Enzyme Kinetics

Introduction

Enzymes are highly specific biological catalysts, which increase the rate of a

reaction by lowering its activation energy. Most enzymes are globular proteins; however
some are RNAs, like ribozymes. Most enzymes require special co-factors called co-

enzymes in order to function. Most co-enzymes are either small organic molecules or

metal ions, like in vitamins. The reactants, which enzymes catalyze, are called substrates

[1]. One of the specific features of enzymes is that they are not consumed in a reaction

and they do not shift the equilibrium of the reaction. Enzymes speed up the reaction so

that equilibrium can be achieved faster. Equilibrium of the reaction is the state at which

reactants are converted into products and products are converted into reactants at a

constant rate. The equilibrium of the reaction is mostly due to the free energy available

for the reaction. A reaction is spontaneous only when the free energy difference (∆ G)

between the products and reactants is negative [1].

There are several major features enzymes employ to catalyze reactions, such as

velocity, high regulation, storability, and high specificity [3, 5]. Enzymes acquire high

specificity from their active sites, which are small three –dimensional clefts that fit

perfectly the substrates [4]. There are two theories of how active site fits a substrate:

lock-and-key and induced fit. In first case the enzyme’s active site is the complementary

share as its substrate. The second case says that a substrate causes a slight change in

active site to fit it better. Saturability depends on enzyme’s structure. That influences

binding of the substrate. The complementary fit of the substrate and active site allows for

saturation over a period of time [4].

Enzymatic activity is depends on allosteric regulation, which controls the amount

of substrate interacting with an enzyme. There are different kinds of allosteric regulation:

irreversible, where the inhibitor dissociates very slowly due to strong interactions.

Competitive inhibitors dissociate at the rate of catalysis, thus reducing the number of
enzymes, which binds substrates and convert them to products. Allosteric enzymes have

sigmoidal curves and do not follow the Michaelis-Menten kinetics [1].

There are several assays that can be used to calculate enzymatic activity. One

possibility is when the activity is stopped after some certain time to quantify the amount

of product formed. Another possible way to measure enzymatic activity is by measuring

some continuous detectable activity like color change, light absorbance, and pH change.

In this experiment the rate of enzyme catalysis was studied. We used alkaline

phosphatase to catalyze the substrate para-nitrophenyl phosphate (pNPP) to form the

products para-nitrophenol (PnP) and phosphate and then measured light absorbance of

the products at 420 nm.

The absorbance was read by the spectrophotometer and was interpreted by using

Michaelis-Menten Kinetics plot (MMK) [2]. It is a graph of the enzyme’s velocity as a

function of substrate concentration. Velocity of an enzyme is measured in micromoles of

product formed per minute and represents the rate of reaction of the enzyme. The

Michaelis-Menten constant (KM) represents the dissociation constant of the enzyme-

substrate complex at which the maximal velocity is half. Maximal velocity (V max ) is the

rate of reaction at which the enzyme is maximally saturated. We will measure K M and V

max using two methods, the Michaelis- Menten plot and Lineweaver-Burk plot. The

Michaelis- Menten plot is not linear degeneration of velocity versus substrate

concentration. KM is determined from this graph; its value is half of V max. The

Lineweaver-Burk plot, however, is linear degeneration that relates the reciprocal of the

velocity to the reciprocal of the substrate concentration. From this graph we can obtain

KM and V max because the x-intercept equals and y intercept equals [1, 3, 5]. The main

purpose of this experiment is to be able to analyze enzyme kinetics by quantifying the maximum

velocity, the specific activity, and the Michaelis-Menten value of alkaline phosphatase enzyme.

Materials & Methods

Standard Curve

In the beginning of the experiment the established amounts of 0.8 µM stock solution were

mixed appropriate amounts of enzyme as in the chart. Spectrophotometer was set at 420

nm the samples were read to obtain a standard curve. The color produced corresponded to

the concentration. The stock concentration of an enzyme was 1 mg/ml.

Tube 0.8 µM PNP(ml) Enzyme(ml) Product(ml) Nanomoles

Blank 0 4 0 0
1 0.1 3.9 0.02 0.08
2 0.2 3.8 0.04 0.16
3 0.3 3.7 0.06 0.24
4 0.4 3.6 0.08 0.32
5 0.5 3.5 0.1 0.4
6 0.6 3.4 0.12 0.48
7 0.7 3.3 0.14 0.56
8 0.8 3.2 0.16 0.64

Assay Optimization

The spectrophotometer was kept at 420 nm and was blanked with 4 ml of provided

buffer. Then different dilutions of the enzyme were measured on the spectrophotometer.

