Lecture 3: Enzyme kinetics

Introduction to enzyme kinetics

 An approach to understanding the mechanism of action of enzymes
 An approach to understanding how mutations may effect function
 An approach to understanding how changes in the physical and
chemical environments change function
 The word kinetics is derived from the Greek word kinetos, which
means “moving”.
 Using mathematical tool, we can describe an enzyme’s catalytic
power, its substrate affinity, and its response to inhibitors, etc.
 Study of this analytical part of the enzyme is known as enzyme
kinetics.
 Enzyme kinetics studies the reaction rates of enzyme-catalyzed
reactions and how the rates are affected by changes in
experimental conditions.
 An essential feature of enzyme-catalyzed reactions is saturation: at
increasing concentrations of substrates the rate increases and
approaches a limit where there is no dependence of rate on
concentration.
 Leonor Michaelis and Maud Menten were among the first scientist
to experiment with enzyme kinetics in a “modern” way,
controlling the pH of the solution etc.
Reaction Rates (reaction velocities): To measure a reaction
rate we monitor the disappearance of reactants or appearance
of products.
e.g., 2NO2 + F2 → 2NO2F

initial velocity =>

[product] = 0,
no back reaction

5
 Reaction Order
 Zero Order. The rate of a zero-order reaction is independent
of the concentration of the reactant(s). Zero-order kinetics
are observed when an enzyme is saturated by reactants.
 First Order. The rate of a first-order reaction varies linearly
on the concentration of one reactant. First-order kinetics are
observed when a protein folds and RNA folds (assuming no
association or aggregation).
 Second Order. The rate of a second-order reaction varies
linearly with the square of concentrations of one reactant (or
with the product of the concentrations of two reactants).
Second order kinetics are observed for formation of double-
stranded DNA from two single-strands.
Figure : Reactions with different orders
with respect to the substrate. In each
case, the concentration of substrate A
is shown reducing from an arbitrary
starting point, over an arbitrary time
period.
(a) Zero-order reaction. Here, the
reduction in concentration of A
shows no dependence on the
substrate concentration.
(b)First-order reaction. In this case,
the rate of the reaction (and so
consumption of A) is directly
proportional to the concentration of
A. The rate slows considerably as A
is consumed.
(c) Second-order reaction. The rate of
reaction is related to the square of
the concentration of A (e.g. two
molecules of A combining to form
some product). The reaction initially
proceeds rapidly, but falls
Why study enzyme kinetics (reaction rates)?
 Quantitative description of biocatalysis
-measure amount or concentration of one enzyme in a mixture by its activity
-measure enzyme purity (specific activity = amount of activity/amount of
protein)
 Understand catalytic mechanism
-info about enzyme active sites and reaction mechanism
 Find effective inhibitors
-study/distinguish different types of inhibitors
-development of specific drugs (enzyme inhibitors)
 Understand regulation of activity
 Measurement of velocity = reaction rate
 Compare enzymes under different conditions, or from different tissues or
organisms
 understand how differences relate to physiology/function of organism
 – e.g., physiological reason for different Km values for hexokinase vs.
glucokinase (discussed later in course)
 compare activity of same enzyme with different substrates (understand
specificity)
 A simple view
 E+S = ES as an equilibrium
 The mechanism: the first step of the reaction is the binding of the
substrate (S) to the enzyme (E) to form and enzyme-substrate
complex (ES) which then reacts to give the product P and free
enzyme E
 The concentrations: the total initial enzyme concentration is e 0 ,
and the complex concentration is x. The substrate (S)
concentration should too be (s0 - x), but since the substrate
concentration is usually very high:
 The conversion is considered an equilibrium with equilibrium
constant Ks
 The slowest step of the reaction is the ES to E+P and hence the
dominant rate for all the reaction is the rate v = k0x.
• 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.
 The initial velocity increases
 Kinetics is the study of reaction rates (time- with [S] at low [S].
dependent phenomena) Initial velocity
 Rates of reactions are affected by
Enzymes/catalysts - Substrates - Effectors
- Temperature - Concentrations
General Observations
 Enzymes are able to exert their influence at very
low concentrations ~ [enzyme] = nM
 The initial rate (velocity) is linear with [enzyme].
 The initial velocity increases with [substrate] at low
[substrate].
 The initial velocity approaches a maximum at high
[substrate].
Effect of substrate concentration on the initial velocity The initial velocity increases
with [S] at low [S].
of an enzyme-catalyzed reaction. [velocity =d[P]/dt, P=product]
The maximum velocity, Vmax, is extrapolated from the plot,
The initial velocity
because V0 approaches but never quite reaches Vmax. The approaches a
maximum at high
substrate concentration at which V0 is half maximal is Km, [S].

