Contribution to Catalysis and to Stability of the Essential Cysteine Residue at the Active Site of D-Glucosaminate Dehydratase from Pseudomonas fluorescens

Abstract

Chemical modification of Purified D-glucosaminate dehydratase (GADH) apoenzyme by N-ethylmaleimide (NEM) and by 7-chloro-4-aminobenzo-2-oxa-1, 3-diazole (NBDCl) resulted in the time- and concentration- dependent inactivation of the enzyme in each case. The inactivation followed pseudo-first-order kinetics and a double-logarithmic plot of the observed pseudo-first-order rate constant against reagent concentration proved evidence for an approximately first-order reaction, suggesting that the modification of a single cysteine residue per mole of enzyme resulted in inactivation. Amino acid analysis of the NEM-inactivated enzyme showed that three moles of cysteine residues among six moles per mole of subunit were modified under these conditions, therefore one of the three cysteine residues modified by NEM may be essential for activity. Pyridoxal 5'-phosphate (PLP) and D-glucosaminate (GlcNA) protected the enzyme against inactivation by NEM and NBDCl. The apoenzyme was inactivated by EDTA and activity of enzyme was restored by incubation with Mn²⁺ in the presence of PLP. Incubation of the EDTA-treated enzyme with NEM inhibited the restoration of activity. These results suggest that one of the cysteine residues of GADH may be chelated to a Mn²⁺ at or near the active site of GADH, contributing to formation of the active enzyme.