Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities.
NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Schmidt
H H
HH
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
Smith
R M
RM
Nakane
M
M
Murad
F
F
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001708:Biopterins / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D002531:Cerebellum / Q000201:enzymology; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry*; D004093:Dihydropteridine Reductase / Q000737:chemistry*; D005415:Flavins / Q000378:metabolism*; D009249:NADP / Q000737:chemistry; D019001:Nitric Oxide Synthase; D051381:Rats; D011919:Rats, Inbred Strains
D002147:Calmodulin; D005415:Flavins; D001708:Biopterins; D009249:NADP; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D004093:Dihydropteridine Reductase; D008058:Dihydrolipoamide Dehydrogenase