Alexandrescu A T, Hinck A P, Markley J L
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Biochemistry. 1990 May 15;29(19):4516-25. doi: 10.1021/bi00471a003.
Staphylococcal nuclease exists in solution as a mixture of two folded (N and N') and two unfolded (U and U*) forms. Earlier workers [Evans et al. (1989) Biochemistry 28, 362] have proposed that the N'/N and U/U* structural differences involve cis/trans isomerization about the Lys116-Pro117 peptide bond with N and U cis and N' and U* trans. The present results show that residue changes throughout the nuclease structure have large effects on the distribution of the N and N'forms. The N'/N ratios at 313 K for nuclease H124L (N'/N = 0.07) and nuclease G79S (N'/N = 12) differ by 2 orders of magnitude. Thermodynamic parameters for equilibria linking the two folded and two unfolded substates were evaluated for seven mutants of nuclease which were found by kinetic assays to have similar enzymatic activities but by NMR spectroscopy to have a wide dispersion of thermal stabilities. Our results indicate that mutational perturbations of the N'/N equilibrium in folded nuclease (delta G for the N in equilibrium N' reaction) are strongly coupled to changes in the stability of the N form (delta G for the N in equilibrium U reaction), but much less so to the stability of the N' form (delta G for the N' in equilibrium U* reaction).
葡萄球菌核酸酶在溶液中以两种折叠形式（N和N'）和两种未折叠形式（U和U*）的混合物存在。早期研究人员[埃文斯等人（1989年），《生物化学》28卷，362页]提出，N'/N和U/U的结构差异涉及赖氨酸116 - 脯氨酸117肽键的顺/反异构化，其中N和U为顺式，N'和U为反式。目前的结果表明，核酸酶结构中各处的残基变化对N和N'形式的分布有很大影响。核酸酶H124L（N'/N = 0.07）和核酸酶G79S（N'/N = 12）在313 K时的N'/N比值相差2个数量级。对核酸酶的七个突变体评估了连接两种折叠亚态和两种未折叠亚态的平衡的热力学参数，通过动力学测定发现这些突变体具有相似的酶活性，但通过核磁共振光谱发现它们的热稳定性有很大差异。我们的结果表明，折叠核酸酶中N'/N平衡的突变扰动（N与N'平衡反应中N的ΔG）与N形式稳定性的变化（N与U平衡反应中N的ΔG）紧密相关，但与N'形式的稳定性（N'与U*平衡反应中N'的ΔG）的相关性要小得多。
