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首页> 外文期刊>Biochemistry >Coulombic forces in protein-RNA interactions: Binding and cleavage by ribonuclease A and variants at Lys7, Arg10, and Lys66
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Coulombic forces in protein-RNA interactions: Binding and cleavage by ribonuclease A and variants at Lys7, Arg10, and Lys66

机译：蛋白质-RNA相互作用中的库仑力：核糖核酸酶A及其在Lys7，Arg10和Lys66上的变体的结合和切割

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摘要

The interactions between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extend beyond the scissile P-O-5' bond. Enzymic subsites interact with the bases and phosphoryl groups of the bound substrate. Those residues interacting with the phosphoryl group comprise the PO, P1, and P2 subsites, with the scissile bond residing in the P1 subsite. Here, the function of the PO and P2 subsites of RNase A is characterized in detail. Lys66 (PO subsite) and Lys7 and Arg10 (P2 subsite) were replaced with alanine residues. Wild-type RNase A and the K66A, K7A/R10A, and K7A/R10A/K66A variants were evaluated as catalysts for the cleavage of poly(cytidylic acid) [poly(C)] and for their abilities to bind to single-stranded DNA, a substrate analogue. The values of k(cat) and K-m for poly(C) cleavage were affected by altering the PO and P2 subsites. The k(cat)/K-m values for poly(C) cleavage by the K66A, K7A/R10A, and K7A/R10A/K66A variants were 3-fold, 60-fold, and 300-fold lower, respectively, than that of wild-type RNase A. These values indicate that the PO and P2 subsites contribute 0.70 and 2.46 kcal/mol, respectively, to transition-state binding. Binding experiments indicate that the PO and P2 subsites contribute 0.92 and 1.21 kcal/mol, respectively, to ground-state binding. Thus, the PO subsite makes a uniform contribution toward binding the ground state and the transition state, whereas the P2 subsite differentiates, binding more tightly to the transition state than to the ground state. In addition, nucleic acid binding to wild-type RNase A is strongly dependent on NaCl concentration, but this dependence is diminished upon alteration of the PO or P2 subsite. The logarithm of Kd is a Linear function of the logarithm of [Na+] over the range 0.018 M less than or equal to [Na+] less than or equal to 0.14 M, with partial derivative log K-d/partial derivative log [Na+] = 2.3 +/- 0.1, 1.8 +/- 0.1, 1.4 +/- 0.1, and 0.9 +/- 0.2 for nucleic acid binding to wild-type RNase A and the K66A, K7A/R10A, and K7A/R10A/K66A variants, respectively. Similar experiments with NaF and the wild-type enzyme yield a log K-d/partial derivative log [Na+] = 2.0 +/- 0.2, indicating that the anion makes only a small contribution to nucleic acid binding. Together these data provide a detailed picture of the contributions of Coulombic interactions to binding and catalysis by RNase A, and illuminate the general role of Coulombic forces between proteins and nucleic acids. [References: 93]
机译：牛胰腺核糖核酸酶A（RNase A）与其RNA底物之间的相互作用超出了易裂的P-O-5'键。酶亚位点与结合的底物的碱基和磷酸基相互作用。与磷酰基相互作用的那些残基包括PO，P1和P2亚位点，易断裂键位于P1亚位点中。在此，详细描述了RNA酶A的PO和P2亚位点的功能。 Lys66（PO亚位点）和Lys7和Arg10（P2亚位点）被丙氨酸残基取代。评价野生型RNase A和K66A，K7A / R10A和K7A / R10A / K66A变异体作为裂解聚胞苷酸[poly（C）]的催化剂，以及它们与单链DNA结合的能力，底物类似物。聚（C）裂解的k（cat）和K-m值受PO和P2亚位点改变的影响。 K66A，K7A / R10A和K7A / R10A / K66A变体切割poly（C）的k（cat）/ Km值分别比野生型低3倍，60倍和300倍这些值表明PO和P2亚位点分别对过渡态结合贡献0.70和2.46 kcal / mol。结合实验表明，PO和P2亚位点分别对基态结合贡献0.92和1.21 kcal / mol。因此，PO亚位点对结合基态和过渡态做出了统一的贡献，而P2亚位点分化，与过渡态的结合比与基态的结合更紧密。另外，与野生型RNase A结合的核酸强烈依赖于NaCl的浓度，但是这种依赖因PO或P2亚位点的改变而减弱。 Kd的对数是[Na +]的对数在小于或等于[Na +]小于或等于0.14 M的范围内的对数的线性函数，偏导数log Kd /偏导数log [Na +] = 2.3 +/- 0.1、1.8 +/- 0.1、1.4 +/- 0.1和0.9 +/- 0.2用于核酸分别与野生型RNase A和K66A，K7A / R10A和K7A / R10A / K66A变体结合。用NaF和野生型酶进行的类似实验得出log K-d /偏导数log [Na +] = 2.0 +/- 0.2，表明该阴离子对核酸结合的贡献很小。这些数据共同提供了库仑相互作用对RNase A结合和催化作用的详细描述，并阐明了蛋白质和核酸之间库仑力的一般作用。 [参考：93]

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