Abstract
The first reaction is catalyzed by phenylalanine hydroxylase and the second reaction, which generates the reduced form of pteridine cofactor (biopterin), is catalyzed by dihydropteridine reductase (l-3). A direct assay of phenylalanine hydroxylase can be achieved by supplying optimal concentrations of reduced pteridine cofactor or an analog of the reduced cofactor (6,7-dimethyl-5,6,7,8-tetrahydropterine) maintained in its reduced state by the addition of NADH or NADPH (4). The assay avoids the need for an accessory NADPH enzyme-generating system and ensures that the only rate-determining component in the hydroxylating reaction is the concentration of phenylalanine hydroxylase. A typical assay for phenylalanine hydroxylase is given in Table 1. The control flasks contain all of the reagents except L-phenylalanine. Incubations are carried out at 37” in a 1Oml Erlenmeyer flask in a Dubnoff shaker, in air, and the reaction is started by
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