IDENTIFICATION, CHARACTERIZATION, AND COMPARISON OF THE CALMODULIN-BINDING DOMAINS OF THE ENDOTHELIAL AND INDUCIBLE NITRIC-OXIDE SYNTHASES

The calmodulin (CaM)-binding regions in bovine en dothelial nitric oxi de synthase (eNOS) and murine inducible nitric oxide synthase (iNOS) a re identified in this study as eNOS residues 493-512 and iNOS residues 501-532. Peptides corresponding to eNOS 493-512 and iNOS 501-532 prod uce a Ca2+-dependent, electrophoretic mobility shift of CaM on 4 m ure a gels. The two peptides are also potent inhibitors of the CaM mediate d activation of neuronal nitric oxide synthase and have dissociation c onstants for CaM binding of 4.0 and 1.5 nM, respectively. Substitution of eNOS and iNOS CaM-binding domains in eNOS/iNOS chimeric proteins p roduces major alterations in the Ca2+ and CaM dependence of the intact enzymes expressed and purified from a baculovirus/Sf9 insect cell sys tem. Replacement of aligned NCS sequence with eNOS 493-512 creates a f unctional, chimeric iNOS that is both Ca2+- and CaM dependent. Replace ment of aligned eNOS sequence with iNOS 501-532 creates a functional, chimeric eNOS that is CaM-independent but that remains Ca2+-dependent. Specific amino acid residues critical for CaM binding by eNOS are als o identified in this study as Phe-498, Lys-499, and Leu-511 in the bov ine eNOS sequence.