Seminar no.

4-5 Basics of enzyme kinetics

Determination and analysis of enzyme kinetic parameters play pivotal roles in clinical
practice and biomedical science. In routine laboratory diagnostics enzymatic activity (a normalized
and standardized measure of in vivo catalytic activity) is determined from serum samples.
Obtained activity values are then compared to reference intervals that represent either healthy or
compromised in vivo enzymatic functions. More detailed characterization of enzyme kinetic
parameters is required for identifying physiological substrates of a given enzyme, or the rate
limiting steps of an entire metabolic pathway. The latter type of analysis is highly important for
selecting which metabolic reaction step should be targeted by therapeutic interventions. Such
studies allow agonists and antagonists, which enhance or inhibit a given biochemical process in
the body, to be rationally designed and tested for in vivo efficacy.
To better understand the molecular basis of the above issues, we will discuss the
mechanistic meaning and experimental estimation of enzyme kinetic parameters in three
consecutive seminars.

In this seminar we will familiarize ourselves with the use of the Michaelis-Menten kinetic model by
solving sample problems. The most common form of the Michaelis-Menten equation is:

[S]
v  Vmax ,
Km  [S]

where v describes initial reaction velocity for an enzymatic reaction (which is assumed here to
convert a single substrate into a single product for simplicity), Vmax is the theoretical maximal
reaction rate, Km is the Michaelis constant and [S] is the “initial” substrate concentration. Exact
definitions of "initial velocity" and other general assumptions of the Michaelis-Menten model are
described in the text book. The maximal reaction rate Vmax is the rate measured at such high
substrate concentrations that essentially saturate the enzyme: Vmax = [E]tkcat, where [E]t is the
total enzyme concentration (typically in M or mM), and kcat is the catalytic turnover number
(typically in 1/s or 1/min), of the enzyme. The Michaelis constant (typically given in M or mM)
corresponds to the substrate concentration that keeps the enzyme 50% saturated, and therefore
supports a half-maximal reaction rate.

In this Seminar reaction rate will be understood as the change in product concentration per unit
time (d[P]/dt; measured, e.g., in mM/min, M/min), but keep in mind that in some applications it
is defined as the change in the number of moles of product per unit time (dnP/dt, measured, e.g.,
in mol/min). The latter definition essentially amounts to multiplying the Michaelis-Menten
equation with the reaction volume: nP  [P]  Volume , so dnP / dt  (d[P]/dt)  Volume .
Correspondingly, as [E]t  Volume  nE,t , in such applications maximal velocity represents
Vmax  nE,t  kcat .

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Seminar no.4-5 - Enzyme kinetics
Calculation #1
We measure the following (initial) reaction rates (v) at different substrate concentrations ([S]):

[S] (M) 1 5 50 100 500 1000 5000

v (M/min) 16.7 50 90.9 95.2 99 100 100

Calculate Vmax and Km values of the enzyme!

Solution:

Calculation #2
An enzyme converts substrate S to product P. If [S]=100 mM the initial reaction rate (v) is 50
M/min. When [S] is increased 10 times (i.e. to 1 M), the reaction rate does not increase any
further. Calculate the expected reaction rate at 1) [S] = 0.5 Km and 2) [S] = 2Km.

Solution:

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Seminar no.4-5 - Enzyme kinetics
Calculation #3
The Km of an enzyme is 10 mM and Vmax is 100 mM/min for a given Substrate A. Calculate the
reaction rates (v1, v2, v3, and v4) of the enzyme at [S]1 = 5 mM, [S]2 = 10 mM, [S]3 = 50 mM, [S]4 =
100 mM of Substrate A. Plot v values against [S] and fit (by hand) to a Michaelis-Menten
hyperbola. Also graph data using the Lineweaver-Burk double reciprocal plot, and fit the points by
a straight line using a ruler. Verify that the x- and y-axis intercepts are consistent with the given
values of Km and Vmax.

Solution:

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Seminar no.4-5 - Enzyme kinetics
Calculation #4
We monitor reaction rates of the enzyme from Calculation #3, but now using a different substrate
(Substrate B). Testing Substrate B at [S]1 = 5 mM and [S]2 = 10 mM, the measured rates are v1 = 50
mM/min, v2 = 60 mM/min. Provide a first estimate of the Km and Vmax values of the enzyme for
Substrate B based on these data! (Compare them to those for Substrate A!)

