Metabolism of Basic Amino Acids in Pseudomonas putida

Abstract

Abstract γ-Guanidinobutyrate amidinohydrolase, the third enzyme of l-arginine catabolism in Pseudomonas putida, has been purified 68-fold from extracts of cultures grown on l-arginine. Synthesis of the amidinohydrolase is induced by growth on l-arginine or γ-guanidinobutyrate, but not by growth on γ-aminobutyrate or on urea plus dl-malate. Hydrolysis of γ-guanidinobutyrate to γ-aminobutyrate and urea proceeds essentially to completion at pH 7 to 11. The purified enzyme specifically requires Mn2+ and exhibits optimum activity at about pH 10 and 50°. The Km for γ-guanidinobutyrate is 32 mm at pH 10. While δ-guanidinovalerate and e-guanidinocaproate also are hydrolyzed, the value of both Km (δ-guanidinovalerate = 206 mm; e-guanidinocaproate = 163 mm) and of Vmax (606, 27, and 11 e.u. per mg for γ-guanidinobutyrate, δ-guanidinovalerate, and e-guanidinocaproate, respectively) suggest that γ-guanidinobutyrate is the natural substrate. The molecular weight of the purified enzyme was estimated to be 178,000 by gel permeation chromatography and 190,000 by sucrose density gradient centrifugation.