Regulation of Rat Liver Phenylalanine Hydroxylase

Effects of phenylalanine and diand tetrahydropterins on presteady-state and steady-state catalytic behavior of rat liver phenylalanine hydroxylase are analyzed. From this and previous work (Shiman, R, Xia, T., Hill, M., and Gray, D. (1994) J. BioZ. Chem. 269, 2464724656), which analyzed binding of the same compounds to the enzyme in the absence of catalysis, a model of phenylalanine hydroxylase regulation is proposed. The mechanism appears novel in that 1) one substrate, phenylalanine, is a positive effector (activator), 2) a second substrate, (6R)-tetrahydrobiopterin (BH,), is a negative effector that blocks phenylalanine activation by forming an inactive BH,.enzyme complex, and 3) the BH,.enzyme complex sequesters BH, and controls its metabolic availability. Reaction progress curves showing regulatory effects of BH,, 7,8-dihydrobiopterin (BH,!, .and phenylalanine are fit by the model with high preclslon. Data are presented that the high affinity pterin-binding site on unactivated phenylalanine hydroxylase is the pterin site that regulates catalysis. Occupancy of this site by BH, or BH, causes non-cooperative, linear inhibition of phenylalanine activation of the enzyme. All inhibitory effects of BH, appear due to its binding at the pterin regulatory site on unactivated enzyme. BH, inhibits by binding at the active site as well as the pterin regulatory site. 6-Methyltetrahydropterin also appears to bind at the pterin regulatory site, but its effect is only seen at high phenylalanine concentrations. Using kinetic constants measured in this and earlier work, quantitative effects of phenylalanine and BH, regulation on the rate of the phenylalanine hydroxylase reaction in vitro and in vivo are calculated. The effects of formation of the BH,.enzyme complex on free BH, concentration, on enzyme activity, and on regulation of the rate of phenylalanine hydroxylation in liver are discussed.