[Note, that due to experimental errors precise estimation of Km and Vmax in practice requires
measurement of velocities at several different substrate concentrations. However, theoretically,
these parameters are defined already by two [S]-v pairs.]

Solution:

Further calculations for home practice:

Calculation #5: For the enzyme in Calculation #4, what would be the reaction rates using Substrate
B at [S]3=50 mM and [S]4=100 mM? Plot these data, together with the rates at [S]1 and [S]2 given in
Calculation #4), into the Michaelis-Menten and Lineweaver-Burk plots drawn in calculation 3).
Mark Km and Vmax, or 1/Km and 1/Vmax, respectively, on the two plots. Compare the curves for the
two Substrates A and B!

Calculation #6: For an enzyme and a given substrate Vmax = 60 M/min. At [S]=15 mM the
measured rate is 50 M/min. What is the estimate of Km based on these data?

Calculation #7: For an enzyme and a given substrate Km = 20 M. At [S]=60 M the measured
reaction rate is 7.5 M/min. What is the estimate of Vmax based on these data?

Calculation #8: For an enzyme and a given substrate at [S]1 = 1M and [S]2 = 2.5 M, the
measured reaction rates are v1 = 12 M/min and v2 =15 M/min, respectively. What are the
estimates of Km and Vmax of the enzyme based on these data?
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Seminar no.4-5 - Enzyme kinetics
Solutions:

Calculation #1: Vmax is 100 M/min, Km = 5 M

Calculation #2: 1) 16.67 M/min, 2) 25 M/min
Calculation #3: v1 ≈ 33 mM/min, v2 = 50 mM/min, v3 ≈ 83 mM/min, v4 ≈ 91 mM/min
1/[S]1 = 0.2 mM-1, 1/[S]2 = 0.1 mM-1, 1/[S]3 = 0.02 mM-1, 1/[S]4 = 0.01 mM-1
1/v1 = 0.03 min/mM, 1/v2 = 0.02 min/mM, 1/v3 = 0.012 min/mM, 1/v4 = 0.011 min/mM
Calculation #4: Km = 2.5 mM, Vmax = 75 mM/min
Calculation #5: v3 = 71.42 mM/min, v4 = 73.17 mM/min
Calculation #6: Km = 3 mM
Calculation #7: Vmax = 10 M/min
Calculation #8: Km = 0.5M, Vmax = 18 M/min

Material: Harper’s pages 79-80

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Seminar no.4-5 - Enzyme kinetics
 Inhibition of enzyme activity

In this seminar we examine regulation of enzyme activity. Since many pharmacological

agents used in clinical practice are enzyme inhibitors, we further narrow our focus on inhibitory
mechanisms. Enzymes can be antagonized by drugs through several distinct mechanisms. In the
simplest case the molecular stucture of the applied drug closely resembles that of the substrate.
Such a compound can bind to the substrate binding site of the enzyme, in competition with the
substrate (hence the name, competitive inhibition). Importantly, binding of the inhibitor does not
initiate any enzymatic reaction. In contrast, in strict noncompetitive inhibition, binding of the
inhibitor does not affect binding of the substrate. However, while the enzyme-inhibitor complex
can still bind the substrate, it does not convert the substrate to product. Noncompetitive
inhibitors bind enzymes at sites distinct from the substrate-binding site and generally bear little or
no structural resemblance to the substrate.

The above two inhibitory mechanisms are in fact two extreme cases, and several
intermediate scenarios do exist, but discussion of such cases is beyond the scope of this seminar.
In the following sections we will therefore only discuss [1] competitive inhibition (inhibitor and
substrate compete for the same binding site) and [2] noncompetitive inhibition (binding of
inhibitor and substrate do not affect each other): in practice, the mechanism of action of many of
inhibitors can be adequately described using one of these two models. We will be using the
following nomenclature:
v, Km, and Vmax stand for parameters measured in the absence of drug (i.e. un-inhibited enzyme),
v', Km', and Vmax' are apparent parameters measured in the presence of drug (i.e. inhibited
enzyme).
[S] and [I] are (initial) substrate and inhibitor concentrations, respectively.
KI is the inhibitory constant (which reflects the dissociation constant of the enzyme-inhibitor
complex)
IC50, commonly used in clinical practice, is the inhibitor concentration which causes 50% inhibition

[1] – Competitive inhibition

Kinetically, competitive inhibition can be described by the below reaction scheme (left). In
the presence of a fixed inhibitor concentration ([I]) the dependence of reaction rate (v') on
substrate concentration ([S]) will remain hyperbolic, described by the below equation (right).

