PMC

Superoxide release occurs in the absence of BH₄ and is increased by L-arginine (L-Arg) upon activation of the resting state enzyme by calcium/calmodulin. In the absence or presence of L-Arg, BH₄ inhibits superoxide release. Together with L-arg BH₄ dose-dependently increases NO and citrulline production.

Superoxide production from BH₄ free-eNOS was detected with EMPO spin trap upon activation of the enzyme with calcium-calmodulin. Incubation of the enzyme with L-arginine prior to activation increases superoxide yield, while L-NAME at 10-fold higher concentration than L-arginine only decreases superoxide yield by ~25%. BH₄ dose dependently inhibits superoxide release. Both BH₄ and L-arginine decrease superoxide to stimulate NO production as confirmed by L-citrulline production.

(Solid arrow) de novo synthesis pathway involving GTPCH, GTP cyclohydrolase 1, PTPS, 6-pyruvoyl tetrahydropterin synthase, AR, aldose reductase, SR, sepiaperin reductase, CR, carbonyl reductase. (Broken arrow) Salvage pathway of tetrahydrobiopterin via SR, sepiapterin reductase and DHFR, dihydrofolate reductase.

(A) The deprotonated carbon-centered radical (^•BH₄) reacts with oxygen to generate a BH₄ peroxyl radical (BH₄OO^•). This radical species reacts with another BH₄ in the propagation reaction to generate the corresponding peroxide (BH₄OOH), which decomposes into qBH₂ and hydrogen peroxide. (B) General base-catalyzed rearrangement of qBH₂ to generate 7,8-BH₂ in neutral solution. (C) NON-enzymatic conversion of qBH₂ to 7,8-dihydropterin (7,8-PH₂) and lactoyl aldehyde (RCHO).

(Upper left) L-Biopterin is produced from BH₄ oxidative metabolism. Only the pterin ring is oxidized, the side chain hydroxyl groups remain in cis-conformation as predicted to be the favorable conformation in solution. (Upper right) D-neopterin is a byproduct in BH₄ synthesis that also presents hydroxyl groups in cis-position. Both biopterin and neopterin are found in biological fluids and their concentration ratios are used as markers in the diagnosis of BH₄ metabolic disorders. (Lower left) Oxidative degradation of BH₄ by biological oxidants (peroxidase/H₂O₂, peroxynitrite, heme proteins) s convert BH₄ into 7,8-dihydro-L-biopterin (7,8-BH₂), 7,8-dihydropterin (7,8-PH₂) and dihydroxanthopterin. Dihydrofolate reductase (DHFR) can reduce 7,8-BH₂ back to BH₄. (Lower righe) BH₄ cofactor activity in the hydroxylation reactions catalyzed by tyrosine and phenylalanine hydrolase (TH, PAH respectively). Upon binding to enzymes BH₄ side chain hydroxyl groups re-orient in a trans-like conformation presumably to increase binding forces. The BH₄ oxidation product, pterin-4a-carbinolamine, generates quinonoid BH₂ (q BH₂) by spontaneous or catalyzed dehydration by pterin carbinolamine dehydratase (PCD) also known as dimerization cofactor of hepatocyte nuclear factor 1 alpha (DCoH). The qBH₂ is either reduced back to BH₄ by NADH-dependent enzyme dihydropterin reductase (DHPR) or rearranges to form 7,8-BH₂.