Purification and Characterization of Methioninase from Pseudomonas putida

Abstract

Methioninase of Pseudomonas putida was purified to homogeneity, as judged by poly aery lamide gel electrophoresis, with a specific activity 270-fold higher than that of the crude extract. 1. The purified enzyme had an S₂₀, w of 8.37, a molecular weight of 160, 000, and an isoelectric point of 5.6. 2. A break in the Arrhenius plot was observed at 40° and the activation energies below and above this temperature were 15.5 and 2.97 kcal per mole, respectively. 3. In addition to L-methionine, various S-substituted derivatives of homocysteine and cysteine could serve as substrates. D-Methionine, 2-oxo-4-methylthiobutanoate, and related non sulfur-containing amino acids were inert. Equimolar formation of α-ketobutyrate and CH3SH was observed with methionine as a substrate. 4. In addition to the protein peak at 278 nm, two absorption maxima were observed at 345 and 430 nm at pH 7.5. Hydroxylamine removed the enzyme-bound pyridoxal phosphate, resulting in almost complete resolution with the concomitant disappearance of both peaks. Reconstruction of the treated enzyme could be achieved by addition of the cofactor; the Km value was calculated to be 0.37 μM. 5. The reported purified enzyme should be designated as L-methionine methanethiol-lyase (deaminating).