Enzyme Catalysis Essay Research Paper Glucose6Phosphate Appears

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