Inactivation of phosphorylated rat tyrosine hydroxylase by ascorbate in vitro.
Tyrosine hydroxylase activity is reversibly controlled by the actions of several protein kinases. Previous studies showed that, following phosphorylation by protein kinase A, physiological concentrations of ascorbate irreversibly inactivate tyrosine hydroxylase. Several studies were performed to establish the mechanism of inactivation. We found that inactivation occurred under oxygen-free conditions. The results of this and other experiments suggest that oxygenated species such as superoxide or hydrogen peroxide were not required for inactivation by ascorbate. Inhibition of tyrosine hydroxylase by low concentrations of ascorbate raised the question concerning the mechanism for maintaining enzyme activity under physiological conditions. We report that tyrosine, N alpha-methyl tyrosine, 3-iodotyrosine, and phenylalanine protected the phosphorylated enzyme against ascorbate inactivation. Catecholamines (dopamine, norepinephrine, and some of their analogues) also protected the enzyme against ascorbate inactivation. We performed studies to assess conformational changes of tyrosine hydroxylase by measuring the extrinsic fluorescence using 8-anilino-1-naphthalenesulfonic acid as a reporter group. Phosphorylation of tyrosine hydroxylase by protein kinase A decreased the extrinsic fluorescence. Treatment of tyrosine hydroxylase with ascorbate produced a further decrease in fluorescence. These results provide evidence for conformational changes following these treatments. In contrast to extrinsic fluorescence, the circular dichroic spectrum of tyrosine hydroxylase failed to change following phosphorylation by protein kinase A or inhibition by ascorbate. The spectrum was consistent with a secondary structure of tyrosine hydroxylase with 55% alpha helix, 20% beta sheet, 2% beta turn, and 23% random coil.
Roskoski
R
R
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70119.
Gahn
L G
LG
Roskoski
L M
LM
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002942:Circular Dichroism; D004298:Dopamine / Q000494:pharmacology; D016166:Free Radical Scavengers; D005609:Free Radicals; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors*
D000817:Anilino Naphthalenesulfonates; D016166:Free Radical Scavengers; D005609:Free Radicals; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D014446:Tyrosine 3-Monooxygenase; D001205:Ascorbic Acid; D010100:Oxygen; D004298:Dopamine