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Calcium Independent process
iNOS, is unlike other isoforms of NOS. The mechanism involves a calcium independent process. [4]
To understand the Ca independent process, we need to investigate the function and binding mode of the CaM cofactors.


FIG. 8 A docking model constructed manually showing possible interactions between CaM complexed with a helical peptide from human eNOS (1N1W) and the rat nNOS reductase domain (1TLL).
The human eNOS peptide, R492 to G511, is bound to CaM in an antiparallel manner. The linker helix of CaM collapses in the middle so that its N-terminal lobe interacts with the peptide C-terminus while its C-terminal lobe interacts with the peptide N-terminus. The core of the peptide forms an alpha-helix making extensive hydrophobic contacts with CaM. The helical wheel analysis of the CaM binding peptides of different NOS isoforms indicated that in addition to the four hydrophobic residues of the classical ‘‘1-5-8-14’’ motif that contributed to the observed CaM binding in eNOS, iNOS has higher number of hydrophobic residues and a unique distribution along the peptide of hydrophobic and polar/charged residues. This may be the basis for the much higher affinity of CaM to iNOS and why the synthetic CaM binding peptide of iNOS binds to apo-CaM in the absence of Ca2+[4].
Kinetic perspective
The mechanism of the NOS is studied on three processes: rates of ferric heme reduction, ferric heme NO dissociation, and ferrous heme NO oxidation.  These have established a kinetic model that can explain the distinct catalytic behaviors of the three NOS isoforms.


FIG. 9 The kinetic model showing both the productive and futile enzyme re-generation cycles. The three reaction rates listed will determine how three NOS isoforms partition into the two regeneration cycles in distinctive manners.
 iNOS has comparable rates for both the productive and futile cycles, while the eNOS shows relative fast Fe(III)-NO dissociation rate and relative slow ferric heme reduction rate.
If we compare the iNOS and nNOS, because of a much faster Fe(II)–NO oxidation rate in iNOS compared to that for nNOS, the Fe(II)–NO buildup is only significant in nNOS. This makes iNOS more productive in the steady state than nNOS in generating NO or other nitrogen oxides even though the intrinsic (single turnover) activity of nNOS is higher than that of iNOS[4].