Two synthetic peptides corresponding to the proximal heme-binding domain and CD1 domain of human endothelial nitric-oxide synthase inhibit the oxygenase activity by interacting with CaM.
Human endothelial nitric-oxide synthase (eNOS) is a complex enzyme, requiring binding of calmodulin (CaM) for electron transfer. The prevailing view is that calcium-activated CaM binds eNOS at the canonical binding site located at residues 493-510, which induces a conformational change to facilitate electron transfer. Here we demonstrated that the CaM enhances the rate of electron transfer from NADPH to FAD on a truncated eNOS FAD subdomain (residues 682-1204) purified from baculovirus-infected Sf9 cells, suggesting more complicated regulatory mechanism of CaM on eNOS. Metabolically (35)S-labeled CaM overlay on fusion proteins spanning the entire linear sequence of eNOS revealed three positive (35)S-CaM binding fragments: sequence 66-205, sequence 460-592, and sequence 505-759. Synthetic peptides derived from these fragments are tested for their effects on CaM binding and eNOS catalytic activities. Peptides corresponding to the proximal heme-binding site (E1, residues 174-193) and the CD1 linker connecting FAD/FMN subdomains (E4, residues 729-757) bind CaM at both high Ca(2+) (Ca(2+)CaM) and low Ca(2+) (apoCaM) concentrations, whereas peptide of the canonical CaM-binding helix (E2, residues 493-510) binds only Ca(2+)CaM. All three peptides E1, E2 and E4 significantly inhibit oxygenase activity in a concentration-dependent manner, but only E2 effectively inhibits reductase activity. Concurrent experiments with human iNOS showed major differences in the CaM binding properties between eNOS and iNOS. The results suggest that multiple regions of eNOS might interact with CaM with differential Ca(2+) sensitivity in vivo. A possible mechanism in regulating eNOS activation and deactivation is proposed.
Chen
Pei-Feng
PF
Vascular Biology Research Center, Department of Internal Medicine, The University of Texas Health Science Center at Houston, TX, USA. pei-fengchen@sbcglobal.net
Wu
Kenneth K
KK
D000595:Amino Acid Sequence; D018949:Antigens, CD1 / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry* / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*
D018949:Antigens, CD1; D002147:Calmodulin; D017931:DNA Primers; D010455:Peptides; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D010105:Oxygenases; D052250:Nitric Oxide Synthase Type III