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. 2002 May 1;363(Pt 3):745–752. doi: 10.1042/0264-6021:3630745

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

Tatyana V Demidkina 1, Maria V Barbolina 1, Nicolai G Faleev 1, Bakthavatsalam Sundararaju 1, Paul D Gollnick 1, Robert S Phillips 1
PMCID: PMC1222527  PMID: 11964175

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 beta-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 beta-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 beta-elimination to occur. Phe-448 may be important to allow the formation of the closed conformation during the reaction.

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Selected References

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