Autoinhibition of endothelial nitric-oxide synthase - Identification of anelectron transfer control element

The primary sequences of the three mammalian nitric- oxide synthase (NOS) i soforms differ by the insertion of a 52-55-amino acid loop into the reducta se domains of the endothelial (eNOS) and neuronal (nNOS), but not inducible (iNOS). On the basis of studies of peptide derivatives as inhibitors of (N O)-N-. formation and calmodulin (CaM) binding (Salerno, J. C., Harris, D. E ., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Rom an, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q. , and Gross, S. S. (1997) J. Biol, Chem. 272, 29769-29777), the insert has been proposed to be an autoinhibitory element. We have examined the role of the insert in its native protein context by deleting the insert from both wild-type eNOS and fi om chimeras obtained by swapping the reductase domain s of the three NOS isoforms. The Ca2+ concentrations required to activate t he enzymes decrease significantly when the insert is deleted, consistent wi th suppression of autoinhibition. Furthermore, removal of the insert greatl y enhances the maximal activity of mild-type eNOS, the least active of the three isoform. Despite the correlation between reductase and overall enzyma tic activity for the wild-type and chimeric NOS proteins, the loop-free eNO S still requires CaM to synthesize (NO)-N-.. However, the reductive activit y of the CaM-free, loop-deleted eNOS is enhanced significantly over that of CaM-free wild-type eNOS and approaches the same level as that of CaM-bound wild-type eNOS. Thus, the inhibitory effect of the loop on both the eNOS r eductase and (NO)-N-.-synthesizing activities may have an origin distinct f rom the loop's inhibitory effects on the binding of CaM and the concomitant activation of the reductase and (NO)-N-.-synthesizing activities. The eNOS insert not only inhibits activation of the enzyme by CaM but also contribu tes to the relatively low overall activity of this NOS isoform.