Purification and Characterization of Methioninase from Pseudomonas putida
1976 年 79 巻 6 号 p. 1263-1272
詳細
1976 年 79 巻 6 号 p. 1263-1272
Methioninase of Pseudornonas putida was purified to homogeneity, as judged by polyacrylamide 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 278nm, two absorption maxima were observed at 345 and 430nm 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 0.37μM. 5. The reported purified enzyme should be designated as L-methionine methanethiollyase (deaminating).
