Juminaga D, Wedemeyer W J, Garduño-Júarez R, McDonald M A, Scheraga H A
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Biochemistry. 1997 Aug 19;36(33):10131-45. doi: 10.1021/bi970711i.
Three tyrosine-to-phenylalanine mutants of ribonuclease A (Y25F, Y92F, and Y97F) are investigated for their enzymatic activities, molecular stabilities, and unfolding/refolding kinetics. These mutants exhibit 80, 90, and 80%, respectively, of the catalytic activity of the wild-type enzyme. Thermal, Gdn.HCl, and pH transition measurements indicate that Y25F and Y97F are less stable than the wild-type protein, whereas the bulk of the thermodynamic and kinetic evidence indicates that Y92F is as stable as the wild-type protein. Differences in molar extinction coefficients indicate that tyrosines 25, 92, and 97 contribute 38, 13, and 39%, respectively, to the absorption difference between the folded and unfolded states, in general agreement with previous studies but possibly indicating the contribution of a fourth tyrosine residue to account for the remaining 10%. Stopped-flow single- and double-jump kinetic experiments were carried out on the wild-type and three mutant proteins. At least one tyrosine residue besides tyrosine 92 contributes to the slow fluorescence-unfolding phase; the likely candidate for this residue is tyrosine 115 which monitors the cis-trans isomerization of the X-Pro114 peptide bond. Tyrosines 25 and 97 are involved in interactions that retard conformational unfolding and accelerate conformational refolding as well as the cis-trans proline isomerization of the slow-refolding phases, presumably by stabilizing the major beta-hairpin structure of RNase A. These interactions may contribute to the strong pH dependence of the folding and unfolding of ribonuclease A. In contrast, tyrosine 92 does not affect the folding and unfolding rates significantly. An improved "box" model of proline isomerization under unfolding conditions was derived from exhaustive fitting of all possible box models. The kinetic data support the identification of Pro93 as the proline whose isomerization distinguishes the slow-refolding species (USII and USI) from the other, faster-refolding species (Uvf, Uf, and Um), implying that Pro93 isomerizes in the slow-refolding reactions USI --> N and IN --> N. Similarly, Pro114 seems to distinguish between the very fast-refolding species Uvf and the fast-refolding species Uf. Lastly, Pro117 seems to distinguish the major slow-refolding species USII from the minor slow-refolding species USI and the medium-refolding species Um from the fast- and very fast-refolding species.
对核糖核酸酶A的三个酪氨酸到苯丙氨酸突变体（Y25F、Y92F和Y97F）的酶活性、分子稳定性以及解折叠/重折叠动力学进行了研究。这些突变体分别表现出野生型酶催化活性的80%、90%和80%。热变性、盐酸胍变性和pH转变测量表明，Y25F和Y97F比野生型蛋白稳定性更低，而大量的热力学和动力学证据表明Y92F与野生型蛋白一样稳定。摩尔消光系数的差异表明，酪氨酸25、92和97分别对折叠态和解折叠态之间的吸收差异贡献38%、13%和39%，这与之前的研究总体一致，但可能表明还有第四个酪氨酸残基贡献了其余的10%。对野生型和三个突变蛋白进行了停流单跳和双跳动力学实验。除了酪氨酸92之外，至少还有一个酪氨酸残基对缓慢的荧光解折叠阶段有贡献；这个残基可能是酪氨酸115，它监测X-Pro114肽键的顺反异构化。酪氨酸25和97参与了阻碍构象解折叠、加速构象重折叠以及缓慢重折叠阶段顺反脯氨酸异构化的相互作用，大概是通过稳定核糖核酸酶A的主要β-发夹结构来实现的。这些相互作用可能导致核糖核酸酶A折叠和解折叠对pH有强烈的依赖性。相比之下，酪氨酸92对折叠和解折叠速率没有显著影响。通过对所有可能的“盒子”模型进行详尽拟合得到了一个改进的在解折叠条件下脯氨酸异构化的“盒子”模型。动力学数据支持将Pro93鉴定为其异构化区分缓慢重折叠物种（USII和USI）与其他更快重折叠物种（Uvf、Uf和Um）的脯氨酸，这意味着Pro93在缓慢重折叠反应USI→N和IN→N中发生异构化。同样，Pro114似乎区分了非常快速重折叠物种Uvf和快速重折叠物种Uf。最后，Pro117似乎区分了主要的缓慢重折叠物种USII与次要的缓慢重折叠物种USI，以及中等重折叠物种Um与快速和非常快速重折叠物种。
