Pterin-dependent hydroxylases catalyze the rate-limiting steps in the biosynthesis of catecholamine neurotransmitters and of serotonin. This gives these enzymes central roles in the health of an individual. Imbalances in catecholamine levels have been implicated in a number of disease states, including hypertension. Imbalances in serotonin levels have been implicated in several neurologic and psychiatric disorders, including depression. Despite the importance of these pathways in health problems of such major importance, very little is known at a molecular level about the enzymes involved. Tyrosine hydroxylase catalyzes the formation of dihydroxyphenylalanine from tyrosine, the first step in catecholamine biosynthesis. Mechanistic, structural, and regulatory studies will be carried out with the recombinant rat enzyme. Oxygen and deuterium isotope effects and alternate substrates will be used to probe individual steps in the catalytic mechanism. X-Ray crystallography, analytical ultracentrifugation, and crosslinking will be used to examine the protein structure. Site-directed mutagenesis will be used to identify critical residues. The effects of phosphorylation on structure, activity, and inhibition by catecholamines will be determined. Tryptophan hydroxylase catalyzes the formation of 5-hydroxytryptophan from tryptophan, the first step in serotonin biosynthesis. Mechanistic studies will be initiated with the recombinant enzyme. Alternate substrates and isotope effects will be used to probe the mechanistic similarities to tyrosine hydroxylase.
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