the Michaelis constant. The concentration of enzyme in an

experiment such as this is generally so low that [S] >> [E]
even when [S] is described as low or relatively low. The
units shown are typical for enzyme-catalyzed reactions and
are given only to help illustrate the meaning of V0 and [S].
(Note that the curve describes part of a rectangular
hyperbola, with one asymptote at Vmax. If the curve were
continued below [S] = 0, it would approach a vertical
asymptote at [S] = –Km.)
Equations describing Enzyme Kinetics
Start with a mechanistic model
Identify constraints and assumptions
Do the algebra ...
Solve for velocity (d[P]/dt)

Simplest enzyme mechanism

- One reactant (S)

- One intermediate (ES)
- One product (P)
The steady-state assumption
 When the enzyme is first mixed with a large
excess of substrate there is an initial period,
lasting just a few microseconds, the presteady
state, during which the ES complex concentration builds
up
 The reaction quickly achieves a steady state
in which [ES] remains approximately constant
over time
Michaelis-Menten Kinetics

1. First step: The enzyme (E) and the

substrate (S) reversibly and quickly form
a non-covalent ES complex.
2. Second step: The ES complex undergoes
a chemical transformation and
dissociates to give product (P) and
enzyme (E).
3. v=k2[ES]
4. Many enzymatic reactions follow
Michaelis–Menten kinetics, even though
enzyme mechanisms are always more
complicated than the Michaelis–Menten
model.
5. For real enzymatic reactions use kcat
instead of k2.
 Initial velocity Vo  When enzyme is mixed with high
Simple reaction
concentration of substrate [S] reaction
A[s] B [P]
k1 goes rapidly to steady state.
E+S ES E+P  Use low starting [S] and increase
k-1 k2  Hold [enzyme] constant
 Measure initial rate of reaction, Vo as
K2 also known as kcat [S] increases Until rate becomes
constant: approaches Vmax
At steady state  For any enzyme it is possible (pretty
o [ES] = (k /k + k ) [E] [S]
1 -1 2 easy) to determine kcat.
 To understand and compare enzymes
we need to know how well the
enzyme binds to S (i.e, what happens
in the first part of the reaction.) k cat
does not tell us anything about how
well the enzyme binds to the
substrate.
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
Michaelis-Menten Kinetics

The time dependence of

everything (in a
Michaelis-Menten
reaction)
 Assumptions
1. k1,k-1>>k2 (i.e., the first step is fast and is always at equilibrium).
2. d[ES]/dt ≈ 0 (i.e., the system is at steady state.)

d [ ES ]
rateof formationof ES
dt
rateof breakdownof ES
0 (at steady state)

3. There is a single reaction/dissociation step (i.e., k 2=kcat).

4. STot = [S] + [ES] ≈ [S]
5, There is no back reaction of P to ES (i.e. [P] ≈ 0). This assumption
allows us to ignore k-2.
We measure initial velocities, when [P] ≈ 0.
Now: we derive the Michaelis-Menten Equation

d[ES]/dt = k1[E][S] –k-1[ES] – k2[ES] =0

(steady state assumption, see previous graph)

solve for [ES] (do the algebra)

