The crucial roles of Asp-314 and Thr-315 in the catalytic activation of molecular oxygen by neuronal nitric-oxide synthase. A site-directed mutagenesis study.
Nitric-oxide synthase (NOS) is a flavohemoprotein that has a cytochrome P450 (P450)-type heme active site and catalyzes the monooxygenation of L-Arg to NG-hydroxy-L-Arg (NHA) according to the normal P450-type reaction in the first step of NO synthesis. However, there is some controversy as to how the second step of the reaction, from NHA to NO and L-citrulline, occurs within the P450 domain of NOS. By referring to the heme active site of P450, it is conjectured that polar amino acid(s) such as Asp/Glu and Thr must be responsible for the activation of molecular oxygen in NOS. In this study, we have created Asp-314-->Ala and Thr-315-->Ala mutants of neuronal NOS, both of which had absorption maxima at 450 nm in the spectra of the CO-reduced complexes and studied NO formation rates and other kinetic parameters as well as the substrate binding affinity. The Asp-314-->Ala mutant totally abolished NO formation activity and markedly increased the rate of H2O2 formation by 20-fold compared with the wild type when L-Arg was used as the substrate. The NADPH oxidation and O2 consumption rates for the Asp-314-->Ala mutant were 60-65% smaller than for the wild type. The Thr-315-->Ala mutant, on the other hand, retained NO formation activity that was 23% higher than the wild type, but like the Asp-314-->Ala mutation, markedly increased the H2O2 formation rate. The NADPH oxidation and O2 consumption rates for the Thr-315-->Ala mutant were, respectively, 56 and 27% higher than for the wild type. When NHA was used as the substrate, similar values were obtained. Thus, we propose that Asp-314 is crucial for catalysis, perhaps through involvement in the stabilization of an oxygen-bound intermediate. An important role for Thr-315 in the catalysis is also suggested.
Sagami
I
I
Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
Shimizu
T
T
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D004330:Drosophila; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D013054:Spectrophotometry, Atomic; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism*
D013912:Threonine; D001224:Aspartic Acid; D019001:Nitric Oxide Synthase; D000409:Alanine; D010100:Oxygen