Anti-pterins as tools to characterize the function of tetrahydrobiopterin in NO synthase

Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H(4)Bip, has been attributed to enzyme-associated H(4)Bip. To elucidate further H(4)Bip 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 H(4)Bip and inhibited H(4)Bip-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 V-max) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H(4)Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H(4)Bip. The lack of intrinsic 4a-pterincarbinolamine dehydratase activity argued against classical H(4)Bip redox cycling in NOS. Rather, H(4)Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation during L-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.