Site-Directed Mutagenesis of Tyrosine-71 to Phenylalanine in Citrobacter freundii Tyrosine Phenol-Lyase: Evidence for Dual Roles of Tyrosine-71 as a General Acid Catalyst in the Reaction Mechanism and in Cofactor Binding

Abstract

Tyr71 is an invariant residue in all known sequences of tyrosine phenol-lyase (TPL). The substitution of Tyr71 in TPL by phenylalanine results in a mutant Y71F TPL with no detectable activity (greater than 3 x 10(5)-fold reduction) for beta-elimination of L-tyrosine. Y71F TPL can react with S-alkylcysteines, but these substrates exhibit kcat values reduced by 10(3)-10(4)-fold, while the kcat/Km values are reduced by 10(2)-10(3)-fold, compared to wild-type TPL. However, for substrates with good leaving groups (S-(o-nitrophenyl)-L-cysteine,beta-chloro-L-alanine, and O-benzoyl-L-serine), Y71F TPL exhibits kcat values 1.85-7% those of wild-type TPL. Y71F TPL forms very stable quinonoid complexes with strong absorbance at 502 nm from L-phenylalanine, tyrosines (L-tyrosine, 3-fluoro-L-tyrosine, and [alpha-2H]-3-fluoro-L-tyrosine), and S-alkylcysteines (S-methyl-L-cysteine, S-ethyl-L-cysteine, and S-benzyl-L-cysteine). The time courses of the formation of quinonoid intermediates in these reactions are biphasic. The slow phase shows a dependence on concentration of PLP and is due to the cofactor binding steps, while the fast phase is due to the amino acid alpha-deprotonation and reprotonation steps. The rate constants for the fast phase of the reactions of Y71F TPL with L-phenylalanine and S-methylcysteine are similar to those for alpha-deprotonation or reprotonation steps in the reactions of wild-type TPL. The PLP binding constant of Y71F TPL is estimated to be 1 mM by spectrophotometric titration, compared to 0.6 microM for wild-type TPL.(ABSTRACT TRUNCATED AT 250 WORDS)

The Conserved Serine-Tyrosine Dipeptide in Leishmania donovani Hypoxanthine-guanine Phosphoribosyltransferase Is Essential for Catalytic Activity

“…This latter mechanism may be similar to that observed for the acid-catalyzed hydrolysis of glucose 1-phosphate (40). Precedence for Tyr hydroxyls participating as general acids in reaction mechanisms has been obtained for the ketosteroid isomerase of Pseudomonas testosteroni (41) and the tyrosine phenol-lyase of Citrobacter freundii (42). The role of a general acid in PRT catalysis has also been proposed for the highly conserved Lys 103 of OPRTs, since mutation of this residue in the S. typhimurium enzyme caused a 1000-fold decrease in the k cat value of the enzyme without affecting the K m values for either the forward or reverse reaction (39).…”

Exploration of the active site of Escherichia coli cystathionine γ‐synthase

“…4 The tyrosine at position 46 of eCGS was targeted for investigation in this study because it is conserved in many members of fold-type I and phenylalanine substitution variants of the corresponding residue in diverse enzymes, including Y56 of eCBL, Y70 of E. coli aspartate aminotransferase, Y121 of murine erythroid aminolevulinate synthase (meALAS), Y71 of Citrobacter freundii tyrosine phenol lyase and Y64 of Treponema denticola cystalysin, have been reported to impact the kinetic parameters of the target enzyme. 6,[13][14][15][16] The 20-fold decrease in k catR and 20 and 12-fold increases in K l-OSHS mR and K l-OSHS mE of eCGS-Y46F (Table I) of the cofactor, rather than a direct interaction with the substrate, as glycine does not possess a side chain. 15 Given that the hydrogen bond between the phosphate moiety of the cofactor and residue Y121 of meALAS, is conserved in eCBL and eCGS, the observed similar increases in the K m values of the corresponding Y56F and Y46F variants, may also reflect the absence of this link rather than a direct interaction with the substrate.…”

Crystals of Tryptophan Indole-Lyase and Tyrosine Phenol-Lyase Form Stable Quinonoid Complexes

“…Recent studies of E. coli H463F Trpase suggest that His-463 is important in Trp specificity; hence it might be the proposed catalytic base either directly or through a hydrogen-bonded network with other residues and/or active site water (33). Both TPL and Trpase have an active site Tyr residue, which is essential for the elimination of the physiological substrates and which may serve as a general acid to transfer a proton to the leaving group (10,33). Thus, to understand the molecular basis of the reaction specificity of these enzymes, it is critical to obtain three-dimensional structures of the quinonoid intermediates by x-ray crystallography.…”

“…Oxindolyl-L-alanine was found previously to inhibit Escherichia coli Trpase with a K i value of ϳ2.5-6 M (3, 16,18) and to form a prominent absorption band at 502 nm, demonstrating the predominant formation of a stable quinon-oid intermediate. The Y71F mutant TPL was found to be inactive for the elimination of L-Tyr, but it binds L-Tyr and L-Phe tightly and forms stable quinonoid complexes with very strong absorption peaks at 502 nm (10). Presently, there is only one structure reported of a quinonoid intermediate of a PLP-dependent enzyme (19), indicating difficulties in the isolation and predominant accumulation of this species.…”