Essential Histidyl Residues in Glucose Oxidase. Chemical Modification of Histidyl Residues with Diethylpyrocarbonate

Abstract

The apoenzyme of glucose oxidase (EC 1.1.3.4) from Aspergillus niger was modified by diethylpyrocarbonate (DEPC). A second-order rate constant of inactivation with respect to the regaining of activity when reconstituted with FAD was 33.7M^-1s^-1 at pH 6.2 and 25°C. This inactivation was reversed up to 60% by the addition of 0.25 M hydroxylamine. It was suggested that histidyl residues having pK_a = 8.2 were responsible for the reconstitution reaction of holo-oxidase from its pH-dependence of inactivation. In addition to these histidyl residues, two SH-groups were particitated in the DEPC modification, and these residues seemed likely to interact with each other. From the studies on the enzyme kinetics, CD spectra, and stoichiometry on the modification of histidyl residues, all data suggested that inactivation of apoenzyme with DEPC was due primarily to the modification of two histidyl residues essential for the binding of FAD and two cysteines near these residues. The present investigation suggested that there were some other histidyl residues necessary for maintaining the proper conformation of native glucose oxidase.