The present study presents evidence for the conclusion that the catalytic activity of dopamine beta-hydroxylase (DBH; dopamine beta-mono-oxygenase, EC 1.14.17.1) is regulated independently by pH and by anions. In the absence of activating anions (i.e. in 50 mM Mes buffer) the activity was essentially zero at low pH (5.1-5.3) when assayed with the artificial electron donors ferrocyanide (0.25 mM), N, N, N', N'-tetramethyl-p-phenylenediamine (TMPD, 2 mM) or N, N-dimethyl-p-phenylenediamine (1 mM) and tyramine (8 mM) as the substrate to be hydroxylated. However, in the presence of activating anions (e.g. 0.05-0.6 M Cl(-) in 50 mM Mes buffer, 0.1 M phosphate buffer or 0.2 M acetate buffer) a high catalytic activity was observed at pH 5.1-5.3. The pronounced effect of anions at this pH may be related to the postulated anion-induced conformational change of DBH [Syvertsen, Melø and Ljones (1987) Biochim. Biophys. Acta 914, 6-18] resulting in a facilitated access of the substrates to the active site(s). The anion-activated DBH was inhibited when assayed with ferrocyanide and activated when assayed with TMPD as electron donors by increasing the pH (5.1 to 6.0). By contrast, in the absence of anions the activity increased from essentially zero at pH 5.1-5.3 to high values at pH 6.0, irrespective of the electron donor used. The data suggest that the conformational change induced by anion activation exposes a negatively charged group at or near the electron-donor-binding site(s) imposing an electrostatic repulsion towards ferrocyanide (four negative charges) and an electrostatic attraction towards the positively charged TMPD, thus explaining the different pH-activity curves obtained for the two electron donors. In contrast to the artificial electron donors, the physiological donor ascorbate [Terland and Flatmark (1975) FEBS Lett. 59, 52-56] supports hydroxylation of tyramine at low pH also in the absence of Cl(-), acetate or phosphate, confirming that ascorbate also functions as an anion activator.
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机译：本研究提供了以下结论的证据：多巴胺β-羟化酶（DBH；多巴胺β-单加氧酶，EC 1.14.17.1）的催化活性受pH和阴离子的独立调节。在没有活化阴离子的情况下（即在50 mM Mes缓冲液中），用人工电子给体亚铁氰化物（0.25 mM），N，N，N'，N'-四羟基对苯二胺（TMPD，2 mM）或N，N-二甲基对苯二胺（1 mM）和酪胺（8 mM）作为要羟基化的底物。但是，在存在活化阴离子（例如在50 mM Mes缓冲液，0.1 M磷酸盐缓冲液或0.2 M乙酸盐缓冲液中的0.05-0.6 M Cl（-））的情况下，在pH 5.1-5.3下观察到高催化活性。阴离子在此pH值下的明显作用可能与DBH假定的阴离子诱导的构象变化有关[Syvertsen，Melø和Ljones（1987）Biochim。生物物理学。 Acta 914，6-18]导致底物易于到达活性位点。阴离子活化的DBH在用亚铁氰化物测定时被抑制，而在TMPD作为电子给体时通过增加pH（5.1至6.0）来活化。相反，在不存在阴离子的情况下，无论所用的电子供体如何，其活性都从pH 5.1-5.3的基本为零增加到pH 6.0的高值。数据表明，由阴离子活化引起的构象变化会在电子供体结合位点处或附近暴露一个带负电的基团，向亚铁氰化物施加静电排斥作用（四个负电荷），向带正电的TMPD施加静电吸引作用，从而解释了两个电子给体获得的不同的pH活性曲线。与人工电子供体相反，生理性供体抗坏血酸[Terland and Flatmark（1975）FEBS Lett。 59，52-56]也支持在低pH下酪胺的羟基化反应，即使在不存在Cl（-），乙酸盐或磷酸盐的情况下，也证实了抗坏血酸盐也具有阴离子活化剂的功能。
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