Jensen W A, Armstrong J M, De Giorgio J, Hearn M T
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Biochemistry. 1995 Jan 17;34(2):472-80. doi: 10.1021/bi00002a011.
The solution stability of phosphoenolpyruvate carboxylase (PEPC) has been determined in the presence of various salts by temperature-accelerated enzyme inactivation and also by using high-performance size-exclusion chromatography. Kosmotropic (water structuring) anions in the Hofmeister series (HPO(4)2-, citrate3-, SO(4)2-, F-, OAc-) and glutamate stabilized the enzyme most effectively, while Cl- (a borderline Hofmeister anion) and Br- (a chaotropic anion) were destabilizing. The effects of the cations on PEPC stability ranged from relatively inert (Na+, K+) to destabilizing ((CH3)4N+, NH4+, Li+). The observed stabilization of PEPC by specific salts has been interpreted in terms of the positive surface tension increment and the water-structuring effects conferred on the solution by the specific stabilizing reagents. Both these effects enhance hydrophobic interactions of proteins and increase the energy required to enlarge the surface area of the solvent cavity in which the protein resides. The destabilization of PEPC by some salts at a concentration of 0.5 M was associated with the dissociation of the tetrameric enzyme into its dimeric and monomeric forms, a process most probably occurring as a result of ion-peptide dipole binding, which promotes protein-solvent interaction and a subsequent reduction in the free energy of cavity formation. The stabilization of enzyme activity by kosmotropic salts depended on the salt concentration with maximum stabilization of PEPC in solution at 52 degrees C observed with 0.6-0.8 M sodium glutamate, 2 M KF, and 2.2 M KOAc. Higher concentrations of these salts resulted in decreased activity. This reduction in activity of PEPC in the presence of high concentrations of kosmotropic salts appears to be associated with irreversible conformational changes of the tetrameric enzyme.
通过温度加速酶失活以及使用高效尺寸排阻色谱法，在各种盐存在的情况下测定了磷酸烯醇式丙酮酸羧化酶（PEPC）的溶液稳定性。霍夫迈斯特序列中的促溶剂（水结构化）阴离子（HPO₄²⁻、柠檬酸根³⁻、SO₄²⁻、F⁻、醋酸根⁻）和谷氨酸对该酶的稳定作用最为有效，而Cl⁻（一种边界霍夫迈斯特阴离子）和Br⁻（一种离液序列高的阴离子）则会使其不稳定。阳离子对PEPC稳定性的影响范围从相对惰性（Na⁺、K⁺）到不稳定（(CH₃)₄N⁺、NH₄⁺、Li⁺）。特定盐对PEPC的稳定作用已根据正表面张力增量以及特定稳定试剂赋予溶液的水结构化效应来解释。这两种效应都增强了蛋白质的疏水相互作用，并增加了扩大蛋白质所在溶剂腔表面积所需的能量。一些浓度为0.5 M的盐使PEPC不稳定，这与四聚体酶解离成其二聚体和单体形式有关，这一过程很可能是由于离子 - 肽偶极结合导致的，该结合促进了蛋白质 - 溶剂相互作用，并随后降低了腔形成的自由能。促溶剂盐对酶活性的稳定作用取决于盐浓度，在52℃下，0.6 - 0.8 M谷氨酸钠、2 M KF和2.2 M KOAc可使溶液中的PEPC达到最大稳定。这些盐的浓度过高会导致活性降低。在高浓度促溶剂盐存在下PEPC活性的降低似乎与四聚体酶的不可逆构象变化有关。
