𝑜𝑔
1 − 𝑌̅
𝑌̅
This is called Hills equation, after its deriver obeyed a graph of 𝑙𝑜𝑔 (1−𝑌̅) against log [S] will be linear with

slope = n and intercept = log Kb. Such a graph is called a Hill plot, and its experimentally determined slope is
known as the Hill coefficient and given the general symbol h.

𝑌
log(1−𝑌)

Slope = Hill coefficient = h

Intercept
Log[S]
𝑌̅
The Hill plot of 𝑙𝑜𝑔 (1−𝑌̅) against log [S] at fixed protein concentration where the binding shows the positive
homotropic cooperativity.

At value of Ȳ below 0.1 and above 0.9, the slope of the Hill plots tends to a value of 1, indicating an absence
of cooperativity, this is because at very low ligand concentration, there is not enough ligand present to fill
more than one site on most protein molecules, regardless of the affinity. Similarly at high ligand
concentration there are extremely few protein molecules present with more than one binding site remaining
to be filled.

Page 7 of 16
The hill coefficient is therefore taken to be slope of the linear, central portion of the graph where cooperativity
effect is expressed to its greatest extent. For system where cooperativity is complete, the hill coefficient h is
equal to the number of binding sites (n). Protein which exhibit only a partial degree of positive cooperativity
may still give a Hill plot which is a linear central section but in such cases h will be less than n and the linear
sections likely to be shorter than that for a system where cooperativity is more nearly complete.

In the case where S is a substrate and the reaction proceed to yield products in such a way that Michaelis-
Menten equilibrium assumption is valid, then initial velocity is proportional to the concentration of enzyme-
bound substrate i.e. 𝑉0 ∝ [𝑀𝑆]

𝑉0 [𝑀𝑆]
= = 𝑌̅
𝑉𝑚𝑎𝑥 [𝑀0 ]

Where [MS] is the number of substrate-bound units present per unit volume, and [𝑀0 ] is the total number of
subunits per unit volume[𝑀0 ] = [𝑀] + [𝑀𝑆]. Under these conditions.

𝑌̅ 𝑉0
=
1 − 𝑌̅ 𝑉𝑚𝑎𝑥 − 𝑉0

𝑉0
So a Hill plot of 𝑙𝑜𝑔 (𝑉 ) against 𝑙𝑜𝑔[𝑆0 ] may be substituted for Fig I. The slope of the two graph will
𝑚𝑎𝑥 −𝑉0
have these same value and meaning. Note that although the relationship
𝑉0
𝑌̅ =
𝑉𝑚𝑎𝑥
May be assumed valid for system for systems involving monomeric enzyme under general steady-state
conditions the same is not true for the more complicated system involving oligomeric enzyme; in the latter
case

𝑉0
𝑌̅ = Only if the binding process is at or very near equilibrium
𝑉𝑚𝑎𝑥

THE ADAIR EQUATION FOR THE BINDING OF LIGAND TO A PROTEIN HAVING TWO
BINDING SITE FOR THAT LIGAND
If we consider the binding of a ligand to a protein having a number of identical binding sites for that ligand
and making no assumptions at all about cooperativity. The intrinsic (or microscopic) binding constant Kb, for
each site is defined as the binding constant which would be measured if all the other site on the protein were
absent. Since all the sites are identical in the example we are considering, each will have the same Kb. However
the actual, or apparent, binding for each step of the reaction will not be the same. In the case of a diametric
protein (M2) having two identical binding sites for ligand (S):

M2 + S M2S
Apparent binding constant = 𝐾𝑏1

M2S + S M2S2
Apparent binding constant = 𝐾𝑏2
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Note that Kbi and Kb2 depend solely on the position in the reaction sequence and do not refer to any particular
binding site.

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑟𝑜𝑡𝑜𝑚𝑒𝑟𝑠 𝑝𝑒𝑟 𝑢𝑛𝑖𝑡 𝑣𝑜𝑙𝑢𝑚𝑒 𝑤ℎ𝑖𝑐ℎ 𝑎𝑟𝑒 𝑏𝑜𝑢𝑛𝑑𝑒𝑑 𝑡𝑜 𝑙𝑖𝑔𝑎𝑛𝑑

𝐹𝑟𝑎𝑐𝑡𝑖𝑜𝑛𝑎𝑙 𝑠𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑌̅ =
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑟𝑜𝑡𝑜𝑚𝑒𝑟𝑠 𝑝𝑒𝑟 𝑢𝑛𝑖𝑡 𝑣𝑜𝑙𝑢𝑚𝑒

[𝑀𝑆] [𝑀𝑆]
= =
[𝑀0 ] [𝑀𝑆] + [𝑀]

However, there are no isolated protomer present: they are part of the dimeric protein. Hence it is necessary to
express 𝑌̅ in terms of the various protein-ligand complexes which are actually present.

