title: The role of cysteine 116 in the active site of the antitumor enzyme L-methionine gamma-lyase from Pseudomonas putida.
abstract: The cysteinyl residue at the active site of L-methionine gamma-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k(cat) toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the gamma-elimination reaction of L-methionine and in substrate recognition in the MGLs.
authors.affiliation: Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.
authors.forename: Daizou
authors.initials: D
authors.lastname: Kudou
authors.forename: Shintaro
authors.initials: S
authors.lastname: Misaki
authors.forename: Masao
authors.initials: M
authors.lastname: Yamashita
authors.forename: Takashi
authors.initials: T
authors.lastname: Tamura
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Esaki
authors.forename: Kenji
authors.initials: K
authors.lastname: Inagaki
mesh_terms: D000970:Antineoplastic Agents; D001665:Binding Sites; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003545:Cysteine; D008715:Methionine; D016297:Mutagenesis, Site-Directed; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D008715:Methionine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase; D003545:Cysteine