3-磷酸甘油醛脱氢酶的胍变性——全位与半位修饰酶的比较

酵母3-磷酸甘油醛脱氢酶在盐酸胍溶液中内源荧光及NAD荧光衍生物的410nm特征荧光发射光谱的变化结果提示,全位及半位修饰羧甲基酶活性部位与NAD共价连接的荧光衍生物的形成,明显受到盐酸胍的干扰,并且前者比后者更为显著.全位及半位修饰光照酶的特征荧光在低胍浓度下较内源荧光降低更为显著,同时伴有最大发射峰先红移后兰移的现象.NAD荧光衍生物的特征荧光在胍溶液中减弱的动力学过程分为快相和慢相,快相一级动力学常数比慢相的大两个数量级,全位及半位修饰酶的特征荧光的减弱快慢相速度常数分别属于同一个数量级.以上结果提示:酶活性部位的构象较整个分子来说更易被变性剂扰乱,柔性强于整个分子;NAD荧光衍生物的形成需要活性部位具有正确的空间几何结构.

COMPARISON OF THE DENATURATION BY GUANIDINIUM CHLORIDE BETWEEN ALL OF-THE SITES AND HALE OF-THE-SITES MODIFIED GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

Under carefully controlled conditions, yeast GAPDH carboxymethylated at only 2 or all 4 of the subunits can be respectively produced fluorescent NAD derivative at 1 or 2 of the decarboxylated subunits by irradiation in presence of NAD+ (Tsou, et al, Biochem. Soc. Trans. 11, 425-429, 1983). The emission peak at 410nm of the fluorescent NAD derivative introduced at the active site of this enzyme shows both a red shift and a marked decrease in intensity at the same GuHCl concentration required to bring about the inactivation and the initial changes in the intrinsic fluorescence of the holoenzyme. The conformation of modified GAPDH carrying the fluorescent NAD derivative at one subunit is relatively less perturbed than that carrying the derivative at two subunits. The kinetics of this emission change is also biphasic with a fast phase approaching that for the fast phase of inactivation. It appears that treatment by low concentration GuHCl leads both inactivation and perturbation of the active site conformation and these are then followed by unfolding of the molecule as a whole.