Enzyme Kinetics

Enzyme Kinetics I
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!

Think of the graph of [S] vs. v in this way:

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.
 Conditions for Michaelis -Menten
Two assumptions must be met for the Michaelis-
Menten equation

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.
Michaelis-Menten Enzyme kinetics
Don't for get the two assumptions - They both lead
to the same equation, the michaelis-menten equation.

What is this awe inspiring equation? 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
Think of what this means in terms of the equilibrium.
Large vs. a small Km
2) When investigating the initial rate (Vo) the Michaelis-
Menten equation is:

V max [S]
Vo =
[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
Prove this mathematically and graphicaly.
 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 must be. 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
 V max [S] 1 Km + [S]
Vo = =
[S] + K m Vo V max [S]

And this can be simplified to:

) . [S]
1 Km 1 1
Vo
= (V max
+
V max

This is the equation for a st raight line

Y = mX + b

Y = 1/Vo and X = 1 / [S]

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
 Enzyme inhibitors
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
 Enzyme inhibitors
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
 Mixed 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
 Enzyme inhibitors
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
Penicillin as a suicide substrate
- suicide substrates are often un competitive
inhibitors that decrease the energy of the
transition state and allow the ES to have lower
energy that that of the EP.

Bacterial cell wall - extensive cross linking of

sugars and peptides

Penicillin (and ampicillin) have a highly reactive

lactam ring which makes a peptide bond very
reactive.
Penicillin as a suicide substrate

Penicillin mimics the peptide alanine residues

and forms a low energy intermediate by
covalently reacting with a serine

In molecular biology, we use this as a tool.

Ampicillin will stop E. coli growth. Bacteria that
have a gene (plasmid) inserted into the
bacteria have lactamase. An enzyme that
hydrolyses the reactive peptide bond found in
amicillin and penicillin
 Competitive Noncompetitive Uncompetitive

Binds active site binds to other than Transition analog

binding site
inhibition reversed binds covalently
by increasing [S] not reversed by ES not E free
increasing
Km app increases with changes both x and
inhibitor (x axis no effect on S y axis (Km and Vm ax)
intercept changes) binding (Km) only
slows down rate
no change in 1/Vm ax (V)

Usually analogs of decreased Vm axapp

substrate (Y axis intercept)

inhibitor binds
both E free and ES
complex
Other Types of Inhibitors
 Allosteric Regulation
An organism must be able to regulate the catalytic
activities of its component enzymes

coordinate many metabolic processes

Respond to changes in the environment

Growth and differentiation

Both Inhibitors and affectors

 Allosteric Regulation
do not follow Michaelis-
Menten kinetics -
instead use a hill plot for
both + and effects

similar to O2 dissociation
of hemoglobin
 Allosteric Regulation
Two ways:

Control enzyme availability

Synthesis of enzyme
Degeneration

Control enzyme activity

Alterations which affect
the substrate binding
affinity
Turn over number
 Allosteric Regulation
Can cause large changes in enzymatic activity

Regulated by covalent modifications

Usually Phosphorylation and de-Phosphorylation of

specific Ser and Tyr residues.
 Phosphorylation
Phosphorylation is the addition of a phosphate (PO4) group to a
protein or other organic molecule.

kinases (phosphorylation) and phosphatases (dephosphorylation)

are involved in this process. Many enzymes and receptors are
switched "on" or "off" by phosphorylation and dephosphorylation.

Reversible phosphorylation results in a conformational change in

the structure in many enzymes and receptors, causing them to
become activated or deactivated. Phosphorylation usually occurs
on serine, threonine, and tyrosine residues in eukaryotic
proteins
 Phosphorylation regulates phosphenol-
pyruvate (PEP) carboxylase

CAM and C4 plants require a

separation of the initial carboxylation
from the following de-carboxylation

Diuranal regulation is used

IN CAM PLANTS:-
Phosphorylation of the serine residue
of phosphenol-pyruvate (PEP)
carboxylase (Ser-OP) yields a form of
the enzyme which is active at night
This is relatively insensitive to malic
acid
Photophorylation regulates phosphenol-
pyruvate (PEP)carboxylase
During the day:

De-Phosphorylation of the serine

(ser-OH) gives a form of the
enzyme which is inhibited by
malic acid

THIS IS THE OPPOSITE WAY

AROUND FOR C4 PLANTS!
 Phosphorylation
The addition of a phosphate (PO4) molecule to a polar R group of an
amino acid residue can turn a hydrophobic portion of a protein into a
polar and extremely hydrophilic portion of molecule.

In this way it can introduce a conformational change in the structure

of the protein via interaction with other hydrophobic and hydrophilic
residues in the protein
Examples:

Phosphorylation of the cytosolic components of NADPH oxidase, plays

an important role in the regulation of protein-protein interactions in
the enzyme

Phosphorylation of the enzyme GSK-3 by AKT (Protein kinase B) as

part of the insulin signaling pathway
 Phosphorylation
There are thousands of distinct phosphorylation sites
in a given cell since:

There are thousands of different kinds of proteins in

any particular cell (such as a lymphocyte).

It is estimated that 1/10th to 1/2 of proteins are

phosphorylated (in some cellular state).

Phosphorylation often occurs on multiple distinct sites

on a given protein.
Oxidative Phosphorylation
 Bisubstrate Reactions

So far:

Simple, single-substrate reactions that obey the

Michaelis-Menten model

However, approx 60% of known biochemical reactions

involve two substrates and yield two products

Either:
transfer reactions moving a functional group from
one substrate to the other
Oxidation/reduction reaction between substrates
 Bisubstrate Reactions

Sequential reactions

All substrates must combine with

the enzyme before the reaction
can occur and products are
released

A - leading substrate
B following substrate

P 1st product leaving enzyme

Q 2nd product leaving enzyme

ie NAD+ and NADH reactions

involving dehydrogenases
 Bisubstrate Reactions
Ping-pong reactions
Group transfer reactions in which one
or more products are released before
all substrates have been added.

Two stage reaction:

A functional group from 1st sub (A) is
transferred to the 1st product (P)
forming a stable enzyme (F) The Ping

The functional group is displaced from

the enzyme by the 2nd substrate (B)
to yield 2nd product (Q), regenerating
the original form of the enzyme (E)
The Pong

ie many reactions involving Trypsin