Anti-pterins as Tools to Characterize the Function of Tetrahydrobiopterin in NO Synthase

Abstract

Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convertl-arginine to nitric oxide andl-citrulline. Interestingly, all NOS also require (6<i>R</i>)-5,6,7,8-tetrahydro-l-biopterin (H₄Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, <i>i.e.</i> that in the absence of exogenous H₄Bip, has been attributed to enzyme-associated H₄Bip. To elucidate further H₄Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors, termed anti-pterins. In contrast to type II anti-pterins, type I anti-pterins specifically displaced enzyme-associated H₄Bip and inhibited H₄Bip-stimulated NOS activity in a fully competitive manner but, surprisingly, had no effect on basal NOS activity. Moreover, for a number of different NOS preparations basal activity (percent of<i>V</i> _max) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H₄Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H₄Bip. The lack of intrinsic 4<i>a</i>-pterincarbinolamine dehydratase activity argued against classical H₄Bip redox cycling in NOS. Rather, H₄Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation duringl-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.