Identification of the active-site residue of .gamma.-cystathionase labeled by the suicide inactivator .beta.,.beta.,.beta.-trifluoroalanine

Abstract

Inactivation of gamma-cystathionase by beta, beta, beta-trifluoroalanine, a suicide inactivator of the enzyme, results in covalent labeling of an amino group of the protein [Silverman, R. B., & Abeles, R. H. (1977) Biochemistry 16, 5515-5520]. We have established that this modified amino function is the epsilon-NH2 group of a lysine residue. A heptapeptide which includes this modified lysine residue was isolated, and its sequence was found to be Cys-Ser-Ala-Thr-Lys-Tyr-Met. The amino acid sequence was the same as that determined for peptides containing the active-site lysine residue which forms a Schiff base with pyridoxal phosphate. Therefore the epsilon-NH2 group of the active-site lysine which binds pyridoxal phosphate is capable of interacting with the beta carbon of trifluoroalanine, and presumably the beta carbon of normal substrates. We therefore propose that this lysine residue may function as a proton-transfer agent in the reactions catalyzed by gamma-cystathionase.

Crystal Structure of the Pyridoxal-5′-phosphate Dependent Cystathionine β-lyase fromEscherichia coliat 1.83 Å

“…To calculate an initial (F o -F c ) electron density map, the coordinates of the CBL structure were used, from which the bicarbonate and the active-site waters had been removed. Clear, continuous density between the cofactor and the Lys210 was observed in this map, indicating a covalent lysine-inactivator-PLP product, as proposed by Silverman & Abeles (1977) and Fearon et al (1982). Part of the omit map, in which the inhibitor was omitted from the final refined structure, is shown in Figure 12(a) and revealed well-defined electron density for the bound inhibitor.…”

Crystal structure of Escherichia coli cystathionine γ-synthase at 1.5 Å resolution

“…In the E.coli CGS, PLP is covalently linked to Lys198 (Martel et al ., 1987) and gives rise to a strong absorption of the holoenzyme at 422 nm and a weaker absorption at 325 nm, originating from the ketoenamine and enolimine forms of the Schiff base, respectively. The sequences of the enzymes from different kingdoms are ∼30% identical, and the highest homologies are found for the ∼12 residues comprising the consensus PLP‐binding site (Fearon et al ., 1982).…”

Specific labeling of the essential cysteine residue of L-methionine .gamma.-lyase with a cofactor analog, N-(bromoacetyl)pyridoxamine phosphate

“…40 45 Leu-His-His-Gl~-Ile-Gly-Glu-Phf-Glu-Phe-Gly-~'ai-Lys-Leu-Arg-His-~al-Asp-Hse (Duchange et al, 1983), and cystathionine y-lyase of rat liver (Fearon et al, 1982). K* indicates the lysine residue to which pyridoxal-P This suggests that the same cysteine residue was modified with NTCB and BAPMP.…”