Influence of reagents reacting with metal, thiol and amino sites of catalytic activity and l-phenylalanine inhibition of rat intestinal alkaline phosphatase

Abstract

Studies on the inactivation of rat intestinal alkaline phosphatase by several metal-binding agents, namely EDTA, 8-hydroxyquinoline, pyridine-2,6-dicarboxylic acid, [alpha][alpha][image]-bipyridyl, o-phenanthroline and sodium cyanide, indicated the functional role of a metal, probably Zn, in the catalysis. The metal ligands lowered stereospecific uncompetitive inhibition of the enzyme by L-phenylalanine by an extent that paralleled the decline in enzyme activity. The thiol reagents p-hydroxymercuribenzoate, iodoacetamide and I inactivated rat intestinal phosphatase. The enzyme could be protected from inactivation by either cysteine or substrate. The L-phenylalanine inhibition remained unchanged only in the presence of moderately inactivating concentrations of the thiol reagents. Inactivation of the enzyme by the amino-group-blocking reagent, O-methyl-isourea, provided ample evidence for the participation in the catalysis of the [image]-amino group of lysine. At the same time, L-phenylalanine inhibition remained unaltered even when the enzyme was strongly inactivated. This [image]-amino-group-blocked enzyme exhibited no change in migration in starch gel, in contrast with enzyme treated with acetic anhydride, formaldehyde or succinic anhydride. The Michaelis constant of the enzyme was enhanced by such modifications, but the optimum pH remained the same. D-Phenylalanine acted as a competitive or "co-operative" activator for intestinal alkaline phosphatase after it had been modified by acetylation.