Mechanisms of Neurotransmitter Biosynthesis

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DESCRIPTION (provided by applicant): Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase make up the family of non-heme iron pterin-dependent aromatic amino acid hydroxylases. Each enzyme catalyzes the hydroxylation of the aromatic side chain of its substrate in a reaction critical for proper functioning of the central nervous system: phenylalanine metabolism, catecholamine biosynthesis, and serotonin biosynthesis. In addition to their physiological importance, these enzymes are unique in that they are the only nonheme iron enzymes whose physiological reaction is the hydroxylation of unactivated aromatic systems. The present proposal addresses several outstanding questions regarding the catalytic and regulatory mechanisms of these enzymes. The common catalytic mechanism will be studied using a combination of rapid reaction methods and spectroscopy, with the goal of characterizing individual iron species in catalysis. The role of protein dynamics in catalysis by tyrosine and phenylalanine hydroxylase will be studied by determining the effects of substrates on the kinetics of peptide bond exchange with solvent. The regulation of phenylalanine and tyrosine hydroxylase is critical to their proper function. The kinetics of peptide bond exchange will be used to evaluate a literature model for allosteric regulation of phenylalanine hydroxylase, while fluorescence methods will be used to evaluate a structural model for the regulation of tyrosine hydroxylase by phosphorylation. The active site iron atom in these enzymes is bound by a 2-Histidine-1-carboxylate facial triad. These two histidines in phenylalanine hydroxylase will be replaced with analogs and the effects on individual kinetic steps determined, using a combination of steady state kinetics, product partitioning, and kinetic isotope effects. The aromatic amino acid hydroxylases play critical roles in the proper functioning of the central nervous system. Loss of phenylalanine hydroxylase results in phenylketonuria; tyrosine hydroxylase is required to make the neurotransmitters dopamine, norepinephrine, and epinephrine; and tryptophan hydroxylase is required to synthesize the neurotransmitter serotonin.