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L11 Enzyme Kinetics
L11 Enzyme Kinetics
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.
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 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.
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.
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
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
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;
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:
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
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
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.
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.