Nakashima A, Hayashi N, Mori K, Kaneko Y S, Nagatsu T, Ota A
Department of Physiology, School of Medicine, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan.
FEBS Lett. 2000 Jan 7;465(1):59-63. doi: 10.1016/s0014-5793(99)01704-4.
Tyrosine hydroxylase (TH), which converts L-tyrosine to L-3, 4-dihydroxyphenylalanine, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by the feedback inhibition of the catecholamine products including dopamine. To rationalize the significant role of the N-terminal sequence Arg(37)-Arg(38) of human TH type 1 (hTH1) in determining the efficiency of feedback inhibition, we produced mutants of which the positively charged Arg(37)-Arg(38) site was replaced by electrically neutral Gly and/or negatively charged Glu and analyzed the degree of inhibition of these mutant enzymes by dopamine. The replacement of Arg by Gly reduced the inhibitory effect of dopamine on the catalytic activity measured in the basic pH range and the replacement of Arg by Glu was enough to abolish the inhibitory effect, although these mutations brought no significant changes to the circular dichroism spectrum. The prediction of the secondary structure of N-terminal residues 1-60 by computer software specified the location of the Arg(37)-Arg(38) sequence in the turn intervening between the two alpha-helices (residues 16-29 and residues 41-59). These results suggest that the positive charge of the amino acid residues at positions 37 and 38 is one of the main factors that maintains the characteristic of the turn and is responsible for the enzyme inhibition by dopamine.
酪氨酸羟化酶（TH）可将L-酪氨酸转化为L-3,4-二羟基苯丙氨酸，是儿茶酚胺生物合成中的限速酶；其活性受包括多巴胺在内的儿茶酚胺产物的反馈抑制调节。为了阐明人1型TH（hTH1）的N端序列Arg(37)-Arg(38)在决定反馈抑制效率方面的重要作用，我们构建了突变体，将带正电荷的Arg(37)-Arg(38)位点替换为电中性的Gly和/或带负电荷的Glu，并分析了多巴胺对这些突变酶的抑制程度。用Gly替换Arg降低了多巴胺在碱性pH范围内对催化活性的抑制作用，而用Glu替换Arg足以消除抑制作用，尽管这些突变对圆二色光谱没有显著影响。通过计算机软件对N端1-60个残基的二级结构预测确定了Arg(37)-Arg(38)序列位于两个α螺旋（残基16-29和残基41-59）之间的转角处。这些结果表明，37位和38位氨基酸残基的正电荷是维持转角特征并导致多巴胺对酶产生抑制作用的主要因素之一。