The master mixture contained 1.5 ml of 30.0 mM PNPP (para-nitrophenyl phosphate)

substrate, 2.4 ml buffer, and 0.1 ml of alkaline phosphatase, which was added last. The
light absorbance of product formed was noted every 10 seconds for the first 2 minutes

and then every 20 seconds after the initial 2 minutes. Afterwards the absorbance is

measured for a 1:2 mixture with half the amount of enzyme. This solution contains

exactly the same reagents as before with only 0.05 ml of alkaline buffer added instead of

0.1mls.The last mixture, a 1:4 everything was kept the same except 0.025 ml of the

enzymes were added. That procedure was performed in order to determine the best

dilution of enzyme to be used when assaying the unknown samples.

Kinetics

The optimal enzyme dilution, 1:4 was employed to acquire light absorbance while

product was formed. Several enzyme concentrations were prepared via serial dilutions,

30mM, 15mM, 7.5mM, 3.75mM, and 1.88mM PNPP. They were prepared as follows,

30mM was formed by mixing 1.5 ml of 30mM PNPP, 2.475 ml buffer, and 0.025 ml of

the enzyme. For 15mM, 0.75mls of previous dilution is used, along with 3.225 ml buffer

and 0.025 ml of the enzyme. For 7.5 mM PNPP, 0.375 ml of previous dilution was used

along with 3.6 ml of buffer and 0.025 ml of enzyme. For 3.75 mM PNPP, 0.188 ml of

previous dilution along with 3.787 ml buffer and 0.025 ml enzyme are mixed. Finally,

1.88 mM PNPP are made by mixing 0.09 ml of previous dilution with 3.885 ml buffer

and 0.025 ml enzyme. NOTE! Enzyme is added last just before reading it on the

spectrophotometer. The setting was kept at 420 nm, and the samples were read every 10

seconds for the first 2 minutes and every 20 seconds after the initial 2 minutes.

Results

Standard Curve
PNP standard curve table

Tube 0.8µM PNP Enzyme Product Nanomoles Absorbance

(ml) (ml) (ml) (nm)
Blank 0 4 0 0 0
1 0.1 3.9 0.02 0.08 0.082
2 0.2 3.8 0.04 0.16 0.147
3 0.3 3.7 0.06 0.24 0.207
4 0.4 3.6 0.08 0.32 0.306
5 0.5 3.5 0.1 0.4 0.399
6 0.6 3.4 0.12 0.48 0.461
7 0.7 3.3 0.14 0.56 0.509
8 0.8 3.2 0.16 0.64 0.546

This table shows the amounts of PNP and alkaline buffer mixed in various dilutions from

smallest to largest to reproduce a PNP standard curve with the equation of the line and

correlation coefficient to be used to test the unknown samples against. The last column

represents absorbance data acquired from spectrophotometer readings at 420 nm. The

standard curve graph is reproduced below. The dilutions were prepared by diluting the

enzyme stock solutions with alkaline phosphatase. The dilutions were prepared with the

help of the following equation C1V1 = C2V2.

Assay Optimization
Raw data acquired from a
Time (sec) Undiluted 1:2 Dilution 1:4 Dilution
Absorbance (nm) Absorbance (nm) Absorbance (nm)
0 0.000 0.000 0.000
10 0.141 0.132 0.067
20 0.371 0.253 0.135
30 0.536 0.421 0.211
40 0.697 0.533 0.309
50 1.092 0.825 0.388
60 1.276 1.195 0.490
70 1.571 1.524 0.580
80 1.940 1.713 0.665
90 2.306 2.021 0.756
100 2.585 2.260 0.861
110 *** 2.547 0.946
120 *** 2.952 1.028
140 *** *** 1.279
160 *** *** 1.562
180 *** *** 2.047
200 *** *** 2.580
220 *** *** 2.716
240 *** *** 2.767
260 *** *** 2.284
280 *** *** 2.976
300 *** *** ***

In order to acquire V max and KM of the alkaline phosphatase, it is imperative to determine

the best concentration at which to apply it. That is why an enzyme is tested at 3 different

concentrations, undiluted, 1:2, and 1:4 dilutions. Then a graph is produced by plotting

absorbance versus time. The dilution, which has some absorbance at most time points

was selected, thus 1:4 is selected.

