Modulation by pterins of the phosphorylation and phenylalanine activation of phenylalanine 4-mono-oxygenase.
The interaction between phenylalanine 4-mono-oxygenase and analogues of the natural cofactor (6R)-tetrahydrobiopterin [(6R)-BH4] was studied. The rate of cyclic AMP-dependent phosphorylation of phenylalanine 4-mono-oxygenase was inhibited only by those pterins [(6R)-BH4, (6S)-BH4 and 7,8-dihydrobiopterin (BH2)] that were able to decrease the potency and efficiency of phenylalanine as an allosteric activator of the hydroxylase. Since BH2 lacks cofactor activity, this was not required to modulate either the phosphorylation or the phenylalanine-activation of the hydroxylase. Half-maximal inhibition of the phosphorylation was observed at 1.9 microM-(6R)-BH4, 9 microM-(6S)-BH4 and 17 microM-BH2. Competition experiments indicated that all three pterins acted through binding to the cofactor site of the hydroxylase. Since the phosphorylation site and the cofactor binding site are known to reside, respectively, in the N- and C-terminal domains of the hydroxylase, the pterins were able to induce an interdomain conformational change. BH2, whose dihydroxypropyl group is not subject to epimerization, and (6S)-BH4 both inhibited the phosphorylation less efficiently than did the (6R)-epimer of BH4. Pterins with different spatial arrangements of the dihydroxypropyl side chain thus appeared to elicit different conformations of the phosphorylation site. The hydroxylase reaction showed a higher apparent Km for (6S)-BH4 than for (6R)-BH4 both when the native and the phenylalanine-activated enzyme were tested. For the activated enzyme Vmax was 40% lower with the (6S)-epimer than the (6R)-epimer, also when the more rapid enzyme inactivation occurring with the former cofactor was taken into account.
D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006900:Hydroxylation; D007700:Kinetics; D010649:Phenylalanine / Q000494:pharmacology*; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation
D001708:Biopterins; D010649:Phenylalanine; C017226:7,8-dihydrobiopterin; D000242:Cyclic AMP; D010651:Phenylalanine Hydroxylase; C003402:sapropterin