Role of tyrosine 114 of L-methionine gamma-lyase from Pseudomonas putida.
L-Methionine gamma-lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of y-family pyridoxal 5'-phosphate dependent enzymes. A mutant form of L-methionine y-lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in kcat for alpha,gamma-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 gamma-position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of kcat for alpha,beta-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 gamma-elimination process for L-methionine is the rate-limiting determination step in alpha,gamma-elimination overall reaction of the Y114F mutant. These results indicate that Tyr114 of L-methionine gamma-lyase is important in y-elimination of the substrate.
Inoue
H
H
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Japan.
Inagaki
K
K
Adachi
N
N
Tamura
T
T
Esaki
N
N
Soda
K
K
Tanaka
H
H
D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003641:Deamination; D017666:Deuterium Oxide / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D014443:Tyrosine; D010649:Phenylalanine; D006859:Hydrogen; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase; D017666:Deuterium Oxide