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 Tur71 in TPL by phenylalanine results in a mutant Y71F TPL with no detectable activity (greater than 3 x 10(5)-fold reduction) for (b)eta-elimination of L-tyrosine. Y71F TPL can react with S-alkylcysteines, but these substrates exhibit k(cat) values reduced by 10(3)-10(4)-fold, while the k(cat)/K-m 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 k(cat), 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-H-2]-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 or-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 a-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 mu M for wild-type TPL. Thus, the mutation of Tyr71 to phenylalanine results in a 1700-fold increase in the K-D for PLP. This difference in PLP binding constants corresponds to a Delta Delta G value of 4.4 kcal/mol at 25 degrees C. Tyr70 in aspartate aminotransferase, which exhibits a similar three-dimensional structure, forms a hydrogen bond with the pyridoxal 5'-phosphate (PLP) phosphate oxygen OP2 [Smith, D., Almo, S., Toney, M., & Ringe, D. (1989) Biochemistry 28, 8161-8167] and is responsible for cofactor binding, but is not essential for catalysis [Toney, M. D., & Kirsch, J. F. (1987) J. Biol. Chern. 262, 12403]. Therefore, in contrast to Tyr70 in aspartate aminotransferase, Tyr71 in Citrobacter freundii TPL plays a dual role, both in cofactor binding in the absence of substrate and also as a general acid catalyst in the elimination of leaving groups from quinonoid intermediates.