Pteridines, fractionation

Kaufman and his associates [73] reinvestigated the role of the two enzyme fractions in phenylalanine hydroxylation. The reaction was carried out in vitro in the presence of extensively purified rat and sheep liver enzymes. Two cofactors were necessary for the overall reaction a pteridine and NADPH. Dihydropteridine [74, 75] has been established as the cofactor of phenylalanine hydroxylase (see Fig. 3-21). 

The search for a specific hydroxylating enzyme was, till recently, fruitless because of technical difficulties. In addition, many early reports were later recognized as misleading. The finding, for instance, that a soluble rat-liver fraction could hydroxylate L-tryptophan at very high concentrations was confirmed. However, this was clearly demonstrated to be due to the presence of phenylalanine hydroxylase (EC. 1.14.3.1), a well-known enzyme which uses tetrahydrobiopterin, a reduced pteridine derivative, as a cofactor. Since the affinity of phenylalanine hydroxylase for tryptophan is very poor and since phenylketonuric patients who totally lack this enzyme seem to be able to synthesize normal amounts of 5-hydroxyindoles, it was concluded that this enzymatic system has no physiological relevance whatsoever in the biosynthesis of S-HT. 

The reaction catalyzed by the sheep liver enzyme resembles that catalyzed by the enzyme dihydrofolic reductase and the question can be raised about the possible identity of the two enzymes. At the moment the question cannot be answered unequivocally, although there are compelling reasons for concluding that they are distinct enzymes. Thus, preparations of the purified sheep liver enzyme have been obtained which have very low dihydrofolic reductase activity, although purified dihydrofolic reductase fractions still have high sheep liver enzyme activity (Kaufman, unpublished). Furthermore, if the dihydrofolic reductase and the sheep liver enzyme were actually the same enzyme, it would be an enzyme with very peculiar specificity requirements it would be specific for 7,8-dihydropteridines when the pteridine had a PABA-glutamate side chain in position 6, but would be specific for the S,6-dihydropteridine if the substituent at position 6 was a methyl group. In view of these considerations, it seems almost certain that the two enzymes are not the same. 

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