Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine.
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.
Takakura
Tomoaki
T
Discovery Research Laboratories, Shionogi & Co., Ltd., 1-3, Kuise Terajima 2-chome, Amagasaki, Hyogo 660-0813, Japan. tomoaki.takakura@shionogi.co.jp
Mitsushima
Kenji
K
Yagi
Shigeo
S
Inagaki
Kenji
K
Tanaka
Hidehiko
H
Esaki
Nobuyoshi
N
Soda
Kenji
K
Takimoto
Akio
A
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
D000964:Antimetabolites, Antineoplastic; D002087:Butyrates; D011994:Recombinant Proteins; C005087:alpha-ketobutyric acid; D008715:Methionine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase