L11. Enzymatic kinetics. Determination of KM and Vmax.

Chemical reactions occur at different speeds, some are spontaneous, with very high
speeds, although never infinite, others occur at speeds so low that they require the presence of an
activating factor - temperature or catalysts to be performed in a timely manner.
Enzymes are proteins that catalyze biochemical reactions in living cells. The enzymes act
by forming a complex with the substrate molecule (ES), decreasing the activation energy
required to convert the substrate into the product. For a simple reaction, with a single substrate,
the relation can be written:

The initial rate of the enzymatic reaction can be determined by measuring the amount of
transformed substrate or product generated in the time unit. Most commonly, the reaction rate is
expressed in µmole generated product in one minute (µmol / min).
The initial rate (v) of the enzymatic reaction represents the instantaneous rate of product
formation (P) or the disappearance of the substrate (S) as a function of time:

Figure 1 shows the speed variation in time for a typical enzymatic reaction and it is
evident that the speed values change with time, decreasing at the end. This is due to factors such
as enzyme denaturation, inhibition by the reaction product, decreased enzyme saturation,
inactivation of coenzyme, and reverse reaction when the reaction product accumulates.
Therefore it is important to determine the speed not at any time of its development, but only at
the very beginning, when all these factors do not significantly interfere. For this reason, the
initial speed should be used in any enzymatic study. In figure 1 the initial speed corresponds to
the slope of the curve at the origin.

Concentration of the reaction product

Concentration

Concentration of the substrate

Time
Figure 1. Dependence of substrate and product concentrations in time
 In practice, the initial velocity can also be calculated by the formula below,
demonstrating that it is measured at the beginning of the reaction, when the initial rate of
disappearance of the substrate or the formation of the product is linear with respect to time:

The representation of the reaction rate variation as a function of the substrate

concentration for a single substrate reaction, enzymatically catalyzed, is shown in Figure 2.

Figure 2. Variation of reaction rate as a function of substrate concentration

The analysis of the curve presented in figure 2 leads to the following observations:
1. In the "a" portion, at a relatively low substrate concentration, the reaction is
approximately first order relative to the substrate (at low substrate concentrations there are many
more enzyme active centers available than the substrate molecules, therefore the substrate is
immediate. transformed, each increase in substrate concentration leads to proportional increases
in reaction rate).
2. In the "b" portion, as the substrate concentration increases, the initial velocity increases
more slowly, so that it is no longer linearly proportional to the substrate concentration; In this
area the reaction is mixed.
3. In the "c" portion of the curve, at further increases of the substrate concentration, the
reaction rate becomes absolutely independent of the substrate concentration and asymptotically
reaches a constant value. During this interval the reaction is of the order of 0, it is said that the
enzyme is saturated with the substrate (the molecules of the substrate exceed the number of
available active centers of the enzyme). At the saturation concentration of the substrate, the
maximum rate (Vmax) of the enzymatic reaction is reached, any addition of substrate will not
increase the reaction rate.
The mathematical expression linking the initial rate of an enzyme-catalyzed reaction to
substrate concentration and certain enzyme characteristics was developed by L. Michaelis, M.L.
Maintained and then extended by E.G. Briggs and J.B.S. Haldane and starts from the balances
formulated in the equations (1). The velocity equation for an enzyme reaction with a single
substrate is known as the Michaelis-Menten Equation and has the formula given in relation (2):
 KM represents the concentration of the substrate for which the rate of the enzymatic
reaction is half the maximum speed (for reactions with a single substrate). At this point (v=Vmax/2
and KM=[S]) it can be shown that the enzyme is half-committed in the ES complex and KM is
the dissociation constant of this activated ES complex.
KM expresses the magnitude of the affinity of an enzyme for the substrates on which it
acts.
For example, an enzyme E and three different substrates S1, S2, and S3 on which it acts.
The representation v = f ([S]) is shown in Figure 3.

Figure 3. Representation of v=f[S]

Comparing the calculated KM values from the graph to Vmax/2 results: KM1 <KM2 <KM3
which leads to the conclusion that the enzyme affinity decreases for substrates S 1, S2, S3 in this
order (in the case of S1, the amount of substrate required to reach the value Vmax/2 is smaller than
for S2 and S3 respectively). Therefore, if an enzyme has a low value for K M it achieves maximum
catalytic efficiency at low substrate concentrations. K M generally has values between 10-1-10-8 M.
Enzymes that have KM values between 10-1-10-2 M have a low affinity for substrates whose
transformation catalyzes, KM enzymes between 10-5-10-8 M have a high affinity for the substrate.
Both KM and Vmax are determined experimentally from the representation V=f([S]) by plotting the
asymptote on the hyperbolic curve (for finding V max) and then calculating KM, as shown in
Figure 4.

Figure 4. Determination of Vmax and KM

The determination of the two constants can be done with a better accuracy by using a
mathematical artifact: if the relation (2) is true, then its reciprocal is true. The reciprocal of the
relation (2), which is obtained by inverting both members of the equality, is known as the
Lineweaver-Burk Equation:
1 Km 1 1
 x 
v V max  S  V max
The representation 1/v = f (1/[S]) leads to a line, from which the intersection with the
coordinate axes can be evaluated with an increased accuracy of both V max and KM, as can be seen
from Figure 5. The obtained line intersects the abscissa at a point representing the value -1/KM
and the ordinate at a point representing the value 1/Vmax.

Figure 5. Representation of Lineweaver-Burk

Practical application
Influence of substrate concentration on the reaction rate, catalyzed by alkaline phosphatase

Principle
Serum alkaline phosphatase (ALP) is an enzyme present in the liver, bones, kidney,
intestine, mammary gland and placenta. The activity of this enzyme is increased in liver
disorders (biliary obstruction and metastasis), in bone disorders (rickets, osteomaly,
spasmophilia, osteoporosis, bone metastases) in neoplasms, chemotherapy and long-term
antibiotic treatment. ALP activity decreases in anemia and malnutrition.
Alkaline phosphatase (ALP) catalyzes the hydrolysis of substrate molecules by removal
of a phosphate group. For example, p-nitrophenylphosphate is such a substrate molecule for
ALP. The colorimetric reaction used is the reaction in which ALP catalyzes the conversion of p-
nitrophenyl phosphate (p-NPP) into p-nitrophenol (yellow compound) and phosphate:

The variation of the absorbance of the sample over time, measured at 405 nm, is directly
proportional to the serum ALP activity.
 Sodium p-nitrophenolate (resulting in basic medium) has a molar absorptivity of 18,496 x
10 M cm-1.
3 -1

The purpose of this experiment is to monitor the influence of substrate concentration

(pNPP) on the reaction rate for the determination of KM and Vmax of the enzymatic reaction.
The method of analyzing the enzymatic activity (reaction rate) is a kinetic method,
tracking in time the variation of absorption of the reaction medium at λ=405 nm.

Materials and equipment:

1. Micropipette of 20-200 µL;
2. Micropipette of 100-1000 µL;
3. 1.5 mL Eppendorf tubes;
4. 1mL spectrophotometer cuvettes;
5. Spectrophotometer.

Reagents:
1. R1: 1M Diethanolamine Buffer, pH = 9.80: Dissolve 100 mg MgCl2 and 97 ml
diethanolamine in 800 ml bidistilled water. Adjust the pH with a 10 M HCl solution to 9.80, then
make up to a final volume of 1 liter with bidistilled water in a graduated flask.
2. R2: Substrate solution - 2mM para-nitrophenyl phosphate solution (pNPP)
3. R3: R3- working reagent is obtained by combining R1 with R2 according to the protocol in the
table below
4. R4: Alkaline phosphatase solution (from blood serum)
S1 S2 S3 S4 S5 S6
R1 (µL) 599 598 597 580 460 250

R2 (µL) 1 2 3 20 140 350

Substrate concentration
(mmole/L)

Procedure:
Each group performs a Michaelis-Menten curve containing six points. Six
increasing concentrations of substrate (S1-S6) are prepared, for which the reaction rates are
determined according to the protocol below.
- In the 6 Eppendorf tubes (denoted by S1-S6) the volumes described in the table for the buffer
solution (R1) and the substrate solution (R2) are pipetted. The solutions R1 and R2 together form
a total volume of 600 µL.
• Set the wavelength to 405 nm at the spectrophotometer.
• Insert a tank with buffer solution into the spectrophotometer and set A=0.
• 600 µL of S1 + 100 µL R4 is pipetted in another cuvette (mixing easily with the pipette
for homogenization).
• Insert the cuvette into the spectrophotometer and read the absorbance A 0 at time 0 (reading
is done when the absorbance value is stabilized).
• Start the stopwatch and after 3 minutes read the absorbance A3 at time 3 minutes.
• The same is done for S2-S6 solutions.
 Determination of reaction rates
For each of the S1-S6 samples, the reaction rate V1-V6 is calculated according to the
equation below.
Speed (µmole/min) = ∆A/min x VF/(VA x ε) x 106/103
∆A / min - variation of sample absorption per minute;
VF - final volume (mL);
VA - volume R3 (serum) (mL);
Ɛ - molar absorption coefficient (M-1 cm-1) = 18496;

Speed (µmol / min) = ∆A / 3 x 0.7 / (0.01 x 18496) x 103

Speed (µmol / min) = ∆A / min x 3.7846

The substrate concentration for [S1]-[S6] is calculated taking into account the volume of
pNPP concentration 2 mM added in each sample and the final mixture (700 µL).
Example of calculation of substrate concentration [S] (mM) for sample S1:

2 mmole pNPP .................. 106 µL

x mmole pNPP .................. 1 µL

x mmole pNPP/sample = 2 x 10-6

2 x 10-6 mmole pNPP ................ 700 µL
y mmoles pNPP ................. 106 μl

y = 2 x 10-6 x 106/700 = 0.00286 mmol / L [S] (mM) = (mmol pNPP / sample x 106) / 700
The results are shown in the table:
Reaction rate 1/Reaction 1/[S]
Substrate [S] (mM) -1 -1
A0 A3 ∆A = A3-A0 (µmoles/min) rate(min*µmol ) (mM )
-2
S1 0.049 0.055 0.006 0.757 x 10 0.00286 132.100 349.65
S2 0.041 0.055 0.014 1.67 x 10-2 0.00571 60.024 163.93
S3 0.037 0.055 0.018 2.27 x 10-2 0.00857 44.038 111.11
S4 0.130 0.183 0.053 6.69 x 10-2 0.05710 14.957 17.51
S5 0.751 0.832 0.081 10.22 x 10-2 0.40000 9.787 2.50
S6 1.537 1.617 0.080 10.09 x 10-2 1.00000 9.909 1.00

In order to investigate the influence of the substrate concentration on the enzymatic

activity, the Michaelis-Menten curve is plotted either according to the parameters [S] and V, or
according to the parameters 1/V = f (1/[S]).

The importance of determining the constant KM

KM represents a measure of the enzyme affinity for a substrate. Thus, KM is inversely
proportional to the affinity for the substrate.
For example, in the physiological control of glucose metabolism, there are two types of
hexokinases, one with high KM (from the liver) and one with low KM for glucose (from glucose-
dependent tissues), both of which contribute to maintaining the steady state of glucose in the
blood.
The reduced resistance of asians to alcoholic beverages is known, a phenomenon that
has biochemical bases. Unlike europeans, asians need a lower amount of alcohol to trigger
vasodilatation that causes blushing and tachycardia. The physiological effects are due to the
acetic aldehyde formed by the action of alcohol dehydrogenase in the liver on the substrate (ethyl
alcohol). Normally, the acetic aldehyde is oxidized by another enzyme called mitochondrial
aldehyde dehydrogenase, which converts it to acetate. In persons of asian origin, an aldehyde
dehydrogenase with high KM (low affinity) for acetaldehyde is present. As such, the acetic
aldehyde is more incompletely metabolized and its accumulation leads to the aforementioned
effects.
 Using the ecuation:
1/V= 34.753 x 1/S + 773.73

