Characterization of a Nonproteolytic Arginine Ester-hydrolyzing Enzyme from Snake Venom

Abstract

Abstract An enzyme that catalyzes the hydrolysis of N-benzoyl-l-arginine ethyl ester and p-toluenesulfonyl-l-arginine methyl ester has been isolated from the venom of Agkistrodon contortrix laticinctus (broadbanded copperhead) by means of DEAE-cellulose chromatography. A high degree of homogeneity is suggested by sedimentation velocity, gel filtration, polyacetate electrophoresis, and isoelectric focusing. The purified enzyme has a sedimentation coefficient of 2.7 S, a diffusion coefficient of 8.3 x 10-7 cm2 per sec, and a molecular weight of 30,000. The isoelectric point, as determined by means of isoelectric focusing, was found to be 9.1. Enzymatic assays showed the preparation to be specific for arginine esters. The Km values determined with N-benzoyl-l-arginine ethyl ester and p-toluenesulfonyl-l-arginine methyl ester are 1.17 x 10-4 and 1.49 x10-3, respectively. The enzyme was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and N-bromosuccinimide. Optimal rates of hydrolysis were observed from pH 7.5 to pH 8.5.