Roskoski R, Gahn L G, Roskoski L M
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70119.
Eur J Biochem. 1993 Dec 1;218(2):363-70. doi: 10.1111/j.1432-1033.1993.tb18385.x.
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.
酪氨酸羟化酶的活性受到几种蛋白激酶的可逆性调控。先前的研究表明，在被蛋白激酶A磷酸化后，生理浓度的抗坏血酸会使酪氨酸羟化酶不可逆地失活。为了确定失活机制，进行了多项研究。我们发现失活在无氧条件下发生。本实验及其他实验结果表明，抗坏血酸导致的失活不需要超氧化物或过氧化氢等含氧物质。低浓度抗坏血酸对酪氨酸羟化酶的抑制引发了关于在生理条件下维持酶活性机制的问题。我们报告酪氨酸、Nα-甲基酪氨酸、3-碘酪氨酸和苯丙氨酸可保护磷酸化的酶免受抗坏血酸的失活作用。儿茶酚胺（多巴胺、去甲肾上腺素及其一些类似物）也可保护该酶免受抗坏血酸的失活作用。我们通过使用8-苯胺基-1-萘磺酸作为报告基团测量外在荧光来评估酪氨酸羟化酶的构象变化。蛋白激酶A使酪氨酸羟化酶磷酸化会降低外在荧光。用抗坏血酸处理酪氨酸羟化酶会使荧光进一步降低。这些结果为这些处理后发生的构象变化提供了证据。与外在荧光不同，酪氨酸羟化酶的圆二色光谱在被蛋白激酶A磷酸化或被抗坏血酸抑制后没有变化。该光谱与酪氨酸羟化酶的二级结构一致，其中α螺旋占55%，β折叠占20%，β转角占2%，无规卷曲占23%。