Vmax  [S]
v' 
K m'  [S]

 [I] 
K m'  K m   1  
 KI 

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Seminar no.4-5 - Enzyme kinetics
The form of this equation is mathematically identical to the Michaelis-Menten equation which
describes the uninhibited enzyme, except for a change in the apparent value of Km. From the
above expression of Km' it is clear that Km' > Km (e.g., if [I]=KI then Km'=2Km). At the same time, Vmax
is not affected by the competitive inhibitor.

The effect of the competitive inhibitor on catalytic activity is best visualized in some form
of substrate-velocity plot:

Note that for the competitive mechanism fractional inhibition (1-v'/v) is smaller at higher
concentrations of the substrate (i.e., the fractional activity of the enzyme (v'/v) approaches 1 at [S]
>> Km'), because the inhibitor is outcompeted by the substrate.

[2] – Noncompetitive inhibition

Kinetically, noncompetitive inhibition can be described by the below reaction scheme (left).
In the presence of a fixed inhibitor concentration ([I]) the dependence of reaction rate (v) on
substrate concentration ([S]) will again remain hyperbolic, described by the below equation (right).

Vmax'  [S]
v' 
K m  [S]
 [I] 
-1
Vmax'  Vmax   1  
 KI 

In this case only the apparent value of Vmax changes relative to the uninhibited situation. From the
above expression of Vmax' it is clear that Vmax' < Vmax (e.g., if [I]=KI then Vmax'=Vmax/2). At the same
time, Km is not affected by the noncompetitive inhibitor.

The effect of the noncompetitive inhibitor on catalytic activity is again best visualized in
some form of substrate-velocity plot:
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Seminar no.4-5 - Enzyme kinetics
Note that for the noncompetitive mechanism fractional inhibition is identical at any substrate
concentration (i.e. v'/v is constant at all [S]), because substrate binding has no effect on inhibitor
binding.

[3] - Example calculations

Calculation #1
The Km of an enzyme is 10 mM. At two substrate concentrations we measure the following
reaction rates in the absence (v) and presence (v') of an inhibitor. We also assessed that Vmax' of
the enzyme in the presence of the inhibitor is 80 mM/min. Determine the type of inhibition.

[S] (mM) 10 150

v (mM/min) 40 75
v' (mM/min) 33 73

Solution

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Seminar no.4-5 - Enzyme kinetics
Calculation #2
At two substrate concentrations we measure the following reaction rates in the absence (v) and
presence (v') of an inhibitor. Identify the mechanism of the inhibitor, assuming that the measured
rates are accurate. [In practice, due to experimental errors, reliable identification of inhibitory
mechanisms requires data measured at several different substrate concentrations. However,
theoretically, the two basic mechanisms can be discerned already by reaction rates measured at
two different substrate concentrations both in the absence and presence of the inhibitor.]

[S] (mM) 5 40
v (mM/min) 40 96
v' (mM/min) 24 80

Solution

Calculation #3
In the above calculation the inhibitor was applied at a concentration of [I] = 4 mM. What is the K I
of the inhibitor? What is the IC50 value of the inhibitor at the above two substrate concentrations?
Solution

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Seminar no.4-5 - Enzyme kinetics
Calculation #4
The following enzyme reaction rates were measured at various substrate concentrations in the
absence (v) and presence (v') of an inhibitor. Determine the mechanism of inhibition, assuming
that the measured rates are accurate

[S] (mM) 0.05 0.1 1 10

v (M/min) 1.05 2 11 20
v' (M/min) 0.79 1.5 8.25 15

Solution

Calculation #5
In the above calculation the inhibitor was applied at a concentration of [I] = 2 mM. What is the K I
of the inhibitor? What is the IC50 value of the inhibitor at the above four substrate concentrations?

Solution

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Seminar no.4-5 - Enzyme kinetics
Further calculations for home practice:

Calculation #6
Solve the problem in Calculation #2 in a graphical way: plot the data for both the uninhibited and
the inhibited enzyme as a Lineweaver-Burk plot, and draw straight lines across the pairs of data
points. Estimate Km, Vmax, Km', and Vmax' from the x- and y-axis intercepts.

