title: Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine.
abstract: L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-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 alpha-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 alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-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 gamma-replacement in the presence of L-methionine at 37 degrees 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 gamma-lyase activity.
authors.affiliation: Discovery Research Laboratories, Shionogi & Co., Ltd., 1-3, Kuise Terajima 2-chome, Amagasaki, Hyogo 660-0813, Japan. tomoaki.takakura@shionogi.co.jp
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Takakura
authors.forename: Kenji
authors.initials: K
authors.lastname: Mitsushima
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yagi
authors.forename: Kenji
authors.initials: K
authors.lastname: Inagaki
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Tanaka
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Esaki
authors.forename: Kenji
authors.initials: K
authors.lastname: Soda
authors.forename: Akio
authors.initials: A
authors.lastname: Takimoto
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000032:analysis / Q000378:metabolism*; D002087:Butyrates / Q000032:analysis; D013437:Carbon-Sulfur Lyases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D018827:Carcinoma, Lewis Lung / Q000628:therapy; D007700:Kinetics; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D011511:Proteus / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000494:pharmacology
chemical_list: D000964:Antimetabolites, Antineoplastic; D002087:Butyrates; D011994:Recombinant Proteins; C005087:alpha-ketobutyric acid; D008715:Methionine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase