Threonine-124 and phenylalanine-448 in Citrobacter freundii tyrosine phenol-lyase are necessary for activity with l-tyrosine

Abstract

Thr-124 and Phe-448 are located in the active site of Citrobacter freundii tyrosine phenol-lyase (TPL) near the phenol ring of a bound substrate analogue, 3-(4′-hydroxyphenyl)propionic acid [Sundararaju, Antson, Phillips, Demidkina, Barbolina, Gollnick, Dodson and Wilson (1997) Biochemistry 36, 6502–6510]. Thr-124 is replaced by Asp and Phe-448 is replaced by His in the crystal structure of a structurally similar enzyme, Proteus vulgaris tryptophan indole-lyase, which has 50% identical residues. Hence, Thr-124 and Phe-448 in TPL were mutated to Ala or Asp, and His, respectively, in order to probe the role of these residues in the reaction specificity for l-Tyr. These mutant enzymes have little or no β-elimination activity with l-Tyr or 3-fluoro-l-Tyr as a substrate, but retain significant elimination activity with S-(o-nitrophenyl)-l-cysteine, S-alkyl-l-cysteines and β-chloroalanine. Furthermore, the binding of l-Tyr and other non-substrate amino acids is not significantly affected by the mutations. The mutant TPLs form intermediates in rapid-scanning stopped-flow experiments with l-Phe, l-Tyr and l-Trp, similar to those seen with wild-type TPL. These results demonstrate that Thr-124 and Phe-448 are necessary for the reaction specificity of TPL for l-Tyr, and probably play a role in the elimination stage of the reaction mechanism. Thr-124 is within hydrogen-bonding distance of the phenolic group of the bound substrate, and may help to orientate the ring for β-elimination to occur. Phe-448 may be important to allow the formation of the closed conformation during the reaction.