Chemical modification studies of Rhizomucor miehei protease: evidence for the role of basic amino acids in enzyme catalysis.
The effect of chemical modification on milk clotting and proteolytic activities of aspartyl protease obtained from Rhizomucor miehei NRRL 3500 was examined in the absence and the presence of its specific inhibitor pepstatin A. The effect on the ratio of milk clotting activity (MC) to proteolytic activity (PA), an index of the quality of milk clotting proteases was also determined. Modification of the enzyme with trinitrobenzenesulfonic acid, diethylpyrocarbonate and phenylglyoxal produced an increase in the ratio of MC/PA, while modification with 2- hydroxy-5-nitrobenzyl bromide did not affect the ratio. Modification with N-acetylimidazole resulted in a marginal increase in MC/PA ratio. Protection using pepstatin A during modification with phenylglyoxal, N-acetylimidazole and 2-hydroxy-5-nitrobenzyl bromide, protected both MC and PA. In the case of modification by diethylpyrocarbonate, pepstatin A protected only MC. Pepstatin A did not protect both the activities on the modification of the enzyme by trinitrobenzene sulfonic acid. These observations indicate the presence of arginine, tyrosine and tryptophan at the catalytic site of the enzyme, for eliciting MC and PA of the enzyme. In general, modification of the positively charged residues increases the MC/PA ratio of the enzyme. In addition the modified lysine residues responsible for the inactivation of the enzyme were not involved in the active site of the enzyme. Thus the lysine residues might have a secondary role in enzyme catalysis. Further, histidine at the catalytic site was found to be exclusively involved in milk clotting activity. The enzyme with modified histidine residues were more susceptible to autocatalysis, indicating that histidine residues protect the enzyme against autolysis.
Department of Biotechnology, Bharathiar University, Coimbatore, India. biotech@as250.bharathi.ernet.in
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D016282:Aspartic Acid Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000187:drug effects / Q000378:metabolism*; D006571:Heterocyclic Compounds / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D007287:Inorganic Chemicals / Q000494:pharmacology; D009930:Organic Chemicals / Q000494:pharmacology; D020103:Rhizomucor
D005656:Fungal Proteins; D006571:Heterocyclic Compounds; D007202:Indicators and Reagents; D007287:Inorganic Chemicals; D009930:Organic Chemicals; D016282:Aspartic Acid Endopeptidases