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Exercise 3.   A Study of Enzyme Specificity. A Kinetic Analysis of Glucose Oxidase

 


A. Introduction

 

A model of the enzyme glyoxalase I.
Image Source: Wikipedia

Enzymes are one of the largest functional groups of proteins in cells. They perform two fundamental and extremely important tasks in the cell: a) they are biological catalysts, that is, they increase the rate of chemical reactions to a useful level, and b) they provide a point where chemical reaction rate can be regulated to meet the moment-to-moment needs of the cell.

One very important characteristic of an enzyme is its specificity, i.e., an enzyme's inherent nature to bind with a specific substrate, or a family of similar substrates, to form a specific product or family of similar products. An enzyme's specificity is based on the 3-D structure and chemistry of its active site, where a few critical R groups are positioned to interact with a few specific functional groups on the substrate.

There are five distinct types or degrees of specificity:

  • Absolute specificity - the enzyme will bind only one specific substrate and catalyze only one reaction.
  • Relative specificity - the enzyme binds with a group of similar substrates and catalyzes a group of similar reactions.
  • Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups.
  • Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure.
  • Stereochemical specificity - the enzyme will act on a particular steric or optical isomer.

Understanding an enzyme's or another protein's specificity is key to understanding the reaction it catalyzes and the cellular process it mediates. The type or degree of specificity of a given enzyme can be determined through a kinetic analysis of the enzyme-catalyzed reaction. In this exercise, we study the structure and function of the enzyme glucose oxidase and review the basic Michaelis-Menten reaction and basic Michaelis-Menten Kinetics. In lab, we will characterize the substrate specificity of the glucose oxidase by determining the substrate binding affinity of glucose oxidase for two monosaccharides, D-glucose and D-xylose.