Abstract
An enzyme which hydrolyzes salicylurate (salicyluric acid) into salicylic acid and glycine was found in a cell-free extract of an intestinal bacterium in rabbit.This enzyme, tentatively named salicylurate-hydrolyzing enzyme, was found to be membrane-bound and was purified from the extract by ammonium sulfate fractionation and column chromatography.Two protein fractions with the enzyme activity were observed on diethylaminoethyl-Toyopearl.The first peak (enzyme I) was further purified by chromatography on carboxymethyl-cellulose and on hydroxyapatite, and its enzymatic properties were characterized.The isoelectric point was 8.7, and the molecular weight was estimated to be 170000 by gel filtration on Sephadex G-150 and 27000 by sodium dodecyl sulfate gel electrophoresis, suggesting that the enzyme exists as a hexamer.The enzyme was strongly inhibited by p-chloromercuribenzoate (PCMB), Hg2+and o-phenanthroline.The activities lost on incubation with PCMB and o-phenanthroline were restored by the addition of 2-mercaptoethanol and Zn2+, respectively.The enzyme catalyzed hydrolysis of N-benzoyl (Bz) amino acids and their derivatives, while it was inert toward n-benzoyl-protected peptides such as p-HO-Bz-Gly-Gly and Bz-Gly-L-His-L-Leu.We conclude that salicylurate hydrolase presented here is a kind of hippurate hydrolase (EC 3.5.1.32).
