REGULATION OF RAT-LIVER PHENYLALANINE-HYDROXYLASE .2. SUBSTRATE-BINDING AND THE ROLE OF ACTIVATION IN THE CONTROL OF ENZYMATIC-ACTIVITY

Activation by phenylalanine and reduction by the cofactor (GR)-tetrahy drobiopterin (BH4) are required for formation of active liver phenylal anine hydroxylase. This work describes effects of the activation and r edox state on substrate and effector recognition of this enzyme, it es tablishes relationships among the pterin and phenylalanine binding sit es on the different forms of the enzyme, and it provides a quantitativ e description of the enzyme's presumptive regulatory and catalytic sit es. BH4, 7,8-dihydrobiopterin (BH2), 6-methyltetrahydropterin, and 5-d eaza-6-methyltetrahydropterin were found to bind to unactivated phenyl alanine hydroxylase with a stoichiometry of 1/enzyme subunit and with hyperbolic kinetics; all appear to compete for the same binding site o n the enzyme, and all appear to bind in the proximity of, but not to, the enzyme's non-heme iron. In the transition from unactivated to acti vated enzyme, phenylalanine and pterin binding is modified, a new site for phenylalanine is formed, and the pterin site is replaced by a sit e of greatly decreased affinity for BH4 and BH2, one which does not ap pear to recognize the dihydroxypropyl side chain of BH4 and BH2. The p terin- and phenylalanine binding sites on activated phenylalanine hydr oxylase appear to be part of the enzyme's active site. Despite large e ffects on substrate binding, neither chelator binding ability nor solv ent accessibility of the iron are affected by activation; activation a ppears to affect the nearby environment of the enzyme's iron but not t he iron itself. Studies of oxidized and reduced phenylalanine hydroxyl ase indicate that the redox state is not a major determinant of pterin and phenylalanine association with enzyme.