The species 𝑀2 consist of two protomer both unbound

The specie 𝑀2 𝑆 consist of one bound and one unbound protomer

The species 𝑀2 𝑆2 consist of two protomers both bound

Therefore the total concentration of ligand-bound protomers present ([𝑀𝑆]) is given by:

[𝑀𝑆] = [𝑀2 𝑆] + 2[𝑀2 𝑆2 ]

Similarly, the total concentration of unbound protomers present [𝑀]

[𝑀] = 2[𝑀2 ] + [𝑀2 𝑆]
Therefore;
[𝑀𝑆] + [𝑀] = [𝑀2 𝑆] + 2[𝑀2 𝑆2 ] + 2[𝑀2 ] + [𝑀2 𝑆]
= 2([𝑀2 ] + [𝑀2 𝑆] + [𝑀2 𝑆2 ])
[𝑀𝑆] [𝑀2 𝑆] + 2[𝑀2 𝑆2 ]
∴ 𝑌̅ = = … … … … … … … … … … … … … … … … … … . (1)
[𝑀𝑆] + [𝑀] 2([𝑀2 ] + [𝑀2 𝑆] + [𝑀2 𝑆2 ])
[𝑀2 𝑆]
𝐾𝑏1 =
[𝑀2 ][𝑆]
[𝑀2 𝑆] = 𝐾𝐵1 [𝑀2 ][𝑆] … … … … … … … … … … … … … … … … … … … … … … … . . … . . (2)
Also
[𝑀2 𝑆2 ]
𝐾𝑏2 =
[𝑀2 𝑆][𝑆]
[𝑀2 𝑆2 ] = 𝐾𝑏2 [𝑀2 𝑆][𝑆] … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … . (3)
Substituting equation (2) into (3)
[𝑀2 𝑆2 ] = 𝐾𝑏1 𝐾𝑏2 [𝑀2 ][𝑆]2 … … … … … … … … … … … … … … … … … … … … … … … … … … … … . . (4)

𝑆𝑢𝑏𝑠𝑡𝑖𝑡𝑢𝑡𝑖𝑛𝑔 𝑓𝑜𝑟 [𝑀2 𝑆] 𝑎𝑛𝑑 [𝑀2 𝑆2 ] 𝑖𝑛 𝑡ℎ𝑒 𝑒𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛 𝑓𝑜𝑟 𝑌̅

𝐾𝑏1 [𝑀2 ][𝑆] + 2𝐾𝑏1 𝐾𝑏2 [𝑀2 ][𝑆]2
𝑌̅ =
2([𝑀2 ] + 𝐾𝑏1 [𝑀2 ][𝑆] + 𝐾𝑏1 𝐾𝑏2 [𝑀2 ][𝑆]2 )

Page 9 of 16
 𝐾𝑏1 [𝑆] + 2𝐾𝑏1 𝐾𝑏2 [𝑆]2
=
2(1 + 𝐾𝑏1 [𝑆] + 𝐾𝑏1 𝐾𝑏2 [𝑆]2 )
This is the Adair equation for the binding of a ligand to a dimeric protein

The Adair Equation for the Binding of Ligand to a Protein Having Three Binding Site for that Ligand
.For a trimeric protein 𝑀3 having three identical binding site for a ligand (S) there are three steps in the bind
process.

