Assay method for antitumor L-methionine γ-lyase: Comprehensive kinetic analysis of the complex reaction with L-methionine

Tomoaki Takakura, Kenji Mitsushima, Shigeo Yagi, Kenji Inagaki, Hidehiko Tanaka, Nobuyoshi Esaki, Kenji Soda, Akio Takimoto

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

L-Methionine γ-lyase (EC 4.4.1.11) is a pyridoxal 5 -phosphate-dependent multifunctional enzyme. Measuring the initial velocity of α-ketobutyrate production by α,γ- elimination of L-methionine catalyzed by L-methionine γ-lyase is not very feasible, because the enzyme simultaneously catalyzes both γ-replacement and α,γ-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to α-ketobutyrate, methanethiol, and ammonia with pyridoxal 5 -phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of α-ketobutyrate production including α,γ- elimination and γ-replacement. The significance of γ-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration γ-replacement in the presence of L-methionine at 37°C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine γ-lyase activity.

Original languageEnglish
Pages (from-to)233-240
Number of pages8
JournalAnalytical Biochemistry
Volume327
Issue number2
DOIs
Publication statusPublished - Apr 15 2004

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Keywords

  • Enzyme activity
  • Enzyme kinetics
  • L-Methionine γ-lyase
  • Progress curve analysis
  • Pyridoxal 5 -phosphate

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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