Kuusk V, McIntire W S
Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121.
J Biol Chem. 1994 Oct 21;269(42):26136-43.
The influence of the monovalent cations on the UV-visible spectra of the methylamine dehydrogenase (MADH) from bacterium W3A1 was investigated. The spectra for the oxidized and 1- and 2-electron-reduced forms, unperturbed by bound cations, were obtained for the enzyme, and the extinction coefficients for these forms were determined. The binding of the following cations was investigated: Li+, Na+, K+, Rb+, Cs+, NH4+, (CH3)3NH+, and (CH3)4N+. It was shown that each cation produced unique spectral changes, some of which were pH-dependent. Except for NH4+, all spectral changes produced by binding of the monovalent cations can be explained by assuming two different binding sites in MADH (type I and type II sites). Na+ and K+ displayed monophasic binding to the type II site, (CH3)3NH+ and (CH3)4N+ displayed monophasic binding to the type I site, and Li+, Rb+, and Cs+ displayed either monophasic or biphasic binding to one or both sites depending on pH. The pH dependence for binding to the two sites is different, i.e. plots of log(Kd) versus pH have negative slopes approximately 1 for the type II site, whereas the negative slope is significantly less than 1 (0.6-0.8) for the type I site. This difference leads to pH-dependent changes in spectral features produced by binding of Li+, Rb+, and Cs+. The spectral changes seen during titrations with NH+4 were unlike those seen for any other cation. The binding of NH+4 was biphasic, and the spectra produced in each phase were unaffected by pH. It is assumed that this cation binds to the tryptophan tryptophylquinone cofactor to produce the iminoquinone in the first phase and then binds to the type I monovalent cation binding site in the second phase. It is suggested that binding of NH+4 (and CH3NH+3) to the type I site is a prelude to binding to the cofactor.
研究了单价阳离子对来自细菌W3A1的甲胺脱氢酶（MADH）紫外可见光谱的影响。获得了该酶在未受结合阳离子干扰的情况下，氧化态、单电子还原态和双电子还原态的光谱，并测定了这些形态的消光系数。研究了以下阳离子的结合情况：Li⁺、Na⁺、K⁺、Rb⁺、Cs⁺、NH₄⁺、(CH₃)₃NH⁺和(CH₃)₄N⁺。结果表明，每种阳离子都会产生独特的光谱变化，其中一些变化与pH值有关。除NH₄⁺外，单价阳离子结合产生的所有光谱变化都可以通过假设MADH中有两个不同的结合位点（I型和II型位点）来解释。Na⁺和K⁺对II型位点表现出单相结合，(CH₃)₃NH⁺和(CH₃)₄N⁺对I型位点表现出单相结合，而Li⁺、Rb⁺和Cs⁺根据pH值对一个或两个位点表现出单相或双相结合。两个位点结合的pH依赖性不同，即log(Kd)对pH的图对于II型位点具有约为1的负斜率，而对于I型位点负斜率明显小于1（0.6 - 0.8）。这种差异导致Li⁺、Rb⁺和Cs⁺结合产生的光谱特征随pH值变化。用NH₄⁺滴定过程中观察到的光谱变化与其他任何阳离子的不同。NH₄⁺的结合是双相的，并且每个阶段产生的光谱不受pH影响。据推测，这种阳离子在第一阶段与色氨酸色氨酰醌辅因子结合产生亚氨基醌，然后在第二阶段与I型单价阳离子结合位点结合。有人提出，NH₄⁺（和CH₃NH₃⁺）与I型位点的结合是与辅因子结合的前奏。
