Threonine-124 and phenylalanine-448 in Citrobacter freundii tyrosine phenol-lyase are necessary for activity with L-tyrosine.
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.
Demidkina
Tatyana V
TV
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
Barbolina
Maria V
MV
Faleev
Nicolai G
NG
Sundararaju
Bakthavatsalam
B
Gollnick
Paul D
PD
Phillips
Robert S
RS
D000595:Amino Acid Sequence; D001665:Binding Sites; D016971:Citrobacter freundii / Q000201:enzymology*; D004926:Escherichia coli; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000502:physiology*; D011732:Pyridoxal Phosphate / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013912:Threonine / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*; D014447:Tyrosine Phenol-Lyase / Q000378:metabolism*
D013912:Threonine; D014443:Tyrosine; D010649:Phenylalanine; D011732:Pyridoxal Phosphate; D014447:Tyrosine Phenol-Lyase