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
The simplest reaction is a single substrate going to a single
product. k -1
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) = = k 2 [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 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 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 ß

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 ß 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