3-磷酸甘油醛脱氢酶胍变性时的活力及构象变化

酵母3-磷酸甘油醛脱氢酶在盐酸胍溶液中的内源荧光及剩余活力的变化结果提示:apo酶及holo酶的活力在胍浓度为0.5M左右可完全丧失.同时伴有内源荧光强度的下降,光谱宽度的增加和335nm最大发射峰的红移(提示了色氨酸残基的暴露).与已经报导的肌肉酶(内源荧光强度在胍浓度为0.4—1.2M范围相对稳定)不同,酵母酶内源荧光在此浓度范围内表现为逐渐降低.在0.7M胍溶液中,内源荧光变化动力学过程只能测出一相,而酶失活动力学过程为快慢两相,快相动力学速度常数至少大于内源荧光降低速度常数三个数量级以上.以上结果提示:低浓度胍可引起该酶的完全失活,活性部位的空间构象比酶分子的构象更易受到变性剂的扰乱;有一个色氨酸残基位于或靠近酶的活性部位.

COMPARISON OF INACTIVATION AND CONFORMATIONAL CHANGES OF YEAST D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE DURING DENATURATION BY GUANIDINIUM CHLORIDE

Changes in intrinsic protein fluorescence of yeast GAPDH have been compared with its inactivation during denaturation in GuHCI solutions. The holo-and the apo-enzymes are completely inactivated at GuHCI concentrations less than 0.5M and this is accompanied by marked decrease in flurescence emission intensity and red Shift of the emission maxima at 335 nm suggesting exposure of aromatic residues. It has been reported before that during the denaturation of the muscle enzyme, following an initial decrease, there is a region of relatvely little fluorescence changes between 0.4-1.2 M GuHCI.For the yeast enzyme, the decrease in emission intensity is continuous with the increase in GuHCI concentration. At 0.7M GuHCI, the fluorescence change is monophasec with a first order rate constant of 1.3×10-4S-1 but the inactivation is a biphasic process with a fast phase rate constant greater than 0.1×S-1. This is 3 order of magnitudes faster than the corresponding unfolding rate. The slow phase inactivation rate, first order constant of 2.1×10-4 S-1 , is close to that of the unfolding rate as measured by fluorescence changes.