Kinetics

The reaction was performed using 5 different substrate concentrations with the 1:4

dilution enzyme concentrations. The table shows the substrate assay raw data and the

following graph represents the linear regression of nM of PNP versus time for each of the

substrate concentrations.
Time(sec) 30mM 15mM 7.5mM 3.75mM 1.88mM
0 0.000 0.000 0.000 0.000 0.000
10 0.061 0.060 0.059 0.040 0.059
20 0.145 0.116 0.098 0.076 0.077
30 0.204 0.181 0.138 0.130 0.094
40 0.226 0.215 0.178 0.173 0.112
50 0.268 0.258 0.234 0.205 0.132
60 0.439 0.314 0.294 0.231 0.154
70 0.469 0.353 0.338 0.244 0.177
80 0.541 0.408 0.378 0.286 0.199
90 0.601 0.456 0.437 0.343 0.225
100 0.684 0.503 0.495 0.413 0.250
110 0.811 0.550 0.526 0.476 0.273
120 0.923 0.588 0.570 0.529 0.294
140 1.278 0.667 0.678 0.655 0.324
160 1.469 0.966 0.814 0.791 0.354
180 1.584 0.929 0.974 0.933 0.392
200 1.854 1.002 1.066 1.039 0.431
220 2.254 1.156 1.186 1.109 0.480
240 2.775 1.290 1.352 1.187 0.544
260 *** 1.419 1.490 1.299 0.611
280 *** 1.597 1.631 1.395 0.652
300 *** 1.875 1.751 1.482 0.689
Part D. Post-Lab Data Analysis and Specific Activity

The Michaelis – Menten data table

PNPP Concentration (mM) Slope (∆ A 420 / min) Nmoles PNP/ min
30mM 0.42 0.46
15mM 0.36 0.39
7.5mM 0.27 0.29
3.75mM 0.26 0.28
1.88mM 0.18 0.19
The concentration slope was acquired by multiplying the slopes acquired off initial

velocities of 5 different dilutions. Those slopes were multiplies by 60 to correct for

minutes since the initial information was in seconds. Then, after acquiring the slope, that
value is plugged as the y value in the original standard curve equation, this acquiring

nmoles PNP/min. The results were graphed.

Then the Lineweaver – Burk plot is constructed, which is a double reciprocal plot of the

Michaelis – Menten plot. Since the data is a reciprocal, then the data is directly acquired

from Michaelis-Menten data set, except it is reciprocated. Since the dilutions were

performed, it has to be corrected for this dilution by using the original concentrations of

30mM, 15mM, 7.5mM, 3.75mM, and 1.88mM. The results were graphed.
 PNPP Conc. Nmoles PNP/min 1 / [S] mM -1 1 / [V0] min/nmoles
(mM) [S]
30mM 0.46 0.03 2.17
15mM 0.39 0.07 2.55
7.5mM 0.29 0.13 3.43
3.75mM 0.28 0.27 3.60
1.88mM 0.19 0.53 5.24

There are two ways to find the values for KM and Vmax of the alkaline phosphatase at its
best dilution. It is known from the double reciprocal graph that y intercept equals and x
intercept equals. Thus employing the formula acquired for the line above, y = 5.7097x +
2.2169. When x = 0, there is a y intercept at 2.2169, then 2.2169 =. Then Vmax = 1 /
2.2169 = 0.451 nanomoles/min. At y = o, we can find the x intercept, then 0 = 5.7097x +
2.2169. Then x = - 2.2169 / 5.7097 = - 0.388 =. Then KM = -1 / - 0.388 = 2.577 mM.
Specific activity is calculated by the following formula. Specific activity = nmoles PNP

formed / min * mg protein = activity PNP / mg protein, where mg protein = (mg/ml) *

(ml). The concentration of the stock solution was 1.0 mg/ml. The amount added in the

reaction was 0.1 ml. Then mg protein = 1.0 mg/ml * 0.1 ml = 0.1 mg protein. Since we

used the 1:4 dilutions, then the concentration we used is 0.25* 1.0 = 0.25. Then the

protein amount 0.1 is multiplies by the corrected concentration, 0.25, to give us the final

enzyme concentration of 0.025mg. Activity is given in nmoles/ min and we needed

µmoles/min to acquire the specific activity, the product formation was corrected for by

dividing nmoles-min by 1000.

30mM. 0.46 nmol/min * 1µmol/1000nmol = 4.6 * 10 -4µmol/min. Specific activity = 4.6 *

10 -4µmol/min * 1/0.0025mg = 0. 184 µmol sec-1 mg-1 * 60 = 11.04 µmol min-1 mg-1
15mM. 0.39 nmol/min * 1µmol/1000nmol = 3.9 * 10 -4µmol/min. Specific activity = 3.9 *
10 -4µmol/min * 1/0.0025mg = 0. 156 µmol sec-1 mg-1 * 60 = 9.36 µmol min-1 mg-1
7.5mM. 0.29 nmol/min * 1µmol/1000nmol = 2.9 * 10 -4µmol/min. Specific activity = 2.9 *
10 -4µmol/min * 1/0.0025mg = 0. 116 µmol sec-1 mg-1* 60 = 6.96 µmol min-1 mg-1
3.75mM. 0.28 nmol/min * 1µmol/1000nmol = 2.8 * 10 -4µmol/min. Specific activity = 2.8
-4 -1 -1 -1 -1
* 10 µmol/min * 1/0.0025mg = 0. 112 µmol sec mg * 60 = 6.72 µmol min mg
-4
1.88mM. 0.19 nmol/min * 1µmol/1000nmol = 1.9 * 10 µmol/min. Specific activity = 1.9
-4 -1 -1 -1 -1
* 10 µmol/min * 1/0.0025mg = 0. 076 µmol sec mg * 60 = 4.56 µmol min mg