Calculation #7
Solve the problem in Calculation #4 in a graphical way: plot the last three data points for both the
uninhibited and the inhibited enzyme as a Michaelis-Menten plot. Fit hyperbolas to both data sets.
Estimate Km, Vmax, Km', and Vmax'. (Simple manual "curve fitting" is sufficient for a rough estimation
of the parameters.)

Calculation #8
For an enzyme Km = 10 mM, and KI = 1 mM for a competitive inhibitor. What is the IC50 value of this
inhibitor at a substrate concentration of 20 mM?

Calculation #9
For an enzyme Km = 10 mM, and KI = 1 mM for a noncompetitive inhibitor. What is the IC50 value of
this inhibitor at a substrate concentration of 20 mM?

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Seminar no.4-5 - Enzyme kinetics
Solutions:

Calculation #1: competitive (Vmax = 80 mM,Km' = 14.2 mM, hence Km' > Km and Vmax' = Vmax)
Calculation #2: competitive (Km = 10 mM, Vmax = 120 mM/min, Km' = 20 mM, Vmax' = 120 mM/min,
hence Km' > Km and Vmax' = Vmax). Note: v'/v depends on [S]!
Calculation #3: KI = 4 mM, at [S] = 5 mM IC50 = 6 mM, at [S] = 40 mM IC50 = 20 mM
Calculation #4: noncompetitive (Km = 1 mM, Vmax = 22 M/min, Km' =1 mM, Vmax' = 16.5 M/min;
hence Km' = Km and Vmax' < Vmax) Note: v'/v does not depend on [S]!
Calculation #5: KI = 6 mM, IC50 = 6 mM. Note: IC50 does not depend on [S]!
Calculation #6: See Calculation #2
Calculation #7: See Calculation #4
Calculation #8: IC50 = 3 mM for [S] = 20 mM
Calculation #9: IC50 = KI = 1 mM

Material: Harper’s pages 80-83

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Seminar no.4-5 - Enzyme kinetics
 Enzyme kinetics of metabolic pathways

In this last seminar on enzyme kinetics we focus on biological systems. We identify

physiologically relevant substrates [1] and pathways [2]. We will also select targets for therapeutic
intervention and discuss aspects related to therapeutic efficacy [3].

The major difficulty trying to understand processes that happen in living organisms is that in vivo
conditions can hardly (if ever) be replicated in isolated systems. This problem is also relevant for
enzymatic assays. In our in vitro studies we try to model in vivo situations as much as we can, but
some degree of uncertainty always remains as to how close the behaviour of the enzyme in our
assay resembles that in the body. Despite this limitation, experimentally estimated Km and kcat
values of isolated enzymes are quite useful for understanding the roles individual enzymes play in
in vivo metabolic networks. In vivo Vmax values are estimated according to Vmax; in vivo  kcat  [E]in vivo ,
where the in vivo enzyme concentration ([E]in vivo) has to be determined by other means. The in
vivo reaction rate (vin vivo) is then obtained as vin vivo  Vmax; in vivo  [S]in vivo / (Km  [S]in vivo ) .

1. Identification of the physiologically relevant substrate

Obtaining Km and kcat (or Vmax, if perfectly pure preparations are not available) values of an enzyme
towards various alternative substrates allows addressing the physiological relevance of each
substrate. As an example, Table 1 summarizes kinetic parameters of brain hexokinase determined
for 1 g of brain tissue by an in vitro assay using either glucose or fructose as substrate. Determine
the physiological substrate of hexokinase in brain tissue, if estimated intracellular concentrations
of glucose and fructose are 10 and 1 M, respectively!

TABLE 1
Substrate Vmax; in vivo Km
(mole*min-1) (M)
glucose 17 10
fructose 25 1000

Solution:

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Seminar no.4-5 - Enzyme kinetics
2. Identification of the physiologically relevant pathway

Once substrates and enzymes in a series of enzymatic reactions are identified we can investigate
which potential metabolic pathways are physiologically relevant (i.e. dominate over alternative
routes). This can be done by comparing metabolic fluxes (vin vivo) along different pathways,
calculated for each enzyme using the Michaelis-Menten equation and in vitro estimates of kcat and
Km, but in vivo estimates of enzyme and substrate concentrations ([E]in vivo, [S]in vivo).

