Removal of a putative inhibitory element reduces the calcium-dependent calmodulin activation of neuronal nitric-oxide synthase.
Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.
Montgomery
H J
HJ
Department of Chemistry and the Guelph Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Romanov
V
V
Guillemette
J G
JG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005486:Flavin Mononucleotide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D017931:DNA Primers; D004791:Enzyme Inhibitors; D005486:Flavin Mononucleotide; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D002118:Calcium