Erythro- 7,8- Dihydroneopterin triphosphate

Determination of GTP cyclohydrolase and D-erythro-7,8-dihydroneopterin triphosphate synthetase... 

FIGURE 40-2 The phenylalanine hydroxylase (PAH) pathway. Phenylketonuria usually is caused by a congenital deficiency of PAH (reaction 1), but it also can result from defects in the metabolism of biopterin, which is a cofactor for the hydroxylase. Enzymes (1) Phenylalanine hydroxylase (2) Dihydropteridine reductase (3) GTP cyclohydrolase (4) 6-pyruvoyltetrahydrobiopterin synthase. BH4, tetrahydrobiopterin DEDT, o-erythro-dihydroneopterin triphosphate QH2, dihydrobiopterin. 

The insolubilization of NAD and AMP and the uses of these supports have already been described, as has the use of hydrazide-agarose for immobilization. Other insolubilized nucleotide affinity columns have also been described. For example, Olsen [101] isolated galactosyltransferase from whey using a UDP-Sepharose affinity column. GTP coupled to a hydrazide Sepharose derivative was used to isolate D-erythro-dihydroneopterin triphosphate synthetase, the first enzyme for folate biosynthesis in Lactobacillus plantarum [102]. ATP-agarose has been used in the purification of heavy meromysin, elution being effected with ATP [103]. 

These oxidation reactions require oxygen (O2) and tetrahydrobiopterin as a cofactor. Thus, as shown in Scheme 13.39, 7,8-dihydroneopterin 3 -triphosphate (generated from guanosine triphosphate [GTP] as seen in Scheme 12.118) is converted to 6-pyruvoyl-5,6,7,8-tetrahydropterin by an elimination reaction and two keto-enol isomerizations. The process is catalyzed by the enzyme 6-pyruvoyltetra-hydropterin synthase (EC 4.2.3.12). Then, via an intermediate, written as an equilibrium between a-hydroxyketones (named dihydrosepiapterin) linked by a common enol, reduction to tetrahydrobiopterin is effected (in two separate steps) by 2 equivalents of NADPH used by the enzyme sepiapterin reductase (EC 1.1.1.153). Tetrahydrobiopterin is the cofactor involved in the National Institutes of Health (NIH) shift (cf. Chapter 6) pathway used by the iron-containing enzyme phenylalanine 4-monooxygenase (EC 1.14.16.1) to convert phenylalanine (Phe, F) to tyrosine (Tyr, Y) and is converted to (6i )-6-(L-erythro-l,2-dihydroxypropyl)-5,6,7,8-tetrahydro-4a-hydroxypterin in the process. 

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