Enzyme Catalysis Essay, Research Paper

Glucose-6-Phosphate Appears to be a Competitive Inhibitor of the Bovine Intestine Enzyme Alkaline Phosphatase

Abstract

This study focuses on the effects of glucose-6-phosphate (G6P) on the kinetics of the enzyme alkaline phosphatase. Because G6P is a common molecule and has a phosphoester bond, it may prevent alkaline phosphatase from hydrolyzing certain substrates. The data collected in this study seem to indicate that G6P is a competitive inhibitor of the enzyme and causes an increased Km for the reaction. Further studies should be done to investigate the initial conclusion.

Introduction

An enzyme is a catalyst that increases the rate at which a biological reaction occurs by providing a surface where the reactants can be bound in such a way that they become more likely to react with each other. The point on the enzyme where a reactant binds is known as the active site (Becker, et. al., 2000). The binding of a reactant, or substrate, to the active site depends on random collisions and by increasing the substrate concentration more collisions occur and the overall rate of the reaction increases. Eventually the reaction will reach a maximum velocity. When reaction velocity is graphed as a function of substrate concentration, many enzymatic reactions give rectangular hyperbolas. This type of graph is referred to as a Michaelis-Menten plot and two important features are the maximum velocity (Vmax) and the substrate concentration at one half of the maximum velocity, Km (Becker, et. al., 2001). Because it is difficult to estimate Vmax and Km from a Michaelis-Menten plot, a Lineweaver-Burk plot can also be used. This plot is a double reciprocal plot (1/v vs. 1/[S] is graphed) and the x and y intercept values are used in determining Km and Vmax.

The velocity of an enzymatic reaction can be affected not only by changing the substrate concentration, but also by the presence of an inhibitor. A competitive inhibitor is one that decreases the velocity of a reaction by competing with the substrate for the active site (Becker, et. al., 2001). The influence of an inhibitor depends on the ratio of the substrate and inhibitor concentrations. If a competitive inhibitor concentration is held constant and substrate concentration is increased, the substrate will eventually be able to overcome the inhibitor and will reach the same Vmax as it would without inhibitor. However, the Km for the reaction will increase because with inhibitor present it takes a higher concentration of substrate to reach one half of the maximum velocity (Becker, et. al., 2001).

This study investigates the kinetics of the enzyme alkaline phosphatase from bovine intestine. A phosphatase is an enzyme that hydrolyzes phosphate esters and has an active site that recognizes many phosphate esters. One such substrate is p-nitrophenylphosphate (pNPP). Phosphatase converts pNPP to p-nitrophenol and the alkaline conditions cause it to dissociate to p-nitrophenolate. By varying pNPP concentration and measuring amount of p-nitrophenolate formed, alkaline phosphatase was found to behave by Michaelis-Menten kinetics. However, there was no inhibitor present and such a situation is unlikely. One molecule that is quite common in the biological world is glucose-6-phosphate (G6P). G6P forms an integral part of the glycolytic pathway and is always present.

Because G6P is a common molecule and has a phosphate ester bond which can by hydrolyzed, this study looks at how the presence of G6P affects the ability of alkaline phosphatase to convert pNPP to p-nitrophenolate. We hypothesize that G6P is a competitive inhibitor of alkaline phosphatase and as such it will increase the Km of the reaction, but have no effect on the Vmax. To study the kinetics of alkaline phosphatase, assays of varying pNPP concentrations with no inhibitor were studied. The amount of pNPP hydrolyzed was determined by measuring the absorbance of the p-nitrophenolate produced. A second set of assays was run with a fixed amount of G6P present. The results were compared by making various plots.

Methods

Assay without G6P: Ten test tubes with pNPP concentrations ranging from 0.025 mM to 0.500 mM were prepared. The necessary amount of pNPP, 0.05M Tris-HCl (a buffer with a pH of 8.7), and water were added to each assay tube. To simulate the environment that the reaction normally occurs in, the assay tubes and alkaline phosphatase were warmed in a 38C water bath. To initiate each reaction, 1mL of alkaline phosphatase was added to the assay tube. A blank assay tube with a 0.500mM pNPP concentration and no enzyme was also prepared. Each reaction was stopped after 3.5 minutes by adding 1 N NaOH, which denatured the enzyme and converted any p-nitrophenol into p-nitrophenolate. The assays had varying degrees of yellow color because of the p-nitrophenolate and the absorbance of each assay was measured at 400nm. The blank was used to correct for absorbance due to pNPP (Becker, et. al., 2001). The corrected absorbances were used to determine the velocity of each reaction (a complete breakdown of the calculations can be found in the Appendix). Additionally, replicates were made of assay 3 and assay 8 to ensure that the reaction ran properly. Because they had absorbances similar to their counterparts (within 2 hundredths) it was assumed the reaction worked. The replicates were not used in the calculation.

Assay with G6P: The above procedure was repeated with only one modification: a fixed amount of 0.1mM G6P was added to each assay tube and to the blank. This concentration was chosen because it was practical to work with and only about two degrees of magnitude higher than the concentration of G6P in a cell.

