Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine, using molecular oxygen and a tetrahydropterin as cosubstrates. The role of tyrosine hydroxylase as the catalyst for this rate-limiting step in catecholamine biosynthesis gives the enzyme a central role in the health of an individual. Imbalances in catecholamine levels have been implicated in a number of disease states. Hypertension is a major health problem in the United States; a number of studies have found altered catecholamine metabolism in hypertensive animals. Neurotic disorders impose an equally large cost upon society; the catecholamine-producing areas of the brain are affected in several neurologic and psychiatric diseases. Finally, a number of drugs, both legal and illegal have been shown to directly effect the activity of tyrosine hydroxylase in vivo. The goals of the research described here are to determine the catalytic mechanism of this important enzyme and the mechanistic and structural effects of short term regulation by phosphorylation. The catalytic mechanism of tyrosine hydroxylase is very poorly understood. The enzyme contains a single ferrous atom per active site and has no visible chromophore. Mechanisms involving electrophilic aromatic substitution, epoxidation, or hydroxyl radical attack have been proposed for this or similar systems. To distinguish among these possibilities: 1) The effect of the electron donating ability of p- substituents of substituted phenylalanines on the partitioning between hydroxylation and unproductive tetrahydropterin oxidation will be determined. 2) Isotope effects on the relative amounts of p- and m- tyrosine produced from phenylalanine will be used to test for an epoxide intermediate. 3) The pterin product from uncoupled turnover will be determined. To probe the role of the active site iron: 1) Rapid quench epr spectroscopy will be used to determine if the active site iron changes valency during catalysis. 2) The effect of nitric oxide on the epr signal of the Fe(II) form of the enzyme will be determined in the presence and absence of substrates and inhibitors. 3) Circular dichroism and low temperature magnetic circular dichroism will be used to determine the coordination number and geometry of the iron atom. Rapid changes in the rate of biosynthesis of catecholamines are controlled by phosphorylation of tyrosine hydroxylase at several seryl residues. Phosphorylation-dephosphorylation cascades are by far the most common means of rapidly and reversibly modifying the activities of enzymes. Our goal is to determine the structural and catalytic changes in this critical enzyme which occur upon phosphorylation. To determine the effect of phosphorylation on catalysis, 1) the effect on the kinetics and the pH dependence of enzyme of phosphorylation at Ser4O will be determined, and 2) site directed mutagenesis will be used to replace Ser4O with other amino acids.
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