Schmid F X, Baldwin R L
Proc Natl Acad Sci U S A. 1978 Oct;75(10):4764-8. doi: 10.1073/pnas.75.10.4764.
Unfolded RNase A is known to contain an equilibrium mixture of two forms, a slow-folding form (U(1)) and a fast-folding form (U(2)). If U(1) is produced after unfolding by the slow cis-trans isomerization of proline residues about X-Pro imide bonds, then the formation of U(1) should be catalyzed by strong acids. Therefore, the rate of formation of U(1) has been measured at different HClO(4) concentrations. After rapid unfolding of the native protein in concentrated HClO(4) at 0 degrees , the slow formation of U(1) was measured by use of refolding assays. Catalysis of its formation was found at HClO(4) concentrations above 5 M. The uncatalyzed reaction follows apparent first-order kinetics but, in the acid-catalyzed range, two reactions are found. The faster reaction produces two-thirds of the slow-folding species and shows acid catalysis above 5 M HClO(4). Catalysis of the slower reaction begins at 8 M HClO(4). The faster reaction shows a 100-fold increase in rate at 10.6 M HClO(4) over the rate of the uncatalyzed reaction of 5 M. The activation enthalpy of the uncatalyzed reaction has been measured in two sets of unfolding conditions: DeltaH(double dagger) is 21.5 kcal/mol (1 kcal = 4.2 x 10(3) J) in 3.3 M HClO(4) and 21.0 kcal/mol in 5 M guanidine HCl, pH 2.5.Both acid catalysis of the formation of U(1) and its high activation enthalpy are consistent with the rate-limiting step being cis-trans isomerization either of X-Pro imide bonds or of peptide bond. The rate of the uncatalyzed reaction is in the range expected for proline isomerization and is 0.1% of that of peptide bond isomerization; thus, the simplest explanation for the formation of U(1) is proline isomerization. Earlier data, showing that the kinetic properties of the U(1) right arrow over left arrow U(2) reaction in refolding conditions differ from those of proline isomerization, can be explained if there is kinetic coupling between early steps in the folding of U(1) and its conversion to U(2).The existence of two acid-catalyzed reactions that are distinguished by the HClO(4) concentration at which catalysis begins suggests that at least two essential proline residues produce slow-folding species of RNase A by isomerization after unfolding. Because protonation of imide bonds is responsible for acid catalysis of proline isomerization, the slower reaction probably involves an imide bond with a low pK. It may be the bond connecting Lys-41 and Pro-42, because the positive charge on Lys-41 could make this bond more difficult to protonate.
已知去折叠的核糖核酸酶A包含两种形式的平衡混合物，一种是慢折叠形式（U(1)），另一种是快折叠形式（U(2)）。如果U(1)是在脯氨酸残基围绕X - Pro亚胺键进行缓慢的顺反异构化而导致去折叠后产生的，那么U(1)的形成应该会被强酸催化。因此，已在不同的高氯酸（HClO₄）浓度下测量了U(1)的形成速率。在0℃下将天然蛋白质在浓HClO₄中快速去折叠后，通过复性分析来测量U(1)的缓慢形成过程。发现在HClO₄浓度高于5 M时会催化其形成。未催化的反应遵循表观一级动力学，但在酸催化范围内，发现有两个反应。较快的反应产生三分之二的慢折叠物种，并且在HClO₄浓度高于5 M时显示出酸催化作用。较慢反应的催化作用在8 M HClO₄时开始。较快的反应在10.6 M HClO₄时的速率比5 M时未催化反应的速率增加了100倍。已在两组去折叠条件下测量了未催化反应的活化焓：在3.3 M HClO₄中，ΔH‡为21.5千卡/摩尔（1千卡 = 4.2×10³焦耳），在5 M盐酸胍（pH 2.5）中为21.0千卡/摩尔。U(1)形成的酸催化作用及其高活化焓都与限速步骤是X - Pro亚胺键或肽键的顺反异构化一致。未催化反应的速率处于脯氨酸异构化预期的范围内，并且是肽键异构化速率的0.1%；因此，对U(1)形成的最简单解释是脯氨酸异构化。早期的数据表明，在复性条件下U(1)⇌U(2)反应的动力学性质与脯氨酸异构化的不同，如果在U(1)折叠的早期步骤与其转化为U(2)之间存在动力学耦合，那么这一点可以得到解释。存在两个由催化开始时的HClO₄浓度区分的酸催化反应，这表明至少有两个必需的脯氨酸残基在去折叠后通过异构化产生核糖核酸酶A的慢折叠物种。由于亚胺键的质子化是脯氨酸异构化酸催化的原因，较慢的反应可能涉及一个pK值较低的亚胺键。它可能是连接赖氨酸 - 41（Lys - 41）和脯氨酸 - 42（Pro - 42）的键，因为赖氨酸 - 41上的正电荷可能使该键更难质子化。
