Analysis of the roles of amino acid residues in the flavoprotein tryptophan 2-monooxygenase modified by 2-oxo-3-pentynoate: characterization of His338, Cys339, and Cys511 mutant enzymes.
The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. His338, Cys339, and Cys511 of the Pseudomonas savastanoi enzyme were previously identified as possible active-site residues by modification with 2-oxo-3-pentynoate ([G. Gadda, L.J. Dangott, W.H. Johnson Jr., C.P. Whitman, P.F. Fitzpatrick, Biochemistry 38 (1999) 5822-5828]). The H338N, C339A, and C511S enzymes have been characterized to determine the roles of these residues in catalysis. The steady-state kinetic parameters with both tryptophan and methionine decrease only slightly in the case of the H338N and C339A enzymes; the decrease in activity is greater for the C511S enzyme. Only in the case of the C511S enzyme do deuterium kinetic isotope effects on kinetic parameters indicate a significant change in catalytic rates. The structural bases for the effects of the mutations can be interpreted by identification of L-amino acid oxidase and tryptophan monooxygenase as homologous proteins.
Sobrado
Pablo
P
Department of Biochemistry and Biophysics, Texas A & M University, College Station, TX 77843-2128, USA.
Fitzpatrick
Paul F
PF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D003553:Cystine / Q000378:metabolism*; D004926:Escherichia coli; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology*; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism
C109822:2-oxo-3-pentynoic acid; D005231:Fatty Acids, Unsaturated; D003553:Cystine; D006639:Histidine; D014364:Tryptophan; D008715:Methionine; D006899:Mixed Function Oxygenases; C023204:tryptophan 2-monooxygenase