The role of cysteine 116 in the active site of the antitumor enzyme L-methionine γ-lyase from Pseudomonas putida

Daizou Kudou, Shintaro Misaki, Masao Yamashita, Takashi Tamura, Nobuyoshi Esaki, Kenji Inagaki

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

The cysteinyl residue at the active site of L-methionine γ-lyase from Pseudomonas putida (MGL Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter kcat toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the γ-elimination reaction of L-methionine and in substrate recognition in the MGLs.

Original languageEnglish
Pages (from-to)1722-1730
Number of pages9
JournalBioscience, Biotechnology and Biochemistry
Volume72
Issue number7
DOIs
Publication statusPublished - 2008

Fingerprint

Pseudomonas putida
Lyases
lyases
active sites
Methionine
Cysteine
cysteine
methionine
Catalytic Domain
Mutagenesis
Enzymes
Substrates
enzymes
mutagenesis
Kinetic parameters
mutants
Catalyst activity
Substitution reactions
substrate specificity
Substrate Specificity

Keywords

  • L-methionine γ-lyase
  • Pyridoxal 5′-phosphate
  • Saturation mutagenesis

ASJC Scopus subject areas

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

Cite this

The role of cysteine 116 in the active site of the antitumor enzyme L-methionine γ-lyase from Pseudomonas putida. / Kudou, Daizou; Misaki, Shintaro; Yamashita, Masao; Tamura, Takashi; Esaki, Nobuyoshi; Inagaki, Kenji.

In: Bioscience, Biotechnology and Biochemistry, Vol. 72, No. 7, 2008, p. 1722-1730.

Research output: Contribution to journalArticle

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AU - Esaki, Nobuyoshi

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