M3 + S M3S (apparent binding constant Kb1)

M3S + S M3S2 Apparent binding constant Kb2

M3S2 + S M3S3 (Apparent binding constant Kb3)

Using biochemical reasoning exactly as for a dimeric protein

𝐾𝑏1 [𝑆] + 2𝐾𝑏1 𝐾𝑏2 [𝑆]2 + 3𝐾𝑏1 𝐾𝑏2 𝐾𝑏3 [𝑆]3

𝑌̅ =
3(1 + 𝐾𝑏1 [𝑆] + 𝐾𝑏1 𝐾𝑏2 [𝑆]2 + 𝐾𝑏1 𝐾𝑏2 𝐾𝑏3 [𝑆]3

Similarly, for tetrameric (𝑀4 ) protein having four identical binding site for a ligand [S], will equally have four
steps in the binding process, with apparent binding constants Kbi, Kb2, Kb3 and Kb4 and the Adair equation
is found to be

𝐾𝑏1 [𝑆] + 2𝐾𝑏1 𝐾𝑏2 [𝑆]2 + 3𝐾𝑏1 𝐾𝑏2 𝐾𝑏3 [𝑆]3 + 4𝐾𝑏1 𝐾𝑏2 𝐾𝑏3 𝐾𝑏4 [𝑆]4
𝑌̅ =
4(1 + 𝐾𝑏1 [𝑆] + 𝐾𝑏1 𝐾𝑏2 [𝑆]2 + 𝐾𝑏1 𝐾𝑏2 𝐾𝑏3 [𝑆]3 + 𝐾𝑏1 𝐾𝑏2 𝐾𝑏3 𝐾𝑏4 [𝑆]4

However, if there is no interaction between the respective binding sites, the Adair equation reduces to

𝐾𝑏[𝑆]
𝑌̅ =
1+𝐾𝑏[𝑆]

The scatchard plot

For systems where a single ligand [S] binds to an oligomeric protein having n identical and non-interacting
binding sites for that ligand.
[𝑀𝑆] 𝐾𝑏 [𝑆]
𝑌̅ = =
[𝑀𝑆] + [𝑀] 1 + 𝐾𝑏 [𝑆]

[𝑀𝑆]
𝐻𝑜𝑤𝑒𝑣𝑒𝑟 𝑌̅ =
[𝑀0 ]

[𝑀𝑆] 𝐾𝑏 [𝑆]
=
[𝑀0 ] 1 + 𝐾𝑏 [𝑆]

[𝑀𝑆] + [𝑀𝑆]𝐾𝑏 [𝑆] = [𝑀0 ]𝐾𝑏 [𝑆]

Page 10 of 16
Collecting like terms

[𝑀𝑆] = [𝑀0 ]𝐾𝑏 [𝑆] − [𝑀𝑆]𝐾𝑏 [𝑆]

[𝑀𝑆] = 𝐾𝑏 [𝑆]([𝑀0 ] − [𝑀𝑆])

Divide both side of the equation by [𝑆]

[𝑀𝑆] 𝐾𝑏 [𝑆]([𝑀0 ] − [𝑀𝑆])

=
[𝑆] [𝑆]

[𝑀𝑆]
= 𝐾𝑏 [𝑀0 ] − 𝐾𝑏 [𝑀𝑆]
[𝑆]

[𝑀𝑆]
∴ 𝐴 𝑔𝑟𝑎𝑝ℎ 𝑜𝑓 𝑎𝑔𝑎𝑖𝑛𝑠𝑡 [𝑀𝑆] 𝑤𝑖𝑙𝑙 𝑏𝑒 𝑙𝑖𝑛𝑒𝑎𝑟
[𝑆]

[𝑀𝑆]
𝑎𝑛𝑑 𝑠𝑖𝑛𝑐𝑒 𝑌̅ = 𝑡ℎ𝑖𝑠 𝑖𝑠 𝑏𝑎𝑠𝑖𝑐𝑎𝑙𝑙𝑦 𝑎 𝑝𝑙𝑜𝑡 𝑜𝑓 𝐸𝑎𝑑𝑖𝑒 𝐻𝑜𝑓𝑠𝑡𝑒 𝑡𝑦𝑝𝑒 𝑤𝑖𝑡ℎ 𝑡ℎ𝑒 𝑎𝑥𝑖𝑠 𝑟𝑒𝑣𝑒𝑟𝑠𝑒𝑑
[𝑀0 ]

[𝑀𝑆]
[𝑆]
Positive cooperstivity

No cooperativity
Negative cooperativity

[MS]
The intercept on the 𝑦 𝑎𝑥𝑖𝑠 = 𝐾𝑏 [𝑀0 ] = 𝐾𝑏 [𝐸0 ] 𝑎𝑛𝑑 𝑎 𝑠𝑙𝑜𝑝𝑒 𝑡ℎ𝑎𝑡 𝑖𝑠 𝑒𝑞𝑢𝑎𝑙 𝑡𝑜 – 𝐾𝑏

𝑎𝑛𝑑 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 𝑜𝑛 𝑥 𝑎𝑥𝑖𝑠 = [𝑀0 ] = 𝑛[𝐸0 ]

The Scatchard plot may be used to determine the type of cooperativity and also the number of binding sites.

Sigmoidal Kinetic and Allosteric Enzymes

Many enzyme are oligomeric proteins made up of several identical subunits or protomers, each protomer can
exist in two conformational forms. The T-form and R-form. The T-form is that which predominates in the
unliganded protein, whereas the R-form predominates in the protein-ligand complexes.

The Monod – Wyman – Changeux (MWC) model

The MWC model is sometimes called symmetrical model because it is based on the assumption that in a
particular protein molecule, all of the protomers must be in the same conformational state: all must be in the
R-form or all in the T- form, no hybrids being found because of supposed unfavourable interaction between
subunits in different conformational states. The two conformational forms of the protein are equilibrium in the
Page 11 of 16
absence of a ligand and the equilibrium is disturbed by the binding of the ligand in effect explaining the concept
of cooperativity.

If a diametric protein having two identical binding sites for substrate or ligand(s) in the absence of ligand,
there will be equilibrium between the two conformational forms of the dimer

R2 T2

The equilibrium constant being termed allosteric constant and given symbol L. the hybrid RT is held to be
unstable and ignored, the ligand can bind to either of the site on the R2 molecule, each having an intrinsic
dissociation constant KR. In the simplest form of the hypothesis, it is assumed that S does not bind to T to any
appreciable extent. Therefore, only processes which need to be considered (apart from any subsequent reaction
to form products are :

R2 T2 [𝑇 ]
(𝑒𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑢𝑚 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 𝐿 = [𝑅2 ])
2

∴ [𝑇2 = 𝐿[𝑇2 ]] … … … … … … … … … … … … … … … … … … … . . (1)

R2 + S R2S
(𝐼𝑛𝑡𝑟𝑖𝑛𝑠𝑖𝑐 𝑑𝑖𝑠𝑠𝑜𝑐𝑖𝑎𝑡𝑖𝑜𝑛 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 𝐾𝑅 )

R2S + S R2S2
(𝐼𝑛𝑡𝑟𝑖𝑛𝑠𝑖𝑐 𝑑𝑖𝑠𝑠𝑜𝑐𝑖𝑎𝑡𝑖𝑜𝑛 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 𝐾𝑅 )

In diagrammatic form this may be within as;

S S S
T2 R2 or R2S2
S
R2S
The concept of Adair equation equally applies here but with extra complication of two conformational forms.
However, we assume that the binding of one molecule of S to R2 does not alter the affinity of the other binding
site for S.

The concentration of bound subunits= [𝑅2 𝑆] + 2[𝑅2 𝑆2 ]

The total concentration of subunits = 2[𝑅2 ] + 2[𝑅2 𝑆] + 2[𝑅2 𝑆2 ] + 2[𝑇2 ]

[𝑅2 𝑆] + 2[𝑅2 𝑆2 ]
𝑌̅ = … … … … … … … … … … … … … . (2)
2([𝑅2 ] + [𝑅2 𝑆] + [𝑅2 𝑆2 ] + [𝑇2 ])

Substitute equation (1) into (2)

[𝑅2 𝑆] + 2[𝑅2 𝑆2 ]
𝑌̅ =
2([𝑅2 ] + [𝑅2 𝑆] + [𝑅2 𝑆2 ] + 𝐿[𝑅2 ])

Page 12 of 16
For the first step of the binding process

R2 + S R2S

[𝑅2 𝑆]
𝐾𝑏1 =
[𝑅2 ][𝑆]

[𝑅2 𝑆] = 𝐾𝑏1 [𝑅2 ][𝑆]

Since there are two unbound site which may be filled in the forward reaction but only one bound ligand to
dissociate in the reverse reaction

2 2
𝐾𝑏1 = 2 × 𝑖𝑛𝑡𝑟𝑖𝑛𝑠𝑖𝑐 𝑏𝑖𝑛𝑑𝑖𝑛𝑔 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 = =
𝑖𝑛𝑡𝑟𝑖𝑛𝑠𝑖𝑐 𝑑𝑖𝑠𝑠𝑜𝑐𝑖𝑎𝑡𝑖𝑜𝑛 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 𝐾𝑅

Hence, substituting for 𝐾𝑏1 𝑖𝑛 𝑡ℎ𝑒 𝑒𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛[𝑅2 𝑆]

2[𝑅2 ][𝑆]
[𝑅2 𝑆] =
𝐾𝑅

For the second step of the binding process

R2S + S R2S2

[𝑅2 𝑆2 ]
𝐾𝑏2 =
[𝑅2 𝑆][𝑆]

[𝑅2 𝑆2 ] = 𝐾𝑏2 [𝑅2 𝑆][𝑆]

Since there are only one unbound site which may be filled in the forward reaction but two bound ligand
molecules to dissociate in the reverse reaction

1 1
𝐾𝑏2 = =
2 × 𝑖𝑛𝑡𝑟𝑖𝑛𝑠𝑖𝑐 𝑏𝑖𝑛𝑑𝑖𝑛𝑔 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 2𝐾𝑅

Hence, substituting for 𝐾𝑏1 and 𝐾𝑏2 in the expression [𝑅2 𝑆2 ]

2 1[𝑅2 ][𝑆]2 [𝑅2 ][𝑆]2

[𝑅2 𝑆2 ] = × =
𝐾𝑅 2𝐾𝑅 (𝐾𝑅 )2

Now substituting for [𝑅2 𝑆] and [𝑅2 𝑆2 ] in the expression for 𝑌̅

[𝑅2 𝑆] + 2[𝑅2 𝑆2 ]
𝑌̅ =
2([𝑅2 ] + [𝑅2 𝑆] + [𝑅2 𝑆2 ] + 𝐿[𝑅2 ])

2[𝑅2 ][𝑆] 2[𝑅2 ][𝑆]2

+
𝐾𝑅 (𝐾𝑅 )2
=
2[𝑅 ][𝑆] [𝑅2 ][𝑆]2
2([𝑅2 ] + 𝐾2 + + 𝐿[𝑅2 ])
𝑅 (𝐾𝑅 )2
Page 13 of 16
Further simplification of the expression gives

[𝑆] [𝑆]
(1 +
𝐾𝑅 𝐾𝑅 )
=
[𝑆] 2
𝐿 + (1 + 𝐾 )
𝑅

This is the Monod–Wyman–Changeux equation for a dimeric protein. It may similarly be shown that for a
protein consisting of a protomer, each with a binding site for the substrate or ligand [S], the MWC equation
is:

[𝑆] [𝑆] 𝑛−1

𝐾𝑅
(1+ 𝐾𝑅
)
𝑌̅ = [𝑆]
𝑛
𝐿+(1+ )
𝐾𝑅

According to this equation, the greater the value of L, the more sigmoidal a plot of Ȳ against [S]. If L = 0, a
hyperbolic curve is obtained, a hyperbolic curve is also obtained, as would be expected for a monomeric
protein i.e. n =1 and for the situation where the substrate can bind equally well to the R and the T
conformational form.

The MWC equation is consistent with sigmoidal binding curve, even though its derivation assumes that the
binding of one molecule of ligand does not affect the affinity for the ligand of the other binding sites on the
molecule. The explanation for the cooperative affects lies in the Rn/Tn equilibrium. When L is large, this
equilibrium is in favour of the Tn form in the absence of ligand. If ligand is introduced, but at very low
concentration there will not be enough present to react significantly with the small amount of Rn present, so
very little formation of RnS, RnS2 and other liganded species of protein will take place. At higher ligand
concentrations however, there will be enough ligand present to force formation of significant amount of RnS,
RnS2, etc. thus some free Rn will be removed from the system, therefore distributing the Rn/Tn equilibrium
and causing more Rn to be formed from Tn. This freshly formed Rn can also react with ligand, resulting in yet
more formation of RnS, RnS2 and other liganded form. Hence, the Tn species can be regarded as a reservoir
of Rn which only becomes available when ligand concentration is high enough to cause the formation of
appreciable amount of protein-ligand complex, there will be a surge in the binding curve in the region of the
critical ligand concentration.

