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)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Chen HY,Demidkina TV,Phillips RSdoi
10.1021/bi00038a023subject
Has Abstractpub_date
1995-09-26 00:00:00pages
12276-83issue
38eissn
0006-2960issn
1520-4995journal_volume
34pub_type
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