The Role of Cysteine 116 in the Active Site of the Antitumor Enzyme L-Methionine γ-Lyase from Pseudomonas putida

Abstract

The cysteinyl residue at the active site of L-methionine γ-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 γ-elimination reaction of L-methionine and in substrate recognition in the MGLs.

• L-methionine γ-lyase
• pyridoxal 5′-phosphate
• saturation mutagenesis