At still her ligand concentration, more of reservoir protein will be utilized and this process will continue until
a ligand concentration is reached which is higher enough to force conversion of all Tn to Rn, at this point the
protein will be fully saturated with ligand. Thus, the overall binding curve will be sigmoidal, a characteristic
of positive homotropic cooperativity; it is thus apparent that MWC model cannot explain negative homotropic
cooperativity.

Page 14 of 16
The MWC Model and Allosteric Regulation
One of the reasons for the introduction of the MWC model was attempt to explain the phenomena of allosteric
inhibition and activation. Compound or regulation of allosteric enzyme also called effector and modifiers
generally do not resemble the substrate in structure, so are likely to bind to the enzyme at separate site and
affect the bind of the substrate by heterotropic cooperativity.

According to MWC model, alloseteric inhibitors bind to the T-form of the enzyme, stabilizing it and thus
increasing the value of L. Allosteric activators have opposite effect, binding to and stabilizing the R-form and
decreasing the value of L, in this case, the binding of the modifier to one of the forms of the enzyme will
disturb the R/T equilibrium and therefore show some degree of sigmoidal character if investigated in the
absence of substrate. Enzyme subject to allosteric control may fit into either two categories, they may be K-
series or V-series enzyme. K-series enzymes are those where the presence of the modifier changes the binding
characteristic of the enzyme for the substrate but does not affect the Vmax of the reaction. V-series enzymes
are those where the presence of a modifier results in a change in Vmax but not in the value of the apparent
Km (or S0) for the substrate.

+ allosteric activator

Ȳ
In the absence of modifier

+ allosteric inhibitor

[S]
The effect of allosteric activator and inhibitors on the binding of a substrate to the K-series enzyme, at fixed
concentration and enzyme.

The Koshland-Nemethy-Filmer (KNF) model

The KNF model differs from the MWC in that it does not exclude hybrids between the two conformational
forms of the protein. Therefore for a dimeric protein where each protomer can exist in R- and T- forms, the
species R2, T2, R2S, R2S2, RTS, RSTS, T2S and T2S2 can all exist. In the KNF linear sequential model the only
protein specie present to any appreciable extent at (or near) equilibrium are T2, T.RS and R2S2.
The reaction sequence may therefore be written as:
T2 + S T.RS (Apparent binding constant Kb1)

T.RS + S R2S2 (Apparent binding constant Kb2)

Page 15 of 16
 𝐾𝑏1 [𝑆] + 2𝐾𝑏1 𝐾𝑏2 [𝑆]2
𝑌̅ = 𝐴𝑑𝑎𝑖𝑟 ′ 𝑠𝑒𝑞𝑢𝑎𝑡𝑖𝑜𝑛
2(1 + 𝐾𝑏1 [𝑆] + 𝐾𝑏1 𝐾𝑏2 [𝑆]2

𝑖𝑓 𝐾𝑏1 = 4𝐾𝑏2, 𝑡ℎ𝑒𝑟𝑒 𝑖𝑠 𝑛𝑜 𝑐𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑣𝑖𝑡𝑦

𝑖𝑓 𝐾𝑏1 < 4𝐾𝑏2, 𝑡ℎ𝑒𝑟𝑒 𝑖𝑠 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 ℎ𝑜𝑚𝑜𝑡𝑟𝑜𝑝𝑖𝑐 𝑐𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑣𝑖𝑡𝑦
𝑖𝑓 𝐾𝑏1 > 4𝐾𝑏2, 𝑡ℎ𝑒𝑟𝑒 𝑖𝑠 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 ℎ𝑜𝑚𝑜𝑡𝑟𝑜𝑝𝑖𝑐 𝑐𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑣𝑖𝑡𝑦
The KNF model was developed from the induced fit theory of Koshland and implies that the substrate or
ligand induces a conformational change to take place 𝑇 → 𝑅 as it binds to the T-form of the protein.

The KNF Model and Allosteric Regulation

In contrast to the MWC model, where the explanation for allosteric regulation is relatively straight forward,
the KNF model allows for possibility that allosteric modification may act in variety of ways, for example the
modifier could bind to the same form of the enzyme as the substrate and cause either the same or a different
conformational change to take place, also the binding of the modifier might or might not prevent the
subsequent binding of the substrate to the same subunit. The overall effect will depend on factors of this type
and also on the various interaction constants involved.

Page 16 of 16