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.
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)
Chen
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HY
Department of Chemistry, University of Georgia, Athens 30602-2556, USA.
Demidkina
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Phillips
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D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D016971:Citrobacter freundii / Q000201:enzymology*; D003545:Cysteine / Q000031:analogs & derivatives; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014447:Tyrosine Phenol-Lyase / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D011732:Pyridoxal Phosphate; D014447:Tyrosine Phenol-Lyase; D003545:Cysteine