Salicylurate-Hydrolyzing Enzyme from Intestinal Bacterium in Rabbit

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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 on hydroxyapatite, and its enzymatic preperties were characterized. The isoelectric point was 8.7, and the molecular weight was estimated to be 170,000 by gel filtration on Sephadex G-150 and 27,000 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).