Plomer J J, Gafni A
Institute of Gerontology, University of Michigan, Ann Arbor 48109.
Biochim Biophys Acta. 1992 Aug 21;1122(3):234-42. doi: 10.1016/0167-4838(92)90398-w.
The denaturation of the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride has been studied using enzymatic activity, intrinsic fluorescence, circular dichroism, and light scattering measurements. Equilibrium experiments at 25 degrees C revealed that between 0.9 and 1.2 M denaturant the enzyme underwent a conformational change, exposing tryptophan residues to solvent, with some loss of secondary structure and a complete loss of enzymatic activity but without dimer dissociation to subunits. This inactive, partially unfolded, dimeric intermediate was susceptible to slow aggregation, perhaps due to exposure of 'sticky' hydrophobic stretches of the polypeptide chain. A second equilibrium transition, reflecting extensive unfolding and dimer dissociation, occurred only at denaturant concentrations above 1.4 M. Kinetics experiments demonstrated that in the denaturant concentration range of 1.7-1.9 M the fluorescence change occurred in two distinct steps. The first step involved a large, very rapid drop in fluorescence whose rate was strongly dependent on the denaturant concentration. This was followed by a small, relatively slow rise in the emission intensity, the rate of which was independent of denaturant concentration. Enzymatic activity was lost with a denaturant-concentration-dependent rate, which was approx. 3-times slower than the rate of the first step in fluorescence change. A denaturation mechanism incorporating several unfolding intermediates and which accounts for all the above results is presented and discussed. While the fully unfolded enzyme regained up to 55% of its original activity upon dilution of denaturant to a concentration that would be expected to support native enzyme, denaturation intermediates were able to reactivate only minimally and in fact were found to aggregate and precipitate out of solution.
利用酶活性、内源荧光、圆二色性和光散射测量等方法，研究了嗜温明串珠菌葡萄糖-6-磷酸脱氢酶二聚体在盐酸胍作用下的变性情况。25℃下的平衡实验表明，在0.9至1.2M变性剂之间，该酶发生了构象变化，色氨酸残基暴露于溶剂中，二级结构有所损失，酶活性完全丧失，但二聚体未解离为亚基。这种无活性、部分展开的二聚体中间体易于缓慢聚集，这可能是由于多肽链中“粘性”疏水片段的暴露。第二个平衡转变反映了广泛的展开和二聚体解离，仅在变性剂浓度高于1.4M时发生。动力学实验表明，在1.7-1.9M的变性剂浓度范围内，荧光变化分两个明显步骤发生。第一步涉及荧光的大幅快速下降，其速率强烈依赖于变性剂浓度。随后是发射强度的小幅相对缓慢上升，其速率与变性剂浓度无关。酶活性以依赖于变性剂浓度的速率丧失，该速率约为荧光变化第一步速率的3倍。本文提出并讨论了一种包含多个展开中间体并能解释上述所有结果的变性机制。当将变性剂稀释至预期能支持天然酶的浓度时，完全展开的酶可恢复高达其原始活性的55%，而变性中间体只能极少程度地重新激活，实际上还会聚集并从溶液中沉淀出来。
