Role of C6 chirality of tetrahydropterin cofactor in catalysis and regulation of tyrosine and phenylalanine hydroxylases.
The chiral specificities of bovine striatal tyrosine hydroxylase (TH) (unphosphorylated and phosphorylated by cAMP-dependent protein kinase) and rat liver phenylalanine hydroxylase (PH) were examined at physiological pH using the pure C6 stereoisomers of 6-methyl- and 6-propyl-5,6,7,8-tetrahydropterin (6-methyl-PH4 and 6-propyl-PH4) and (6R)- and (6S)-tetrahydrobiopterin (BH4). Both PH and phosphorylated TH have substantially higher Vmax values with the unnatural (6R)-propyl-PH4 than the natural (6S)-propyl-PH4 (approximately 6- and 11-fold, respectively). However, the Km's are also higher such that Vmax/Km is almost unaffected by C6 chirality. Unphosphorylated TH has equal Km values for both isomers of 6-propyl-PH4, but has about a 6 times greater Vmax with the unnatural isomer, making it the fastest cofactor yet for this form of the enzyme. With the shorter 6-methyl group, chiral differences are still recognized by phosphorylated TH but hardly at all by PH. Inhibition of both PH and TH by amino acid substrate which occurs with (6R)-BH4 as cofactor is also observed with (6S)-propyl-PH4 but not with (6S)-BH4, (6R)-propyl-PH4, or (6R)- or (6R,S)-methyl-PH4. The Km for (6S)-BH4 with phosphorylated TH is nearly 3 times higher than with (6R)-BH4, but Vmax is unchanged. With unphosphorylated TH, (6S)-BH4 produces very low decelerating rates, which was shown not to be due to irreversible inactivation of the enzyme. The Km for (6R)-BH4 with either hydroxylase is 10 times higher than for the equivalently configured (6S)-propyl-PH4. Comparison of these two cofactors reveals that the 1' and 2' side-chain hydroxyl groups of the natural cofactor promote different regulatory functions in PH than in TH.
Bailey
S W
SW
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.
Dillard
S B
SB
Ayling
J E
JE
D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D006878:Hydroxides / Q000378:metabolism; D017665:Hydroxyl Radical; D007536:Isomerism; D007700:Kinetics; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000187:drug effects; D010766:Phosphorylation; D011622:Pterins / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D014443:Tyrosine / Q000737:chemistry; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000187:drug effects
D006878:Hydroxides; D011622:Pterins; C011552:tetrahydropterin; D017665:Hydroxyl Radical; D014443:Tyrosine; D010651:Phenylalanine Hydroxylase; D014446:Tyrosine 3-Monooxygenase