STAPHYLOCOCCAL NUCLEASE - PROPOSED MECHANISM OF ACTION BASED ON STRUCTURE OF ENZYME-THYMIDINE 3',5'-BISPHOSPHATE-CALCIUM ION COMPLEX AT 1.5-A RESOLUTION
STAPHYLOCOCCAL NUCLEASE - PROPOSED MECHANISM OF ACTION BASED ON STRUCTURE OF ENZYME-THYMIDINE 3',5'-BISPHOSPHATE-CALCIUM ION COMPLEX AT 1.5-A RESOLUTION
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DOI：
10.1073/pnas.76.6.2551
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发表时间：
1979-01-01
影响因子：
11.1
通讯作者：
LEGG, MJ
中科院分区：
文献类型：
--
作者：
COTTON, FA;HAZEN, EE;LEGG, MJ
金黄色葡萄球菌核酸酶(EC 3.1.4.7)-胸腺嘧啶核苷3‘’，5‘-二磷酸-Ca~(2+)(酶-抑制剂)复合体的结构通过大量的补充数据和基于电子密度图修正过程的相精化方案扩展到1.5。通过将这种结构与酶的已知性质相关联，提出了一种作用机制，即与5‘’-CH2O(H)离开基团相一致的与Glu-43结合的水分子对Ph的亲核攻击。Glu-43的羧酸盐通过充当从攻击的水分子中提取质子的一般碱来促进这种攻击。通过钙离子和磷酸盐O原子之间的离子相互作用以及Arg-35和Arg-87的胍离子与磷酸盐O原子的4H键的极化，进一步促进了亲核攻击。这些相互作用也可能通过降低三角双锥过渡态的能量来催化反应。核酸底物的水解通过裂解5‘’sbd.P.sbd.O键产生自由的5‘’-羟基和末端的3‘’-磷酸单酯基团进行。在抑制剂络合物中，唯一能向剩余的5‘’-O提供质子的一般酸基是Arg-87的胍离子。目前缺乏直接结构支持的替代质子给体可能是Tyr-113的酚羟基或水分子。尤其值得注意的是，反应物在活性中心的位置的精密度和刚性，以及可能的鸟粪离子与Arg-35和Arg-87的双重结合和催化作用。
The structure of the staphylococcal [Staphylococcus aureus] nuclease (EC 3.1.4.7)-thymidine 3'',5''-bisphosphate-Ca2+ (enzyme-inhibitor) complex was extended to 1.5 .ANG. resolution by using much additional data and a phase refinement scheme based on an electron-density map modification procedure. By correlating this structure with the known properties of the enzyme, a mechanism of action is proposed that involves nucleophilic attack on Ph by a water molecule, which is bound to Glu-43, in line with the 5''-CH2O(H) leaving group. The carboxylate of Glu-43 promotes this attack by acting as a general base for the abstraction of a proton from the attacking water molecule. Nucleophilic attack is further faciliatated by polarization of the phosphodiester by an ionic interaction between a Ca2+ ion and a phosphate O atom and by 4 H bonds to phosphate O atoms from guanidinium ions of Arg-35 and Arg-87. These interactions may also catalyze the reaction by lowering the energy of a trigonal bipyramidal transition state. The hydrolysis of nucleic acid substrate proceeds by cleavage of the 5''.sbd.P.sbd.O bond to yield a free 5''-hydroxyl group and a terminal, 3''-phosphate monoester group. In the inhibitor complex the only general acid group found in a position to donate a proton to the leaving 5''-O is the guanidinium ion of Arg-87. Alternative proton donors, presently lacking direct structural support, could be the phenolic hydroxyl group of Tyr-113 or a water molecule. The precision and rigidity of the location of the reactants at the active site and the probable dual binding and catalytic roles of the guanidinium ions of Arg-35 and Arg-87 are especially noteworthy.