Role of tyrosine 114 of L-methionine gamma-lyase from Pseudomonas putida

Biosci Biotechnol Biochem. 2000 Nov;64(11):2336-43. doi: 10.1271/bbb.64.2336.

Abstract

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.

MeSH terms

  • Carbon-Sulfur Lyases / chemistry
  • Carbon-Sulfur Lyases / genetics
  • Carbon-Sulfur Lyases / metabolism*
  • Catalysis
  • Deamination
  • Deuterium Oxide / metabolism
  • Hydrogen / metabolism
  • Magnetic Resonance Spectroscopy
  • Mutagenesis, Site-Directed
  • Phenylalanine / genetics
  • Phenylalanine / metabolism
  • Pseudomonas putida / enzymology*
  • Pseudomonas putida / genetics
  • Tyrosine / genetics
  • Tyrosine / metabolism*

Substances

  • Tyrosine
  • Phenylalanine
  • Hydrogen
  • Carbon-Sulfur Lyases
  • L-methionine gamma-lyase
  • Deuterium Oxide