[ES] = [E][S] k1/(k-1 + k2)

Define KM (Michealis Constant)

KM = (k-1 + k2)/k1 => [ES] = [E][S]/KM

rearrange to give KM = [E][S]/[ES]

KM = [E][S]/[ES]
 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.
 Michaelis-Menten Kinetics

The Enzyme-Substrate Complex (ES)

The enzyme binds non-covalently to the substrate to form a non-covalent ES

complex
the ES complex is known as the Michaelis complex.
A Michaelis complex is stabilized by molecular interactions (non-covalent
interactions).
Michaelis complexes form quickly and dissociate quickly.
KM is the substrate concentration required to reach half-
maximal velocity (vmax/2).
Simple Enzyme-Catalyzed Reaction:
• Measurement of velocity:
– V = rate of appearance of product =
change in [P] per unit time
– units of velocity (V)?
_______________
Plot of [P] vs. time
• Experimentally,
forward velocity
VF = slope of plot of [P] vs. time
because
VF = kF[S] = Δ[P]/Δt
• Determining initial velocity, Vo
• •Why we measure initial velocity, Vo, the slope of [P]
vs. [time] at very early time after mixing enzyme
with substrate
• Problem:
• – As S is converted to P, concentration of S decreases,
so forward velocity gets slower and slower.
• – Furthermore, as [P] increases, rate of reverse reaction
(P --> S) becomes significant, and eventually, when VF
= VR, reaction is at equilibrium: d[P]/dt = 0.
• Solution:
–Measure V at very early times in reaction, before [S]
decreases signficantly (so [P] = ~0).
–Velocity measured immediately after mixing E + S, at
beginning of reaction (initial velocity), is called Vo.
Vo vs. [S] plots
Simple uncatalyzed S P reaction shows linear
dependence of Vo on [S]: Enzyme-catalyzed reactions
show a hyperbolic dependence of Vo on [S]:
1. Rate is saturable: there's a maximum rate (velocity =
Vmax) for any single concentration of enzyme.
2. Rate (velocity) is proportional to [E total]: at any single
concentration of [S], Vo = c[E] (double [E] double
Vo).
3. Half-maximal velocity occurs at a specific substrate
concentration, independent of [E].
Km = substrate concentration
that gives Vo = 1/2 Vmax.
Dependence of Vo on [S] in enzyme-
catalyzed reaction:
Plot of Vo vs. [S]) for an enzyme that
follows Michaelis-Menten kinetics
• plots as a rectangular hyperbola
• approaches Vmax (= maximum velocity) at
high [S]
• 3 parts of [S] concentration range
1. At very low [S]: Vo is proportional to [S];
doubling [S] 􀋠 double Vo.
2. In mid-range of [S], Vo is increasing less
as [S] increases (where Vo is
around 1/2 Vmax).
KM = [S] that gives
Vo = 1/2 Vmax.
3. At very high [S], Vo is independent of [S]:
Vo = Vmax
 Michaelis-Menten Enzyme kinetics
 Don't forget 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....
Effect of [substrate] on RX Velocity
 Michaelis-Menten Equation
Rules for using the M&M equation:
The reaction must be first order and [S] >> E

Understanding Km – the Michaelis Constant • KM is the Michaelis constant

– KM is constant for any given enzyme/substrate pair Independent of substrate
or enzyme concentration
– units are in terms of concentration
Km is a constant derived from rate constants
Km is a measure of ES binding; relative measure of the affinity of a substrate
for an enzyme (how well it binds) and also informs about the rate of a reaction.
The binding constant is appoximated by Km
Small Km means tight binding; large Km means weak binding.
– In the simplest assumption, the rate of ES breakdown to product (k2) is the
rate-determining step of the reaction
 Significance of KM
When V= ½ Vmax, what is [S]?

The KM of an enzyme is the

substrate concentration at which
the reaction occurs at half of the
maximum rate.
Higher KM = lower the affinity = higher [S] required to reach ½
Vmax
Clinical Insight
Variations in KM can have Physiological Consequences
The physiological consequence of KM is illustrated by the sensitivity
of some persons to ethanol. Such persons exhibit facial flushing and
rapid heart rate (tachycardia) after ingesting even small amounts of
alcohol. In the liver, alcohol dehydrogenase converts ethanol into
acetaldehyde.
Alcohol dehydrogenase
CH3CH2OH + NAD+ CH3CHO + NADH + H+
Ethanol Acetaldehyde

Normally, the acetaldehyde, which is the cause of the symptoms

when present at high concentrations, is processed to acetate by
aldehyde dehydrogenase.
Aldehyde dehydrogenase
CH3CHO + NAD+ + H2O CH3COO- + NADH + 2 H+
 Most people have two forms of the aldehyde dehydrogenase, a low KM
mitochondrial form and a high KM cytoplasmic form. In susceptible
persons, the mitochondrial enzyme is less active owing to the substitution
of a single amino acid, and so acetaldehyde is processed only by the
cytoplasmic enzyme.
 Because the cytoplasmic enzyme has a high KM, it achieves a high rate of
catalysis only at very high concentrations of acetaldehyde. Consequently,
less acetaldehyde is converted into acetate; excess acetaldehyde escapes
into the blood and accounts for the physiological effects.
 Km and Vmax Values Are Important Enzyme Characteristics
 The Km values of enzymes range widely. For most enzymes, KM lies
between 101 and 107 M. The KM value for an enzyme depends on the
particular substrate and on environmental conditions such as pH,
temperature, and ionic strength. The Michaelis constant, Km, as already
noted, is equal to the concentration of substrate at which half the active
sites are filled. Thus, KM provides a measure of the substrate concentration
required for significant catalysis to take place.
 For many enzymes, experimental evidence suggests that the KM value
provides an approximation of substrate concentration in vivo, which in
turn suggests that most enzymes evolved to have significant activity at the
substrate concentration commonly available.
 We can speculate about why an enzyme might evolve to have a KM value
that corresponds to the substrate concentration normally available to the
enzyme.
 If the normal concentration of substrate is near KM, the enzyme will
display significant activity and yet the activity will be sensitive to changes
in environmental conditions—that is, changes in substrate concentration.
 At values below KM, enzymes are very sensitive to changes in substrate
concentration but display little activity.
 At substrate values well above KM, enzymes have great catalytic activity
but are insensitive to changes in substrate concentration.
 Thus, with the normal substrate concentration being approximately KM,
the enzymes have significant activity (1/2 Vmax) but are still sensitive to
changes in substrate concentration.
 TURNOVER NUMBER (kcat) – CATALYTIC CONSTANT
- How fast ES complex proceeds to E + P
- Number of catalytic cycles that each active site undergoes per unit time
- Rate constant of the reaction when enzyme is saturated with substrate
- First order rate constant (sec-1 ) turnover number = kcat = Vmax/[ET]
[ET] = total enzyme concentration