Substrate Conc. Nmol/min PNP µmol/min PNP Specific Activity

nM
30mM 0.46 4.6 * 10 -4 11.04
15mM 0.39 3.9 * 10 -4 9.36
7.5mM 0.29 2.9 * 10 -4 6.96
3.75mM 0.28 2.8 * 10 -4 6.72
1.88mM 0.19 1.9 * 10 -4 0. 076

Discussion

The reasoning behind doing the standard curve was that the PNP standard curve provided

a defined means of testing the unknown experimental results against in order to

determine the appropriate protein concentration. For example in order to determine the

amount of product formed it is imperative to know some trend in concentrations, where

the unknowns will fall in.

The assays were performed with varying enzyme concentrations in order to

determine the most optimal enzyme concentration dilution to be used to test the unknown

samples, and that would be the most optimal dilution for the spectrophotometer to detect

most data from. Even though, according to results, the undiluted linear regression had the

best correlation coefficient, the 1:4 dilution was chosen because the spectrophotometer

output was most consistent at all time points unlike the other dilutions. The assays with

different substrate concentrations were performed in order to demonstrate the relationship

between initial and maximal reaction velocities and substrate amount. The initial

velocities were determined by choosing an early time point at which the graphs were still

linear and the points were reconstructed de novo atop the already graphed lines and the

line of best fit was generated. The equation of each line was produced, thus providing the

appropriate slope. The slope values were then plugged into the standard curve equation

for y value, thus converting to nanomoles of PNP and results were graphed.

Major sources of error were produced by spectrophotometer readings on parts of

the varying substrate quantification. The first was where the reading was stopped too

early because samples were read every ten seconds for five minutes until the investigator

had no more space on graph. Also there were experimental errors, producing values that

did not fit the general trends. The best example would be the Michealis-Menten graph,

where the supposed Vmax value was too high due to the velocity of 30mM substrate

concentration value being higher than predicted. The Lineweaver-Burke graph did look
similar to what was expected. Since this graph produced the x and y intercepts the real

Vmax and Km values could be acquired, so the error on the Michealis-Menten graph was

determined. The KM and Vmax values that were calculated from the Lineweaver-Burk plot

did not correlate with those from Michaelis-Menten values.

The specific activity was worked out in order to provide the ratio of substrate-

enzyme activity due to the amount of enzyme present in the mixture. The data acquired

from these calculations emphasizes that the substrate concentration affects enzymatic

activity rates. With the known reaction rates can be compared from assay mixtures with

various volumes. The provided information signifies that alkaline phophatase lowers the

transition state of para-nitrophenyl phosphate reaction in order to generate the para-

nitrophenyl product at a rate dependant on the substrate concentration.

References

1) Berg, Jeremy M., Tymoczko, John L., Stryer, Lubert. (2007) Enzymes: Basic
Concepts and Kinetics. In Biochemistry. Sixth Edition. W.H. Freeman and
Company, New York, pp. 205 – 240.
2) Biology Department. Northeastern University. Begley, Gail S. (Editor). (2006).
Lab 5: Analyzing Enzyme Kinetics. In Biochemistry. A Laboratory Manual for
Bio U324. Biology Department. Northeastern University. Begley, Gail S. (Editor),
Boston, pp. EK1 – EK8.
3) Halford, S.E. (1971) Escherichia coli Alkaline Phosphatase. An Analysis of
Transient Kinetics. Biochemistry Journal (1971), Vol. 125, pp. 319 – 327.
4) Mandecki, Wlodek, Shallcross, Mary Ann, Sowadski, Janusz, and Tomazic-Allen,
Susan (1991) Mutagenesis of conserved residues within the active site of
Escherichia coli alkaline phosphatase yields enzymes with increased kcat. Protein
Engineering Design & Selection Journal (1991), Vol. 4, No. 7, pp. 801-804.
5) Bloch, Will and Schlesinger, Milton J. (1973) The Phosphate Content of
Escherichia coli Alkaline Phosphatase and Its Effect on Stopped Flow Kinetic
Studies. The Journal of Biological Chemsitry (1973), Vol. 248, pp. 5794 – 5805.