Gorren A C, Duine J A
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Biochemistry. 1994 Oct 11;33(40):12202-9. doi: 10.1021/bi00206a024.
The catalytic parameters of Thiobacillus versutus methylamine dehydrogenase (MADH) with the physiological substrates methylamine and amicyanin show a pH profile that is quite different from the one found in commonly used assays with artificial electron acceptors. The optimum at pH 7.5, observed for kcat in the latter case, is absent with amicyanin as the reoxidizing substrate. With amicyanin kcat scarcely depends on pH; the same is true for the maximal rate of reduction of MADH by methylamine (kred). Conversely, both the specificity constant (kcat/Km) of MADH for amicyanin and the apparent second-order rate constant for the reduction of MADH by methylamine (kassocapp) increase very sharply with pH. MADH has a high- and a low-affinity binding site for monovalent cations. Cation binding to the high-affinity site, which only binds the larger cations (Cs+, Rb+, and NH4+), is accompanied by a red shift in the absorbance spectrum, whereas cation binding to the low-affinity site, which, less specifically, favors binding of the smaller cations, leads to a bleaching of the visible spectrum with a concomitant increase in the near-UV. Cation binding to either site strongly affects the reactivity of MADH. The reduction of MADH by methylamine is inhibited by monovalent cations, whereas the oxidation of reduced MADH by amicyanin is strongly stimulated. For the former reaction it was established that cations affect only kassocapp, not kred. Some speculations about the molecular basis for the effects of pH and cations are presented.
硫杆菌甲基胺脱氢酶（MADH）与生理底物甲胺和蓝铜蛋白的催化参数显示出的pH曲线，与常用的人工电子受体检测中发现的pH曲线有很大不同。在后一种情况下，观察到的kcat在pH 7.5时的最佳值，在以蓝铜蛋白作为再氧化底物时并不存在。对于蓝铜蛋白，kcat几乎不依赖于pH；甲胺对MADH的最大还原速率（kred）也是如此。相反，MADH对蓝铜蛋白的特异性常数（kcat/Km）以及甲胺还原MADH的表观二级速率常数（kassocapp）都随pH急剧增加。MADH对单价阳离子有一个高亲和力和一个低亲和力结合位点。阳离子与仅结合较大阳离子（Cs+、Rb+和NH4+）的高亲和力位点结合时，吸收光谱会出现红移，而阳离子与低亲和力位点结合时，该位点对较小阳离子的结合不太特异，会导致可见光谱漂白，同时近紫外光增加。阳离子与任何一个位点的结合都会强烈影响MADH的反应性。甲胺对MADH的还原受到单价阳离子的抑制，而蓝铜蛋白对还原型MADH的氧化则受到强烈刺激。对于前一个反应，已确定阳离子仅影响kassocapp，而不影响kred。本文还对pH和阳离子影响的分子基础进行了一些推测。
