Encinas M Victoria, González-Nilo Fernando D, Andreu José M, Alfonso Carlos, Cardemil Emilio
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, 33, Santiago, Chile.
Int J Biochem Cell Biol. 2002 Jun;34(6):645-56. doi: 10.1016/s1357-2725(01)00175-3.
ATP-dependent phosphoenolpyruvate (PEP) carboxykinases are found in plants and microorganisms, and catalyse the reversible formation of PEP, ADP, and CO(2) from oxaloacetate plus ATP. These enzymes vary in quaternary structure although there is significant sequence identity among the proteins isolated from different sources. To help understand the influence of quaternary structure in protein stability, the urea-induced unfolding of free- and substrate-bound tetrameric Saccharomyces cerevisiae PEP carboxykinase is described and compared with the unfolding characteristics of the monomeric Escherichia coli enzyme [Eur. J. Biochem. 255 (1998) 439]. The urea-induced denaturation of S. cerevisiae PEP carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism (CD) spectra, and 1-anilino-8-naphthalenesulfonate (ANS) binding. The unfolding profiles were multi-steps, and formation of hydrophobic structures were detected. The data indicate that unfolding and dissociation of the enzyme tetramer are simultaneous events. Ligand binding, most notably PEP in the presence of MnCl(2), conferred a marked protection against urea-induced denaturation. A similar protection effect was found when N-iodoacetyl-N'-(5-sulfo-1-napthyl)ethylene diamine (1,5-I-AEDANS) was covalently bound at Cys(365), within the active site region. Refolding experiments indicated that total recovery of tertiary structure was only obtained from samples previously unfolded to less than 30%. In the presence of substrates, complete refolding was achieved from samples originally denatured up to 50%. The unfolding behaviour of S. cerevisiae PEP carboxykinase was found to be similar to that of E. coli PEP carboxykinase, however all steps take place at lower urea concentrations. These findings show that, at least for monomeric and tetrameric ATP-dependent PEP carboxykinases, quaternary structure does not contribute to protein conformational stability.
依赖ATP的磷酸烯醇式丙酮酸（PEP）羧激酶存在于植物和微生物中，催化草酰乙酸与ATP可逆形成PEP、ADP和CO₂。这些酶的四级结构各不相同，尽管从不同来源分离的蛋白质之间存在显著的序列同一性。为了帮助理解四级结构对蛋白质稳定性的影响，本文描述了尿素诱导的游离和底物结合的四聚体酿酒酵母PEP羧激酶的去折叠过程，并将其与单体大肠杆菌酶的去折叠特性进行了比较[《欧洲生物化学杂志》255 (1998) 439]。通过监测酶活性、蛋白质固有荧光、圆二色性（CD）光谱和1-苯胺基-8-萘磺酸盐（ANS）结合，研究了尿素诱导的酿酒酵母PEP羧激酶的变性。去折叠过程是多步的，并且检测到了疏水结构的形成。数据表明，酶四聚体的去折叠和解离是同时发生的事件。配体结合，尤其是在MnCl₂存在下的PEP，对尿素诱导的变性具有显著的保护作用。当N-碘乙酰基-N'-（5-磺基-1-萘基）乙二胺（1,5-I-AEDANS）共价结合在活性位点区域内的Cys(365)时，也发现了类似的保护作用。复性实验表明，只有从未折叠至小于30%的样品中才能完全恢复三级结构。在底物存在的情况下，最初变性高达50%的样品能够实现完全复性。发现酿酒酵母PEP羧激酶的去折叠行为与大肠杆菌PEP羧激酶相似，然而所有步骤都在较低的尿素浓度下发生。这些发现表明，至少对于单体和四聚体依赖ATP的PEP羧激酶来说，四级结构对蛋白质构象稳定性没有贡献。
