Abstract

The pH dependence of kcat/Km for the papain-catalyzed hydrolysis of ethyl hippurate, N-alpha-benzoyl-L-citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-alpha-benzoyl-L-arginine was determined below pH 6.4. The value of kcat/Km was observed to be modulated by two acid ionizations rather than a single ionization as previously believed. For the five substrates studied, the average pK values for the two ionizations are 3.78 +/- 0.2 and 3.95 +/- 0.1 at T/2 0.3, 25 degrees C. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme.

Highlights

  • Citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-a-benzoyl-L-a&nine was determined below pH 6.4

  • The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme

  • These investigators found that at r/2 0.3, 25”C, the pH dependence of kcat/Km for the papain-catalyzed hydrolysis of N-a-benzoyl-DL-arginine p-nitroanilide depends on two acid ionizations, each having a pK

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Summary

Introduction

Citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-a-benzoyl-L-a&nine was determined below pH 6.4. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme. Cys-25, which is acylated and deacylated during the catalytic cycle, three other ionizable groups are close to the active site of papain. Provided there is no change in the rate-determining step with pH, a pH-&/K, profile should yield true macroscopic ionization constants for groups on the free enzyme or substrate that modulate catalytic activity. The bell-shaped pH-Lt/K, profiles observed for reactions catalyzed by papain are usually attributed to control of catalytic activity by the state of protonation of two groups with pK values of about 4 and 8.5 [3]. Active enzyme is envisaged as having one group protonated and the other group unprotonated

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