Gorren A C, Moenne-Loccoz P, Backes G, de Vries S, Sanders-Loehr J, Duine J A
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Biochemistry. 1995 Oct 10;34(40):12926-31. doi: 10.1021/bi00040a002.
The nonconvertible substrate analogues di-, tri-, and tetramethylammonium are bound with fairly high affinity to oxidized methylamine dehydrogenase (MADHox) from Thiobacillus versutus and induce the same red-shift in the optical absorbance spectrum of MADHox as do the monovalent cations Cs+, Rb+, and NH4+. Like the monovalent cations, trimethylamine also competitively inhibits the reduction of MADHox by methylamine. Rapid-scan experiments show that within the first few milliseconds of the reaction between MADHox and methylamine a red-shifted intermediate is formed as well. Taken together these experiments demonstrate the existence of a common binding site on MADHox for the substrate CH3NH3+, the substrate analogues (CH3)2NH2+, (CH3)3NH+, and (CH3)4N+, and the monovalent cations Cs+, Rb+, and NH4+. Therefore we conclude that, prior to conversion, methylamine is noncovalently bound to MADHox as a cation. The resonance Raman spectra of MADHox in the absence and presence of Cs+, NH4+, and (CH3)3NH+ are very similar, except for the C=O stretching frequencies of the o-quinone carbonyls of the tryptophyltryptophanquinone (TTQ) active center, which show 5-30 cm-1 downshifts. From these Raman results and the X-ray crystal structure, we conclude that the CH3NH3+ binding site is in close proximity to the O6 carbonyl oxygen of the TTQ.
不可转化的底物类似物二甲基铵、三甲基铵和四甲基铵与来自维氏硫杆菌的氧化型甲胺脱氢酶（MADHox）具有相当高的亲和力，并在MADHox的光吸收光谱中诱导出与单价阳离子Cs⁺、Rb⁺和NH₄⁺相同的红移。与单价阳离子一样，三甲胺也竞争性抑制甲胺对MADHox的还原。快速扫描实验表明，在MADHox与甲胺反应的最初几毫秒内也会形成一个红移中间体。综合这些实验证明，MADHox上存在一个共同的结合位点，用于结合底物CH₃NH₃⁺、底物类似物(CH₃)₂NH₂⁺、(CH₃)₃NH⁺和(CH₃)₄N⁺以及单价阳离子Cs⁺、Rb⁺和NH₄⁺。因此我们得出结论，在转化之前，甲胺以阳离子形式非共价结合到MADHox上。在不存在和存在Cs⁺、NH₄⁺和(CH₃)₃NH⁺的情况下，MADHox的共振拉曼光谱非常相似，除了色氨酸 - 色氨酸醌（TTQ）活性中心的邻醌羰基的C = O伸缩频率显示出5 - 30 cm⁻¹的下移。根据这些拉曼结果和X射线晶体结构，我们得出结论，CH₃NH₃⁺结合位点靠近TTQ的O6羰基氧。
