A RESIDUE TO RESIDUE HYDROGEN-BOND MEDIATES THE NUCLEOTIDE SPECIFICITY OF RIBONUCLEASE-A
A RESIDUE TO RESIDUE HYDROGEN-BOND MEDIATES THE NUCLEOTIDE SPECIFICITY OF RIBONUCLEASE-A
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DOI：
10.1006/jmbi.1995.0500
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发表时间：
1995-09-22
影响因子：
5.6
通讯作者：
RAINES, RT
中科院分区：
文献类型：
--
作者：
DELCARDAYRE, SB;RAINES, RT
牛胰腺核糖核酸酶A（RNase A）催化结合在酶的B1亚位点中的残基后RNA的P-O-5'键的切割。该亚位点与胞苷的结合比与尿苷的结合紧密30倍，并且比与腺嘌呤的结合紧密>10（5）倍。结构研究表明，Thr 45的羟基可以直接与结合核苷酸的碱基相互作用。相比之下，Asp 83的羧酸酯基团不能直接与结合的底物相互作用，但可以接受来自Thr 45的羟基的氢键。为了评估Thr 45-Asp 83氢键在催化中的作用，制备了T45 G、D83 A和T45 G/D83 A RNA酶A，并测定了它们催化各种底物裂解的能力。结果表明Asp 83的侧链增强了尿苷结合在B1亚位点的反应的催化作用，但这种增强依赖于Thr 45的侧链。相比之下，Asp 83的侧链不有助于催化与B1亚位点中的胞苷的反应。热力学循环从动力学参数的裂解聚（U）表明，Thr 45-Asp 83相互作用有助于1.2千卡/摩尔的过渡态稳定，这是0.9千卡/摩尔大于其贡献的基态稳定。因此，像许多残基-底物相互作用一样，这种残基与残基的相互作用随着反应接近过渡态而变得更强，从而增强催化作用。(C)1995年学术出版社
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