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Mutagenesis of serine 40 of tyrosine hydroxylase: implications for the enzyme's regulatory mechanism
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The enzyme tyrosine hydroxylase (TYH) catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA), utilizing tetrahydropterin and molecular oxygen. This is the rate-limiting step in the production of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. TYH is regulated both by phosphorylation of serine residues in the regulatory domain and by binding of catecholamines. Phosphorylation at serine 40 serves to activate the enzyme by increasing the catecholamine dissociation rate, thus lowering the catecholamine binding affinity. To understand more about how phosphorylation exerts this effect, amino acid substitutions were made at serine 40 of TYH. Tyrosine hydroxylase having serine 40 replaced by glutamate, aspartate, alanine, valine, or threonine was expressed and purified, and the kinetic parameters and catecholamine binding affinities of the proteins were characterized. Substitutions at serine 40 were found to have little effect on the K[m] and V[max] values. The DOPA and dopamine binding affinities of the mutant enzymes were compared to those of wild type and phosphorylated TYH by measuring K[a] values. The K[a] for phosphorylated enzyme was an order of magnitude lower than that of wild type enzyme when DOPA was used, but three orders of magnitude lower when dopamine was used. Based on catecholamine binding affinities, S40T TYH behaved more similarly to wild type enzyme than did any of the other mutant enzymes. The S40E and S40D mutations decreased catecholamine binding affinity, but the effect varied depending on whether DOPA or dopamine was used. The dopamine binding affinity of S40E TYH was as low as that of phosphorylated TYH, while that of S40D TYH was an order of magnitude larger than these were. Using DOPA, however, S40D TYH had a lower K[a] than did S40E TYH, but it was still 2-fold greater than the value for phosphorylated TYH. When alanine or valine was substituted for serine 40, there was only a 2 to 4-fold decrease in K[a] when DOPA was used and a 60-fold decrease when dopamine was used. From these results it was concluded that a combination of factors, such as charge and hydrogen bonding ability, interact to produce the activation effect seen with phosphorylation.
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Includes bibliographical references (leaves 41-42).
Issued also on microfiche from Lange Micrographics.
McCulloch, Ruth Irene (2001). Mutagenesis of serine 40 of tyrosine hydroxylase: implications for the enzyme's regulatory mechanism. Master's thesis, Texas A&M University. Available electronically from
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