Let us consider the following metabolic scheme:

Here metabolic intermediate C might be produced either by enzyme E1 (from substrate A) or by

enzyme E2 (from substrate B). C is then converted to intermediate D by enzyme E3, and finally D is
converted into product P by enzyme E4. Which of the two potential pathways (A  C  D  P or
B  C  D  P) is the predominant route that determines metabolic flux in a living cell,
considering the in vitro and in vivo parameters summarized in Table 2?

TABLE 2

Measured in vivo Measured in vitro CALCULATED

Enzyme [E]in vivo [S]in vivo kcat Km Vmax; in vivo vin vivo
(mM) (mM) (min-1) (mM) (mM/min) (mM/min)
E1 0.0005 1 70 0.1
E2 0.00002 0.02 2000 0.5
E3 0.0002 0.05 500 0.1
E4 0.000003 0.12 25000 0.15

Solution (fill in last two columns in Table 2):

Note that under steady-state conditions the net reaction rate of each step in a linear (i.e. un-
branched) metabolic pathway has to be identical, otherwise some intermediates would
accumulate others would become depleted: that steady-state rate of substance flow is called the
metabolic flux of the pathway. When two pathways merge, or a pathway branches into two, the
sum of the fluxes in the two arms must equal the total flux in the common pathway. Verify that
these concepts apply in the above example!
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Seminar no.4-5 - Enzyme kinetics
3. Identification and pharmacological modulation of the rate limiting step

3.1. As a first exercise, identify the rate limiting step of the following reaction chain, using the
parameters given in Table 3 below, and fill in the missing parameters in the table!

TABLE 3

[S]in vivo Km Vmax; in vivo vin vivo

Enzyme
(M) (M) (M/min) (M/min)
E1 90 10 3
E2 5 8.1
E3 21 10.8

Solution:

3.2. As a second exercise, calculate the effects on metabolic flux (i.e., the rate of production of D)
of three pharmacological inhibitors that act by decreasing V max;in vivo for E1, E2, or E3, respectively,
by 50%! Fill in the missing values in Tables 4-5-6! Assume that the concentration of source
compound A is kept constant by complex metabolic regulation.

Solutions:
TABLE 4: Parameters in the presence of Inhibitor 1
[S]in vivo Km Vmax; in vivo vin vivo
Enzyme
(M) (M) (M/min) (M/min)
E1 90 10 1.5
E2 5 8.1
E3 21 10.8

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Seminar no.4-5 - Enzyme kinetics
 TABLE 5: Parameters in the presence of Inhibitor 2
[S]in vivo Km Vmax; in vivo vin vivo
Enzyme
(M) (M) (M/min) (M/min)
E1 90 10 3
E2 5 4.05
E3 21 10.8

TABLE 6: Parameters in the presence of Inhibitor 3

[S]in vivo Km Vmax; in vivo vin vivo
Enzyme
(M) (M) (M/min) (M/min)
E1 90 10 3
E2 5 8.1
E3 21 5.4

Observe that a 50% inhibition of the rate limiting enzyme E1 causes a 50% reduction in metabolic
flux, and a consequent reduction in the concentrations of the downstream metabolic
intermediates B and C (Table 4). In contrast, flux remains unaffected by a 50% inhibition of
enzymes E2 or E3: in the latter cases the corresponding substrates accumulate to levels that are
sufficient to maintain unaltered flux (Tables 5 and 6). In general, as long as V max; in vivo of an enzyme
remains higher than the steady-state flux of the pathway (i.e., the enzyme does not become rate
limiting), accumulation of the corresponding substrate can compensate for a reduction in
enyzmatic activity. Thus, effective pharmacological inhibition of a pathway must target the rate
limiting step.

Note, that in our simple reaction sequence all steps are assumed irreversible. In reality, enzymatic
reactions are reversible, and changes in the concentrations of reaction intermediates will also
affect the rates of the reverse steps. This ensures that the concentration of no intermediate will
fall to zero or rise to infinity. (E.g., in our scheme >75% inhibition of enzyme E3 would cause [C] to
rise to infinity!) Nevertheless, this simple scheme adequately demonstrates some key features of a
rate limiting step.

Material: Harper’s pages 83, 85

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Seminar no.4-5 - Enzyme kinetics