A comparative study of Drosophila phenylalanine hydroxylase with a natural and a synthetic tetrahydropterin as cofactor.
1. Phenylalanine hydroxylase activity has been analyzed in Drosophila melanogaster using as cofactors the natural tetrahydropteridine 5,6,7,8-tetrahydrobiopterin (H4Bip) and the synthetic one 5,6-dimethyl-5,6,7,8-tetrahydropterin (H4Dmp). 2. The apparent Vmax and KM for substrate and cofactor showed that the enzyme has two times more affinity for the substrate when H4Bip is the cofactor in the reaction. Similarly to what was found with purified rat liver phenylalanine hydroxylase, H4Bip was the most effective cofactor, leading to 4-5 times more activity than that obtained with H4Dmp. 3. With the natural cofactor H4Bip, no activation of the enzyme with Phe was necessary (in contrast to mammalian phenylalanine hydroxylase), and this tetrahydropteridine inhibits phenylalanine hydroxylase activity when the enzyme is exposed to it before phenylalanine addition. With the synthetic H4Dmp, both types of preincubations led to an increase of phenylalanine hydroxylase activity. 4. The enzyme is highly unstable compared to mammalian phenylalanine hydroxylase, even at -20 degrees C. 5. Thorax and abdomen extracts caused significant inhibition of phenylalanine hydroxylase activity from third instar larvae or newborn adult head extracts, when assayed with the synthetic cofactor H4Dmp. This inhibition did not happen with H4Bip. The presence of the pteridine 7-xanthopterin in adult bodies was not the cause of this inhibition.
Bel
Y
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Departamento de Genética, Facultad de Biología, Universitat de València, Spain.
Jacobson
K B
KB
Ferré
J
J
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology*; D007700:Kinetics; D010651:Phenylalanine Hydroxylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011621:Pteridines / Q000138:chemical synthesis / Q000378:metabolism; D011622:Pterins; D013379:Substrate Specificity
D003067:Coenzymes; D011621:Pteridines; D011622:Pterins; D001708:Biopterins; C078628:5,6-dimethyl-5,6,7,8-tetrahydropterin; D010651:Phenylalanine Hydroxylase; C003402:sapropterin