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首页> 外文期刊>Journal of Molecular Biology >A RESIDUE TO RESIDUE HYDROGEN BOND MEDIATES THE NUCLEOTIDE SPECIFICITY OF RIBONUCLEASE A
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A RESIDUE TO RESIDUE HYDROGEN BOND MEDIATES THE NUCLEOTIDE SPECIFICITY OF RIBONUCLEASE A

机译：残基氢键残基介导核糖核酸酶A的核苷酸特异性

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摘要

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O-5' bond of RNA after residues bound in the enzyme's B1 subsite. This subsite binds to cytidine 30-fold more tightly than to uridine and >10(5)-fold more tightly than to adenine. Structural studies had suggested that the hydroxyl group of Thr45 can interact directly with the base of a bound nucleotide. In contrast, the carboxylate group of Asp83 cannot interact directly with bound substrate hut can accept a hydrogen bond from the hydroxyl group of Thr45. To assess the role of the Thr45-Asp83 hydrogen bond in catalysis, T45G, D83A and T45G/D83A RNase A were prepared and their abilities to catalyze the cleavage of various substrates were determined. The results indicate that the side-chain of Asp83 enhances catalysis of reactions in which uridine is bound in the B1 subsite, but that this enhancement relies on the side-chain of Thr45. In contrast, the side-chain of Asp83 does not contribute to catalysis of reactions with cytidine in the B1 subsite. Thermodynamic cycles derived from kinetic parameters for the cleavage of poly(U) indicate that the Thr45-Asp83 interaction contributes 1.2 kcal/mol to transition state stabilization, which is 0.9 kcal/mol greater than its contribution to ground state stabilization. Thus, like many residue-substrate interactions, this residue to residue interaction enhances catalysis by becoming stronger as the reaction approaches the transition state. (C) 1995 Academic Press Limited [References: 49]
机译：牛胰核糖核酸酶A（RNase A）催化结合在酶B1亚位点上的残基后，切割RNA的P-O-5'键。该亚位点与胞苷的结合力比尿苷高30倍，与腺嘌呤的结合力> 10（5）-倍。结构研究表明，Thr45的羟基可直接与结合核苷酸的碱基相互作用。相反，Asp83的羧酸盐基团不能直接与结合的底物相互作用，但可以接受来自Thr45羟基的氢键。为了评估Thr45-Asp83氢键在催化中的作用，制备了T45G，D83A和T45G / D83A RNase A，并确定了它们催化各种底物裂解的能力。结果表明，Asp83的侧链增强了尿苷结合在B1亚位点上的反应的催化作用，但是这种增强依赖于Thr45的侧链。相反，Asp83的侧链对B1亚位点中胞嘧啶核苷的反应没有催化作用。从动力学参数得出的裂解Poly（U）的热力学循环表明，Thr45-Asp83相互作用对过渡态稳定的贡献为1.2 kcal / mol，比对基态稳定的贡献大0.9 kcal / mol。因此，就像许多残基-底物相互作用一样，随着反应接近过渡态，该残基对残基的相互作用通过变得更强而增强了催化作用。 （C）1995 Academic Press Limited [参考号：49]

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