Structure and mechanism of tryptophan indole-lyase and tyrosine phenol-lyase.
Tyrosine phenol-lyase (TPL) and tryptophan indole-lyase (Trpase) catalyse the reversible hydrolytic cleavage of L-tyrosine or L-tryptophan to phenol or indole, respectively, and ammonium pyruvate. These enzymes are very similar in sequence and structure, but show strict specificity for their respective physiological substrates. We have mutated the active site residues of TPL (Thr(124), Arg(381), and Phe(448)) to those of Trpase and evaluated the effects of the mutations. Tyr(71) in Citrobacter freundii TPL, and Tyr(74) in E. coli Trpase, are essential for activity with both substrates. Mutation of Arg(381) of TPL to Ala, Ile, or Val (the corresponding residues in the active site of Trpase) results in a dramatic decrease in L-Tyr beta-elimination activity, with little effect on the activity of other substrates. Arg(381) may be the catalytic base with pK(a) of 8 seen in pH-dependent kinetic studies. T124D TPL has no measureable activity with L-Tyr or 3-F-L-Tyr as substrate, despite having high activity with SOPC. T124A TPL has very low but detectable activity, which is about 500-fold less than wild-type TPL, with L-Tyr and 3-F-L-Tyr. F448H TPL also has very low activity with L-Tyr. None of the mutant TPLs has any detectable activity with L-Trp as substrate. H463F Trpase also exhibits low activity with L-Trp, but retains high activity with other substrates. Thus, additional residues remote from the active site may be needed for substrate specificity. Both Trpase and TPL may react by a rare S(E)2-type mechanism.
Phillips
Robert S
RS
Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA. rsphillips@chem.uga.edu
Demidkina
Tatyana V
TV
Faleev
Nicolai G
NG
D001665:Binding Sites; D006860:Hydrogen Bonding; D007700:Kinetics; D013329:Structure-Activity Relationship; D014368:Tryptophanase / Q000737:chemistry* / Q000378:metabolism; D014447:Tyrosine Phenol-Lyase / Q000737:chemistry* / Q000378:metabolism
D014368:Tryptophanase; D014447:Tyrosine Phenol-Lyase