Role of tyrosine 114 of L-methionine gamma-lyase from Pseudomonas putida

• Published in: Bioscience, biotechnology, and biochemistry Vol. 64; no. 11; pp. 2336 - 2343
• Main Authors: Inoue, H. (Okayama Univ. (Japan). Faculty of Agriculture), Inagaki, K, Adachi, N, Tamura, T, Esaki, N, Soda, K, Tanaka, H
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 2000; Japan Society for Bioscience Biotechnology and Agrochemistry
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbon-Sulfur Lyases - chemistry; Carbon-Sulfur Lyases - genetics; Carbon-Sulfur Lyases - metabolism; Catalysis; CHEMICAL STRUCTURE; Deamination; Deuterium Oxide - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; general acid catalyst; Hydrogen - metabolism; L-methionine γ-lyase; LYASES; Magnetic Resonance Spectroscopy; METHIONINE; Mutagenesis, Site-Directed; MUTATION; Phenylalanine - genetics; Phenylalanine - metabolism; PSEUDOMONAS PUTIDA; Pseudomonas putida - enzymology; Pseudomonas putida - genetics; reaction mechanism; site-directed mutagenesis; Tyrosine - genetics; Tyrosine - metabolism; Pseudomonadales; Methionine γ-lyase; Tyrosine; Enzyme; Active site; Site directed mutagenesis; Carbon-sulfur lyases; Reaction mechanism; Bacteria; Pseudomonadaceae; Lyases; Pseudomonas putida
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.64.2336

L-Methionine γ-lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of γ-family pyridoxal 5′-phosphate dependent enzymes. A mutant form of L-methionine γ-lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in k cat for α,γ-elimination of L-methionine, while the K m remained the same as the wild type enzyme. The Y114F mutant had the reduced k cat by only 28- and 16-fold for substrates with an electron-withdrawing group at the γ-position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of k cat for α,β-elimination and deamination substrates. The hydrogen exchange reactions of substrate and the spectral changes of the substrate-enzyme complex catalyzed by the mutant enzyme suggested that γ-elimination process for L-methionine is the rate-limiting determination step in α,γ-elimination overall reaction of the Y114F mutant. These results indicate that Tyr114 of L-methionine γ-lyase is important in γ-elimination of the substrate.