Role of Tyrosine 114 of L-Methionine γ-lyase from Pseudomonas putida

Hiroyuki Inoue, Kenji Inagaki, Naoki Adachi, Takashi Tamura, Nobuyoshi Esaki, Kenji Soda, Hidehiko Tanaka

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

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 kcat for α,γ-elimination of L-methionine, while the Km remained the same as the wild type enzyme. The Y114F mutant had the reduced kcat 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 kcat 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.

Original languageEnglish
Pages (from-to)2336-2343
Number of pages8
JournalBioscience, Biotechnology and Biochemistry
Volume64
Issue number11
Publication statusPublished - Nov 2000

Fingerprint

Pseudomonas putida
Lyases
lyases
Methionine
Tyrosine
tyrosine
methionine
Enzymes
Substrates
mutants
enzymes
Homoserine
pyridoxal phosphate
homoserine
deamination
Deamination
Pyridoxal Phosphate
enzyme substrates
Phenylalanine
Phosphates

Keywords

  • General acid catalyst
  • L-methionine γ-lyase
  • Reaction mechanism
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Food Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Chemistry (miscellaneous)
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

Role of Tyrosine 114 of L-Methionine γ-lyase from Pseudomonas putida. / Inoue, Hiroyuki; Inagaki, Kenji; Adachi, Naoki; Tamura, Takashi; Esaki, Nobuyoshi; Soda, Kenji; Tanaka, Hidehiko.

In: Bioscience, Biotechnology and Biochemistry, Vol. 64, No. 11, 11.2000, p. 2336-2343.

Research output: Contribution to journalArticle

Inoue, H, Inagaki, K, Adachi, N, Tamura, T, Esaki, N, Soda, K & Tanaka, H 2000, 'Role of Tyrosine 114 of L-Methionine γ-lyase from Pseudomonas putida', Bioscience, Biotechnology and Biochemistry, vol. 64, no. 11, pp. 2336-2343.
Inoue, Hiroyuki ; Inagaki, Kenji ; Adachi, Naoki ; Tamura, Takashi ; Esaki, Nobuyoshi ; Soda, Kenji ; Tanaka, Hidehiko. / Role of Tyrosine 114 of L-Methionine γ-lyase from Pseudomonas putida. In: Bioscience, Biotechnology and Biochemistry. 2000 ; Vol. 64, No. 11. pp. 2336-2343.
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