 kcat: For the simplest possible mechanism, where ES is the

only intermediate, and dissociation is fast, then kcat=k2, the
first order rate constant for the catalytic step.
 If dissociation is slow then the dissociation rate constant also
contributes to kcat.
 If one catalytic step is much slower than all the others (and
than the dissociation step), than the rate constant for that step
is approximately equal to to kcat.
 kcat is the “turnover number”: indicates the rate at which the
enzyme turns over, i.e., how many substrate molecules one
catalytic site converts to products per second or number of
reaction processes each active site catalyzes per unit time
 If there are multiple catalytic steps (see trypsin) then each of
those rate constants contributes to kcat.
 The microscopic meaning of kcat depends on the details of the
mechanism.
 The maximal rate, Vmax, reveals the turnover number of an enzyme,
which is the number of substrate molecules that an enzyme can convert
into product per unit time when the enzyme is fully saturated with
substrate.
 The turnover number is equal to the rate constant k2, which is also called
kcat. If the total concentration of active sites, [E]T, is known,
 kcat = Vmax / [E]T
 Measure of how quickly an enzyme can catalyze a specific reaction
 For M-M systems kcat = k2
For example, a 10-6M solution of carbonic anhydrase catalyzes the formation of 0.6
M H2CO3 per second when the enzyme is fully saturated with substrate.
Hence, k2 is 6 x 105 s-1. This turnover number is one of the largest known. Each
catalyzed reaction takes place in a time equal to, on average, 1/k2, which is 1.7 µs
for carbonic anhydrase. The turnover numbers of most enzymes with their
physiological substrates fall in the range from 1 to 104 per second
 Significance of kcat/KM
 kcat/KM is the catalytic efficiency. It is used to rank enzymes. A big
kcat/KM means that an enzyme binds tightly to a substrate (small K M),
with a fast reaction of the ES complex.
 kcat/KM is an apparent second order rate constant
v=kcat/KM[E]0[S]
 kcat/KM can be used to estimate the reaction velocity from the total
enzyme concentration ([E]0). kcat/KM =109 => diffusion control.
 kcat/KM is the specificity constant. It is used to distinguish and describe
various substrates.
 When the substrate concentration is much greater than k M, the rate of
catalysis approaches Vmax.
 However, in the cell, most enzymes are not normally saturated with
substrate.
 Under physiological conditions, the amount of substrate present is
usually between 10% and 50% Vmax. Thus, the [S]/ kM ratio is typically
between 0.01 and 1.0.
 Thus, when [S]<< kM, the enzymatic velocity depends on the values of
kcat/ kM, [S], and [E]T. Under these conditions, kcat/ kM is the rate
constant for the interaction of S and E.
 The rate constant kcat/ kM is a measure of catalytic efficiency because it
takes into account both the rate of catalysis with a particular substrate
(kcat) and the strength of the enzyme–substrate interaction (kM).
 For instance, by using kcat/ kM values, we can compare an enzyme’s
preference for different substrates
 When [S] << kM, the enzymatic rate is much less than kcat because most
of the active sites are unoccupied. We can derive an equation that
characterizes the kinetics of an enzyme under these more-typical cellular
conditions:
 V0 =kcat / kM [E][S]
 When [S] << kM, almost all active sites are empty. In other words, the
concentration of free enzyme, [E], is nearly equal to the total
concentration of enzyme [E]T; so V0 = kcat/ kM [S][E]T
• Rate constant of rxn E + S → E + P
• Specificity constant
• Gauge of catalytic efficiency
• Catalytic perfection ~ 108 ─ 109 M-1 s-1 or a diffusion-limited enzyme catalyses a
reaction so efficiently that the rate limiting step is that of substrate diffusion into the
active site, or product diffusion out
Lineweaver-Burk (double reciprocal plot)
There are limitations in the quantitative (i.e. numerical) interpretation of this
type of graph, known as a Michaelis plot. The V max is never really
reached and therefore Vmax and hence KM values calculated from this
graph are somewhat approximate.
 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 m a x [S] 1 Km + [S]
Vo = =
[S] + K Vo V m a x [S]
m

And this can be simplified to:

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

This is the equation for a straight line

Y = mX + b

Y = 1/
Vo and X = 1 / [S]
The velocity of an enzyme-
catalyzed reaction was
measured at several substrate
concentrations. Calculate Km
and Vmax for the reaction
1/[S] (M-1) 1/Vo (mM-1.s)

0.25 0.75

0.5 1.20

1.0 1.71

2.0 2.18

4.0 2.53
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
Not all enzymes fit the simple Michaelis-Menten Model
1. Multisubstrate Reactions
2. Multistep Reactions

3. Non-hyperbolic reactions