7-8-dihydrobiopterin and tetrahydropterin

The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 can support NO synthesis. Here, we characterize pterin-free inducible NOS (iNOS) and iNOS reconstituted with eight different tetrahydro- or dihydropterins to elucidate how changes in pterin side-chain structure and ring oxidation state regulate iNOS. Seven different enzyme properties that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxidation state (i.e., they required fully reduced tetrahydropterins) and were independent of side chain structure: NO synthesis and the ability to increase heme-dependent NADPH oxidation in response to substrates. In contrast, five properties were exclusively dependent on pterin side-chain structure or stereochemistry and were independent of pterin oxidation state: pterin binding affinity, and its ability to shift the heme iron to its high-spin state, stabilize the ferrous heme iron coordination structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer. These results clarify how structural versus redox properties of the pterin impact on its multifaceted role in iNOS function. In addition, the data reveal that during NO synthesis all pterin-dependent steps up to and including heme iron reduction can take place independent of the pterin ring oxidation state, indicating that the requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.

Topics: Animals; Arginine; Biopterins; Catalysis; Dimerization; Electron Transport; Enzyme Induction; Heme; Iron; Kinetics; Mice; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidation-Reduction; Pterins; Spectrophotometry

The cofactor product of the aromatic amino acid hydroxylases, 4a-hydroxy-6(R)-tetrahydrobiopterin, requires dehydration before tetrahydrobiopterin can be regenerated by dihydropteridine reductase. Carbinolamine dehydration occurs nonenzymatically, but the reaction is also catalyzed by 4a-hydroxytetrahydropterin dehydratase. This enzyme has the identical amino acid sequence to DCoH, the dimerization cofactor of the transcription regulator, HNF-1 alpha. The catalytic activity of rat liver dehydratase was characterized using a new assay employing chemically synthesized 4a-hydroxytetrahydropterins. The enzyme shows little sensitivity to the structure or configuration of the 6-substituent of its substrate, with Km's for 6(S)-methyl, 6(R)-methyl, 6(S)-propyl, and 6(R)-L-erythro-dihydroxypropyl all between 1.5 and 6 microM. Turnover numbers at 37 degrees C are 50-90 s-1 at pH 7.4 and 2.5-3-fold lower at pH 8.4. Both 4a(R)- and 4a(S)-hydroxytetrahydropterins are good substrates. The quinoid dihydropterin products are strong inhibitors of the dehydratase with KI's about one half of their respective Km's, but no inhibition was observed with 7,8-dihydropterins or tetrahydropterins. The enzyme contains no metals and no phosphorus. Reaction mechanisms which involve either acid and/or base catalysis are discussed. 4a-Hydroxy-6(R)-tetrahydrobiopterin was determined not to be a product inhibitor of phenylalanine hydroxylase. It is concluded that the dehydratase (which was found to be 6 microM in rat liver) is essential in vivo to prevent rearrangement of 4a-hydroxy-6(R)-tetrahydrobiopterin and to maintain the supply of tetrahydrobiopterin cofactor for the hydroxylases under conditions where the nonenzymatic rate would be inadequate.

Topics: Animals; Biopterins; Catalysis; Cattle; Humans; Hydro-Lyases; Hydrogen-Ion Concentration; Kinetics; Metals; NAD; Osmolar Concentration; Phenylalanine Hydroxylase; Phosphorus; Pterins; Rats; Substrate Specificity; Thermodynamics

The substrate specificity of dihydrofolate reductase from cells of different origin has been thought to be quite narrow, and unconjugated dihydropterins such as 6-methyl-dihydropterin are known to be very poor substrates. We have reinvestigated the substrate specificity of several dihydropterins and, in addition, have observed that in a new series of unconjugated dihydropterins of the general structure 6-CH2O(CH2)nCH3 several compounds are excellent substrates for the bovine liver enzyme, but none of them bind as well as dihydrofolate. The substrate activity (apparent Vmax) of these compounds increases from 17 to 110% that of the natural substrate, dihydrofolate, as n is increased from 0 to 3. In contrast, these unconjugated dihydropterins are very poor substrates for the Escherichia coli enzyme.

Topics: Animals; Biopterins; Cattle; Folic Acid; Kinetics; Liver; Pteridines; Pterins; Substrate Specificity; Tetrahydrofolate Dehydrogenase