Abstract:
:Inducible nitric oxide synthase (iNOS) catalyzes the NADPH-dependent formation of nitric oxide (NO) and citrulline from L-arginine and O2. In addition to serving as substrate, L-arginine alters the enzyme's heme iron spin equilibrium, increases its NADPH oxidation, and promotes assembly of active dimeric iNOS from inactive monomers. To understand what structural aspects of L-arginine are important for causing these effects, we have studied the interactions of iNOS with several L-arginine and guanidine analogs. Very few analogs supported NO synthesis even when bound to iNOS at saturating or near-saturating levels. In contrast, almost all analogs shifted the heme iron spin equilibrium and either increased or decreased NADPH oxidation by iNOS. The guanidine analogs displayed the same pattern of effects as their amino acid counterparts but exhibited a lower affinity except for analogs containing S-alkylisothiourea or aminoguanidine groups. Most analogs also promoted iNOS dimerization, with hydroxyguanidine and S-ethylisothiourea promoting more dimerization than L-arginine itself. Although the analog concentrations required to promote dimerization of monomers were somewhat higher than those required for binding to dimeric iNOS, they followed the same rank order. The degree of dimerization promoted by each analog did not correlate to its binding affinity, its causing a high- or low-spin shift in heme iron spin state, or to its increasing or decreasing NADPH oxidation. Together, we conclude that the enzyme's high degree of substrate specificity only applies to NO synthesis, in that a number of "inactive" structural analogs still bind to iNOS and affect its heme chemistry and structure in the absence of supporting NO synthesis. These latter affects are mediated through binding of the guanidinium portion of L-arginine and its analogs to a single site within iNOS and are relatively independent of the amino acid portion of the molecule.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Sennequier N,Stuehr DJdoi
10.1021/bi952844esubject
Has Abstractpub_date
1996-05-07 00:00:00pages
5883-92issue
18eissn
0006-2960issn
1520-4995pii
bi952844ejournal_volume
35pub_type
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