Analysis of the role of the active site residue Arg98 in the flavoprotein tryptophan 2-monooxygenase, a member of the L-amino oxidase family.
The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. We have previously identified tryptophan 2-monooxygenase as a homologue of L-amino acid oxidase [Sobrado, P., and Fitzpatrick, P. F. (2002) Arch. Biochem. Biophys. 402, 24-30]. On the basis of the sequence comparisons of the different LAAO family members, Arg98 of tryptophan 2-monooxygenase can be identified as an active site residue which interacts with the carboxylate of the amino acid substrate. The catalytic properties of R98K and R98A tryptophan 2-monooxygenase have been characterized to evaluate the role of this residue. Mutation of Arg98 to lysine decreases the first-order rate constant for flavin reduction by 180-fold and the second-order rate constant for flavin oxidation by 26-fold, has no significant effect on the K(d) value for tryptophan or the K(i) value for the competitive inhibitor indoleacetamide, and increases the K(i) value for indolepyruvate less than 2-fold. Mutation of this residue to alanine decreases the rate constants for reduction and oxidation an additional 5- and 2-fold, respectively, and increases the K(d) value for tryptophan and the K(i) value for indolepyruvate by 31- and 17-fold, respectively, while having an only 2-fold effect on the K(i) value for indoleacetamide. Both mutations increase the value of the primary deuterium isotope effect with tryptophan as a substrate, consistent with a later transition state. Both mutant enzymes catalyze a simple oxidase reaction, producing indolepyruvate and hydrogen peroxide. The pH dependences of the V/K(trp) values for the mutant enzymes show that the anionic form of the substrate is preferred but that the zwitterionic form is a substrate. The results are consistent with the interaction between Arg98 and the carboxylate of the amino acid substrate being critical for correct positioning of the substrate in the active site for efficient catalysis.
Sobrado
Pablo
P
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Fitzpatrick
Paul F
PF
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000693:Anaerobiosis; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050830:L-Amino Acid Oxidase; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D010084:Oxidation-Reduction; D014364:Tryptophan / Q000737:chemistry; D014757:Viper Venoms / Q000201:enzymology* / Q000235:genetics
D005420:Flavoproteins; D014757:Viper Venoms; D014364:Tryptophan; D001120:Arginine; D006899:Mixed Function Oxygenases; C023204:tryptophan 2-monooxygenase; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase