An enzyme-catalyzed reaction of substrate S to product P, can be

written
E
S P
Actually, the enzyme and substrate must combine and E recycled after
the reaction is finished, just like any catalyst.
Because the enzyme actually binds the substrate the reaction can be
written as:
k1 k2
E + S <-> ES -> P + E
k- 1

The simplest reaction is a single substrate going to a single product.

Rate or velocity of the reaction depends on the formation of the ES
 The P -> ES is ignored
 The equilibrium constant Keq is based on the idea that the
reaction is limited to the formation of the ES complex and that
only K1 and K-1 are involved because the thermodynamics of
the reversal of K2 cause it to be minimal

k1
Keq =
K-1
How fast an enzyme catalyzes a reaction is it's rate. The rate of the reaction is in
the number of moles of product produced per second

d[P]
rate (v) = = k2 [ES]
dt
The relationship between the concentration of a substrate and the rate
of an enzymatic reaction is described by looking at the
concentration of S and v
 When the reaction is first order - the rate is dependent on [S]
 When the reaction is zero order, there is no relationship between v
and S
 A second order is between 1st and 0 order, where the relationship
between V and [S] is not proportional to [S]

Initial
Velocity
(Vi or V)

[ Substrate]
 To study enzymes, first order kinetics must be followed!

The velocity increases as the substrate concentration is

increased up to a point where the enzyme is "saturated"
with substrate.
At this point the rate of the reaction (v) reaches a maximal
value and is unaffected by further increases in substrate
because all of the enzyme active site is bound to substrate
For the most part enzyme reactions are treated as if
there is only one substrate and one product.
 If there are two substrates, one of them is held at a
high concentration (0 order) and the other substrate is
studied at a lower concentration so that for that
substrate, it is a first order reaction.
 This leads us to the M and M equation.
  Conditions for Michaelis -Menten

 Two assumptions must be met for the Michaelis-

Menten equation
 Equilibrium -the association and dissociation of the
substrate and enzyme is assumed to be a rapid
equilibrium and Ks is the enzyme:substrate
dissociation constant.
 Steady state - the enzyme substrate complex ES is at a
constant value.
 That is the ES is formed as fast as the enzyme releases the
product. For this to happen the concentration of substrate
has to be much higher than the enzyme concentration. That
is why we only study the initial velocity. Later in the
reaction the substrate concentration is relatively lower and
the rate of product starts to be limited by diffusion and not
the mechanism of the enzyme.
The Michaelis-Menten kinetic model explains several aspects of the
behavior of many enzymes. Each enzyme has a Km value that is
characteristic of that enzyme under certain conditions.
Graphical model of the representation of the M&M eq.
◦ Reaction velocity (V) vs concentration of substrate [S]
◦ - as [S] increases, velocity increases and eventually levels off =
V max
◦ 1st order vs zero order rates of reaction - back to the two
assumptions
◦ There are two important values for each enzyme that are
described by the M&M equation; V max and Km (Michaelis-
Menten constant)
 Graphically, these are shown as 1/2 V max = Km can not reach real
V max so....
 Mathematical model of the representation of the M&M eq. -
For the reaction:
k1 k2
E + S <->ES -> P + E
k- 1

1) The Michaelis constant Km is:

K-1 + K2
Km =
K1
2) When investigating the initial rate (Vo) the Michaelis-Menten equation is:

V [S ]
max
V o =
[S] + K
m

Graphical representation is a hyperbola. Think of the difference between O2 binding

of myoglobin and hemoglobin.
 When [S] << Km, the velocity is dependent on [S]
 When [S] >> Km, the initial velocity is independent of [S]

 When [S] = Km, then Vo = 1/2 V max

 Km is a measure of the affinity of the enzyme for it's
substrate and also informs about the rate of a
reaction. The binding constant is appoximated by
Km

 Rules for using the M&M equation:

 The reaction must be first order and [S] >> E (two
assumptions)
Turnover Number - kcat - the direct measure of the catalytic
production of product.
The larger the kcat is, the more rapid the catalytic events at the enzyme's
active site.
The number of times a binding and reaction event "turns over"
- When the [S] << Km so that most of the enzyme is in the free state [E]t =
[E]free then V = kcat / Km [E][S]
- This is a second order rate constant between the substrate and the free
enzyme. This is a good measure of efficiency and specificity.
- When the kcat/Km is near very high, the fastest the enzyme can catalyze a
reaction is the diffusion rate of a molecule!

- 108 - 109 / M . sec

Lineweaver-Burk (double reciprocal plot)
 Vmax and Km are not likely to be determined
by increasing [S]
 Instead the [S] vs. Vo data are transformed to
a plot of their reciprocal of each value.
 1/[S] vs. 1/Vo
So What?
 Km - relates to affinity ; Vmax relates to efficiency
 Km tell how much substrate to use in an assay
 If more than one enzyme share the same substrate, KM
also will determine how to decide which pathway the
substrate will take
Vmax tells about pathways
 Rate limiting enzyme in pathway
 Km and Vmax can be used to determine effectiveness of
inhibitors and activators for enzyme studies and clinical
applications
Competitive inhibition
Inhibitor is similar to substrate and
both bind to or near active site.
compete’ for binding
inhibitor is unreactive - EI state
Lineweaver Burke intersect at the Y
axis
Competitive Inhibition
Noncompetitive inhibitor
inhibitor binds distal to active site
effects enzyme rate not affinity
binds E in E S or E
Reversible
Lineweaver Burke intersect at the Y
axis
Noncompetitive Inhibition
 Inhibitor binds to enzyme site that
involves both S binding and
catalysis

 binds E in E S or E

 Forget the alpha business

Mixed Inhibition
Uncompetitive inhibitor
binds covalently in the transition
state
suicide inhibitor
binds to the ES complex
lowers affinity and velocity
lineweaver Burke plots are parallel
Uncompetitive Inhibition