REGULATION OF RAT-LIVER PHENYLALANINE-HYDROXYLASE .3. CONTROL OF CATALYSIS BY (6R)-TETRAHYDROBIOPTERIN AND PHENYLALANINE

Effects of phenylalanine and di and tetrahydropterins on presteady-sta te and steady-state catalytic behavior of rat liver phenylalanine hydr oxylase are analyzed. From this and previous work (Shiman, R, Xia, T., Hill, M., and Gray, D. (1994) J. Biol. Chem. 269, 24647-24656), which analyzed binding of the same compounds to the enzyme in the absence o f catalysis, a model of phenylalanine hydroxylase regulation is propos ed. The mechanism appears novel in that 1) one substrate, phenylalanin e, is a positive effector (activator), 2) a second substrate, (6R)-tet rahydrobiopterin (BH4), is a negative effector that blocks phenylalani ne activation by forming an inactive BH4 enzyme complex, and 3) the BH 4-enzyme complex sequesters BH4 and controls its metabolic availabilit y. Reaction progress curves showing regulatory effects of BH4, 7,8-dih ydrobiopterin (BH2), and phenylalanine are fit by the model with high precision. Data are presented that the high affinity pterin-binding si te on unactivated phenylalanine hydroxylase is the pterin site that re gulates catalysis. Occupancy of this site by BH4 or BH2 causes non-coo perative, linear inhibition of phenylalanine activation of the enzyme. All inhibitory effects of BH4 appear due to its binding at the pterin regulatory site on unactivated enzyme. BH2 inhibits by binding at the active site as well as the pterin regulatory site. 6-Methyltetrahydro pterin also appears to bind at the pterin regulatory site, but its eff ect is only seen at high phenylalanine concentrations. Using kinetic c onstants measured in this and earlier work, quantitative effects of ph enylalanine and BH4 regulation on the rate of the phenylalanine hydrox ylase reaction in vitro and in vivo are calculated. The effects of for mation of the BH4 enzyme complex on free BH4 concentration, on enzyme activity, and on regulation of the rate of phenylalanine hydroxylation in liver are discussed.