Data Analysis: A Michaelis-Menten plot and a Lineweaver-Burk plot were prepared for each set of assay conditions.

Results

The assays performed with G6P present had lower velocities at each concentration than did the assays without G6P (Figure 1).

Figure 1

Figure 1. Michaelis-Menten plot for the relationship between the velocity of the reaction and the concentration of pNPP. For each concentration, the assays without G6P present (blue) have higher velocities than the assays with G6P (pink).

The percent decrease in velocity for each concentration is shown in Table 1.

Table 1. The percent decrease in velocity of the G6P assays as compared to the velocities of the assays without G6P. The decrease in velocity is shown for each of the ten concentrations assayed.

[S] in mM % decrease in velocity

0.025 39.3

0.050 26.2

0.075 22.8

0.100 13.4

0.125 14.8

0.150 11.4

0.200 11.1

0.300 13.0

0.400 7.5

0.500 5.8

The Lineweaver-Burk plot for both assays shows a linear relationship between 1/v and 1/[S] (Figure 2).

Figure 2

Figure 2. Lineweaver-Burk plot for the assays with and without G6P. The line for no G6P has an equation of y=0.7505x + 17.463 and an R2 value of 0.998. The line with G6P has an equation of y=1.5121x + 15.979 and an R2 value of 0.9947. For both assays, the equation of the line was used to determine Vmax and Km values (see Appendix).

From this plot, the assay without G6P was determined to have a Vmax of 0.0573 umole/min and a Km of 0.043 mM. When G6P was present, Vmax was 0.0626 umole/min and Km was 0.095 mM. The inhibitor constant, Ki, for G6P was calculated to be 0.083 (see Appendix).

Discussion

Although the data do not completely support our hypothesis, they do seem to suggest that G6P is a competitive inhibitor of alkaline phosphatase. The presence of G6P caused the Km to increase, as hypothesized, but did not give the same Vmax as did the reactions without G6P. The Vmax was actually calculated to be higher with G6P. At first, this calculation does not seem to make sense because an inhibitor should never increase the velocity of a reaction. However, the Vmax was calculated by determining the y intercept on the Lineweaver-Burk plot and the intercept is very dependent on the slope and thus the farthest data points on the graph. Because the plot is a double-reciprocal plot, the farthest data points correspond to the smallest concentrations, which have the most uncertainty. In short, the Vmax determination is quite dependent on those data points that are the most uncertain. This most likely explains why the presence of G6P gave a higher Vmax.

In spite of the apparent contradiction in maximum velocities, the data support the idea that G6P is a competitive inhibitor in several ways. First, for every concentration of pNPP studied, the velocity of the reaction was slower in the presence of G6P. Second, the Km for the reaction with G6P is considerable larger than the Km of the reaction without G6P. Some of this difference is probably due to the different Vmax values, but if the reaction with G6P is assumed to have the same maximum velocity as the reaction without it, it can be seen from Figure 1 that at a velocity of .029 (+ Vmax) the reaction with G6P gives a higher concentration. This suggests that G6P competitively inhibits the reaction because a larger concentration of pNPP is required to reach + Vmax. One feature of a competitive inhibitor is that, with enough substrate, it can be overcome. Table 1 shows the general trend of what happens as the ratio of substrate to G6P increases. When G6P was high as compared to pNPP, a 39.3% decrease in velocity was observed. In general, as the amount of G6P got lower as compared to pNPP, the amount the velocity decreased also got smaller. At a pNPP concentration of 0.500 mM the decrease in velocity was only 5.8%. This trend suggests that the effects of G6P were being overcome and if the pNPP concentration were high enough, the percent decrease in velocity would eventually become 0 and both reactions would reach the same maximum velocity.

Because the data suggested that G6P was a competitive inhibitor, Ki was calculated and found to be 0.083. A high inhibitor constant means that the enzyme-inhibitor complex dissociates at a faster pace because the inhibitor does not bind very tightly to the active site (Becker, et. al., 2001). Thus, a higher Ki value corresponds to a smaller effect on reaction velocity. Since G6P appears to be a competitive inhibitor, its presence probably affects reactions in the body. It may be used as a control mechanism to regulate the hydrolysis of pNPP in the intestine. G6P may also inhibit phosphatases other than alkaline phosphatase.

The largest weakness in this experiment was the Vmax value calculated for the reaction with G6P. In further experiments, a greater number of small substrate concentrations should be tested, and since smaller concentrations have the most uncertainty, at least one replicate should be included for each concentration. Also, the G6P concentration tested was higher than that found in a typical cell, so the inhibition effects may have been exaggerated. The effects of lower concentrations should be tested. In general, there was a lot of human error that could have occurred. Volumes required for the dilutions were very exactly and it was sometimes necessary to estimate with the pipette. Timing was also critical in this experiment. The best way to deal with human inconsistencies would be to perform the experiment several times.

In conclusion, this study suggests that glucose-6-phosphate is a competitive inhibitor of alkaline phosphatase from bovine intestine. To further substantiate and investigate this conclusion, additional studies using lower substrate concentration and more replicates should be performed.

