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

Abstract

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.

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

“…The pterinbased analogue 2-amino-4,6-dioxo-3,4,5,6,8,8a,9, 10-octahydrooxazolo[1,2-f]-pteridine (PHS-32) was synthesized as described [17]. All other chemicals, reagents and solvents were of the highest purity available and were from Merck AG (Darmstadt, Germany), Sigma Chemicals (Deisenhofen, Germany) or vasopharm BIOTECH (Wu$ rzburg, Germany).…”

“…Sf 9 cells were transfected with recombinant human NOS-I and the resulting enzyme was purified by 2h,5h-ADP-Sepharose and CaM-Sepharose affinity chromatography [17,41]. The yield of this purification method was 25-35 mg of protein with a specific activity of up to 303 nmol of -citrulline\min per mg. Purified NOS-I was stored at k80 mC in 50 µl aliquots containing 10 % (v\v) glycerol until the day of use.…”

“…Native enzyme was purified from pig cerebellum with previously described methods [17,20,45] by DEAE ion-exchange chromatography and 2h,5h-ADP-Sepharose affinity chromatography (yield of 1.0-1.4 mg of protein from 1 kg of tissue, with a specific activity of up to 463 nmol of -citrulline\min per mg). The amount of enzyme-bound H % Bip was determined by reversephase HPLC coupled with fluorimetric detection (λ ex l 352 nm, λ em l 438 nm), with authentic reagent H % Bip as a standard [17,25,42]. Native NOS-I (0.8-1.0 µg) was incubated in 50 mM Tris\HCl buffer, pH 7.2, containing 1 mM CaCl # , 10 µM FAD, 5 µM FMN, 250 µM CHAPS, 1 mM NADPH for 15 min at either 4 or 37 mC.…”

“…Interestingly, the stabilizing effects of H % Bip might be isoform-dependent. For NOS-I, H % Bip is required for dimer stabilization [17,24,25] but not for subunit assembly [26,27] ; for NOS-II and III, the current evidence that supports a role for H % Bip in subunit assembly is inconsistent [23,[28][29][30][31][32][33] even when the enzymes have been solved at the crystal level [16,34].…”

“…as an oxygen acceptor and electron donor catalysing the initial N-hydroxylation of -arginine. However, the current consensus in the literature does not support such a classical catalytic role [13][14][15][16][17], although some studies can be found to the contrary [18]. Alternatively, protective and stabilizing effects of H % Bip have been described, including (1) the stabilization of the NOS mRNA product [19], (2) protection of the NOS catalytic centre from oxidative damage [16,20], (3) prevention of the formation of superoxide (O # − d) [21] and H # O # [22] by coupling reductive oxygen activation to -arginine oxidation, and (4) promotion of subunit assembly from monomers and stabilization of dimers in the presence of chemical denaturants [23,24] or under native conditions during -arginine turnover [17,25].…”