MECHANISTIC STUDIES OF TYROSINE HYDROXYLASE

  • Fitzpatrick, Paul F (PI)

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date5/1/927/31/13

Funding

  • National Institutes of Health: $273,786.00
  • National Institutes of Health: $157,493.00
  • National Institutes of Health: $318,775.00
  • National Institutes of Health: $250,142.00
  • National Institutes of Health: $238,744.00
  • National Institutes of Health: $325,756.00
  • National Institutes of Health: $241,610.00
  • National Institutes of Health
  • National Institutes of Health: $297,307.00
  • National Institutes of Health: $28,790.00
  • National Institutes of Health: $78,612.00
  • National Institutes of Health: $237,751.00
  • National Institutes of Health: $274,221.00
  • National Institutes of Health: $337,591.00
  • National Institutes of Health: $234,833.00
  • National Institutes of Health
  • National Institutes of Health: $277,914.00
  • National Institutes of Health: $299,552.00
  • National Institutes of Health: $155,716.00
  • National Institutes of Health: $11,087.00
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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