References

Becker, Wayne, Metzenberg, Robert, and Dehring, Anne. 2001. Enzyme catalysis. In Burgess, A.B., editor. Cellular biology laboratory manual, University of Wisconsin-Madison.

Becker, Wayne M., Kleinsmith, Lewis J., and Hardin, Jeff. 2000. The world of the cell, 4th ed. Addison Wesley Longman, San Francisco, CA.

Appendix

A sample calculation for a pNPP concentration of 0.025mM. A total volume of 5 mL was used for each assay, including 0.5mL of buffer and 1.0mL of alkaline phosphatase. The stock [pNPP] was 2 mM.

Determining volume of pNPP to add

M1V1=M2V2 (2)V1=0.025*5 V1=0.0625 mL

Correcting the Absorbance

Corrected=Actual (vol. pNPP in assay/vol. pNPP in 0.0500mM)*Absorbance of blank

= 0.228 (0.0625/1.25)*0.035

= 0.226

Determining Substrate Hydrolyzed

Corrected Absorbance=millimolar extinction coefficient*length of pathway*concentratio

millimolar extinction coefficient=18.4 liter/mmole*cm length of pathway=1cm

concentration= corrected absorbance/(18.4*1)

= .226/(18.4)

= 0.0123 mmol/L

umoles hydrolyzed=concentration*final volume in assay tube

= 0.0123*0.006

= 0.0737 umole

Determining Velocity

velocity=umoles hydrolyzed/length of reaction

= 0.0737/3.5

= 0.0211 umole/min

Assays without G6P

Assay mL pNPP [S] mM mL water Actual A Corrected A umoles hydrolyzed velocity (umole/min) 1/[S] 1/v

1 0.0625 0.0250 3.4375 0.228 0.226 0.0737 0.0211 40.0 47.39

2 0.1250 0.0500 3.3750 0.350 0.332 0.1083 0.0309 20.0 32.36

3 0.1875 0.0750 3.3125 0.400 0.395 0.1288 0.0368 13.3 27.17

4 0.2500 0.1000 3.2500 0.424 0.417 0.1360 0.0389 10.0 25.71

5 0.3125 0.1250 3.1875 0.460 0.451 0.1471 0.0420 8.0 23.51

6 0.3750 0.150 3.1250 0.581 0.471 0.1536 0.0439 6.7 22.78

7 0.5000 0.200 3.0000 0.515 0.501 0.1634 0.0467 5.0 21.41

8 0.7500 0.300 2.7500 0.575 0.554 0.1807 0.0516 3.3 19.38

9 1.0000 0.400 2.5000 0.588 0.560 0.1826 0.0522 2.5 19.16

10 1.2500 0.500 2.2500 0.610 0.575 0.1875 0.0536 2.0 18.66

Blank 1.2500 0.500 3.2500 0.035

Assays with G6P

Assay mL pNPP [S] mM mL G6P mL water Actual A Corrected A umoles hydrolyzed velocity (umole/min) 1/[S] 1/v

1 0.0625 0.0250 0.0500 3.3875 0.139 0.137 0.0447 0.0128 40.0 78.13

2 0.1250 0.0500 0.0500 3.3250 0.248 0.245 0.0799 0.0228 20.0 43.86

3 0.1875 0.0750 0.0500 3.2625 0.310 0.305 0.0995 0.0284 13.3 35.21

4 0.2500 0.1000 0.0500 3.2000 0.369 0.362 0.1118 0.0337 10.0 29.67

5 0.3125 0.1250 0.0500 3.1375 0.392 0.384 0.1252 0.0358 8.0 27.93

6 0.3750 0.150 0.0500 3.0750 0.428 0.418 0.1363 0.0389 6.7 25.71

7 0.5000 0.200 0.0500 2.9500 0.459 0.446 0.1454 0.0415 5.0 24.10

8 0.7500 0.300 0.0500 2.7000 0.502 0.482 0.1572 0.0449 3.3 22.27

9 1.0000 0.400 0.0500 2.4500 0.544 0.518 0.1689 0.0483 2.5 20.70

10 1.2500 0.500 0.0500 2.2000 0.575 0.542 0.1767 0.0505 2.0 19.80

Blank 1.2500 0.500 0.0500 3.3000 0.033

Determining Vmax and Km

From a Lineweaver-Burk plot, the y intercept = 1/Vmax and the x-intercept = -1/Km

Without G6P: y=0.7505x+17.563

y intercept = 17.563 x intercept = -17.563/0.7505

Vmax = 1/17.563 = -23.27

= 0.0573 umole/min Km = -1/-23.27

= 0.043 mM

With G6P: y=1.5121x+15.979

y intercept = 15.979 x intercept = -15.979/1.5121

Vmax = 1/15.979 = -10.57

= 0.0626 umole/min Km = -1/-10.57

= 0.095 mM

Determining Ki

x intercept = -1/ ((1+I/Ki)*Km)

where x intercept is from graph with G6P, I = concentration of inhibitor, and Km is without G6P

-10.57= -1/((1+0.1/Ki)*0.043)

(1+0.1/Ki)*-0.454=-1

1+0.1/Ki = 2.20

0.1/Ki = 1.20

Ki = 0.083