Identification of residues critical for enzymatic activity in the domain encoded by exons 8 and 9 of the human inducible nitric oxide synthase

Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has bee n implicated in the pathogenesis of several diseases including airway infla mmation of asthma. iNOS is active only as a homodimer. We previously demons trated that the region encoded by exons 8 and 9 is critical for dimerizatio n. In this study, alanine-scanning mutagenesis was used to identify critica l amino acids in that region by expression of mutant proteins in human embr yonic kidney 293 cells. All iNOS mutants yielded iNOS protein as detected b y Western analysis. Four iNOS mutants with alanine replacing Trp(260), Asn( 261), Tyr(267), or Asp(280) did not generate NO. Dimer formation was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 4 degreesC , followed by immunoblotting. Wild-type iNOS migrated both as monomers and dimers. iNOS mutants with alanine replacing Trp(260), Asn(261) Or Tyr(267) however, migrated only as monomers, suggesting that their inability to prod uce NO is related to a defect in dimer formation. Interestingly, the Asp(28 0) mutant retained the ability to dimerize, indicating that it represents a n inactive form of an iNOS dimer. These data identify four amino acids in e xons 8 and 9 critical for iNOS activity, three of which also influence dime rization. These residues are strictly conserved among all NOS isforms and a cross species. Thus all NOS isoforms share general structural similarities, including specific amino acids critical for dimerization and catalytic act ivity. These data increase our understanding of the structural elements cri tical for NO synthesis and lay the groundwork for future studies aimed at d ownregulation of iNOS activity.