Enzyme Kinetics

Enzyme Kinetics I
An enzyme-catalyzed reaction of substrate S to product P, can
be written
E

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 MichaelisMenten 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 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
1) The Michaelis constant Km is:k - 1

Km =

K-1 + K2
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:

Vo =

V max [S]
[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
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

Vo =

V max [S]
[S] + K

1
Vo

Km + [S]

V max [S]

And this can be simplified to:

1
Vo

Km

(V

max

).

1
[S]

1
V max

This is the equation for a straight 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 Burk 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

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 action mechanism

Bacteria constantly remodel their peptidoglycan cell walls,
simultaneously building and breaking down portions of the cell
wall as they grow and divide.
-Lactam antibiotics inhibit the formation of
peptidoglycan cross-links in the bacterial cell wall; this is
achieved through binding of the four-membered lactam ring of penicillin to the enzyme DD-transpeptidase.
As a consequence, DD-transpeptidase
cannot catalyze formation of these cross-links, and an
imbalance between cell wall production and degradation
develops, causing the cell to rapidly die.
This weakens the cell wall of the bacterium

Penicillin structure

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 than
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 ampicillin
and penicillin

Competitive
Binds active site
inhibition reversed
by increasing [S]
Kmapp increases with
inhibitor (x axis
intercept changes)

Noncompetitive
binds to other than
binding site
not reversed by
increasing

no change in 1/V max

no effect on S
binding (K m) only
slows down rate
(V)

Usually analogs of
substrate

decreased Vmax app

(Y axis intercept)
inhibitor binds
both E free and ES
complex

Uncompetitive
Transition analog
binds covalently
ES not E free
changes both x and
y axis (Km and Vmax)

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 MichaelisMenten 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 phosphenolpyruvate (PEP) carboxylase

Some plants require a separation of the initial
carboxylation from the following decarboxylation
Diurnal regulation is used
IN CAM PLANTS:Phosphorylation of the serine residue of
phospho enol-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 phosphenolpyruvate (PEP)carboxylase

During the day:
De-Phosphorylation of the serine (serOH) 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

So far:

Bisubstrate Reactions

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