Thorpe C, Williams C H
J Biol Chem. 1976 Dec 10;251(23):7726-8.
A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently (Thorpe, C., and Williams, C.H. (1976) J. Biol. Chem. 251, 3553-3557), in which 1 of the 2 cysteine residues generated on reduction of the intrachain active center disulfide bridge is selectively alkylated with iodoacetamide. This monolabeled enzyme exhibits a spectrum of oxidized bound flavin. The addition of 1 mM NAD+ to this derivative at pH 8.3 causes a decrease in absorbance of approximately 50% at 448 nm, with a concomitant increase at 380 nm. These spectral changes are complete within 3 ms and are reversible. NAD+ titrations generate isosbestic points at 408, 374, and 327 nm; allowing values for the apparent dissociation constant for NAD+ and the extent of bleaching at infinite ligand to be obtained from double reciprocal plots. Between pH 6.1 and 8.8, the apparent KD decreases from 320 to 35 muM, whereas the extrapolated delta epsilon 448 values remain approximately constant at 1/2 epsilon 448. Direct measurement of NAD+ binding by gel filtration at pH 8.8 indicates that the spectral changes are associated with a stoichiometry of 1.2 mol of NAD+ bound/2 mol of FAD. The modified protein is a dimer containing 1 FAD and 1 alkylated cysteine residue/subunit; the native enzyme is also dimeric. The visible spectrum of the species absorbing at 380 nm, approximated by correction for the residual oxidized FAD, shows a single maximum at 384 nm, epsilon 384 = 8.7 mM-1cm-1. Comparison of this spectrum with that of model compounds of known structure suggests that it may represent a reversible covalent flavin adduct induced on binding NAD+.
最近有人描述了一种黄素蛋白猪心脂酰胺脱氢酶的衍生物（索普，C.，和威廉姆斯，C.H.（1976年）《生物化学杂志》251卷，3553 - 3557页），其中在链内活性中心二硫键还原时产生的2个半胱氨酸残基中的1个被碘乙酰胺选择性烷基化。这种单标记酶呈现出氧化结合黄素的光谱。在pH 8.3时向该衍生物中加入1 mM NAD⁺会导致在448 nm处吸光度降低约50%，同时在380 nm处吸光度增加。这些光谱变化在3毫秒内完成且是可逆的。NAD⁺滴定在408、374和327 nm处产生等吸收点；通过双倒数作图可以得到NAD⁺的表观解离常数以及在无限配体时的漂白程度的值。在pH 6.1至8.8之间，表观KD从320 μM降至35 μM，而外推的Δε448值在1/2 ε448处大致保持恒定。在pH 8.8时通过凝胶过滤直接测量NAD⁺结合表明，光谱变化与每2摩尔FAD结合1.2摩尔NAD⁺的化学计量比相关。修饰后的蛋白质是一种二聚体，每个亚基含有1个FAD和1个烷基化的半胱氨酸残基；天然酶也是二聚体。通过对残留氧化FAD进行校正近似得到的在380 nm处吸收的物种的可见光谱在384 nm处有一个单一最大值，ε384 = 8.7 mM⁻¹cm⁻¹。将该光谱与已知结构的模型化合物的光谱进行比较表明，它可能代表在结合NAD⁺时诱导形成的可逆共价黄素加合物。