When
x=0, 1/Vmax = 0.775 x 103, Vmax = 0,13 μmoles/min
y = 0 -1/KM= -22,263, KM = 4,46 x10-5 M

Influence of temperature on the enzymatic activity of serum alkaline phosphatase

Principle
Being proteins, enzymes are sensitive to temperature. There is a temperature value, called
optimal temperature, at which enzymatic activity is the highest. Usually, the optimal temperature
is between 35 and 40°C. Temperature values higher than 50-60°C diminish or inhibate the
enzymatic activity, due to denaturation of proteic structure.

The used colorimetric reaction is the one in which ALP catalyses transformation
of p-NPP in p-nitrophenol (yellow) and phosphate. The intensity of colour resulted during the
reaction is proportional with ALP activity. The activity of ALP will be monitored at: 0°C (ice),
37°C and 95°C.

Reagents, ml 0°C 37°C 95°C

pNPP solution 2mM 1 1 1
Maintain at corresponding temperature for 10 min
ALP solution 0,2 0,2 0,2
Maintain at corresponding temperature for 15 min
NaOH 1 1 1
 When the reaction takes place at 37°C, the colour is more intense compared to the other 2
test tubes. The intensity of yellow colour is proportional with ALP activity.

Influence of pH on ALP activity

Principle
Enzymes have activity over a limited pH domain. Each enzyme has a pH value, called
optimum pH, at which its activity is maximum. The optimum pH value is in relationship to
reaction medium parameters (ionic strength, temperature, substrate concentration). pH variations
modify enzymatic activity, reducing it until the inactivation of enzyme.

The intensity of yellow colour obtained after the hydrolysis of p-NPP is proportional with
ALP activity. The activity of ALP will be observed at different pH values, using various buffer
solutions: diethalnolamine buffer (pH=9.8), barbiturate buffer (pH=7) and acetate buffer (pH=4).

Reagents, ml pH 9.8 pH 7 pH 4
pNPP 2mM 1 1 1
Buffer pH =10 2 - -
Buffer pH = 7 - 2 -
Buffer pH = 4 - - 2
ALP solution 0,20 0,20 0,20
Incubate at 37°C for 15 minutes
NaOH 1M 1 1 1

It can be observed that the most intense colour was obtained in the test tube where the pH
value was 9.8.

Influence of effectors on enzymatic reaction rates

Enzymes can alter their catalytic activity if in the reaction medium are found different
types of compounds. They can bind either to the active site, or at other aminoacid residues. As a
consequence, the conformation of the enzyme will be altered, so the enzymatic activity will be
affected. On the other hand, when complexing agents are added to metalo-enzymes, they will
bind the metal ion found in the enzymes’ structure. Thus, the enzymatic conformation and
activity will be modified.
ALP has Zn ions as enzymatic cofactors. By adding EDTA, the Zn ions will be chelated,
so the enzymatic activity will be affected. The colour reaction that will be monitored is the
transformation of p-NPP in p-nitrophenol (yellow compound), catalysed by ALP.

Reagents, ml Without EDTA With EDTA

pNPP 10mM 1 1
EDTA solution - 0,20
H2O 0,20 -
ALP solution 0,20 0,20
Incubate at 37°C for 15 minutes
NaOH 1M 1 1
 Based on the obtained results, the influence of EDTA on ALP activity will be
commented. In the test tube where EDTA was absent, the yellow colour is more intense
compared to the other one. This is because EDTA chelated the Zn ion, so the enzymatic activity
of ALP was affected.