Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases.
The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.
Spratt
Donald E
DE
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada.
Taiakina
Valentina
V
Guillemette
J Guy
JG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002413:Cations, Divalent / Q000378:metabolism; D002614:Chelating Agents / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D051381:Rats
D002147:Calmodulin; D002413:Cations, Divalent; D002614:Chelating Agents; D010455:Peptides; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D002118:Calcium