The exo- or endonucleolytic preference of bovine pancreatic ribonuclease A depends on its subsites structure and on the substrate size
The exo- or endonucleolytic preference of bovine pancreatic ribonuclease A depends on its subsites structure and on the substrate size
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DOI：
10.1110/ps.ps.13702
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发表时间：
2002-01-01
期刊：
影响因子：
8
通讯作者：
Nogués, MV
中科院分区：
文献类型：
--
作者：
Cuchillo, CM;Moussaoui, M;Nogués, MV
用反相高效液相色谱法研究了牛胰核糖核酸酶A（RNase A）对寡胞苷酸底物的裂解方式。通过RNA酶A消化poly（C）获得范围从二核苷酸到七核苷酸的寡胞苷酸。它们通过MALDI-TOF质谱鉴定;证实它们全部对应于通式结构（CP）（n）C >p，其中C >p表示2 '，3'-环状磷酸酯。这证实了所提出的RNA酶A的机制，其中所谓的水解（或第二）步骤实际上是转磷酸化（第一步）的逆转的特殊情况。对寡核苷酸底物的切割模式表明，天然酶对内切或外切没有特别的偏好，而酶的突变体对内切或外切没有特别的偏好。（K7 Q/R10 Q-RNase A）缺乏p（2）（在3'侧与主要磷酸结合位点p（1）相邻的磷酸结合亚位点）显示出明显的核酸外切模式;缺乏p（0）（在5 ′侧与主要磷酸结合位点p（1）相邻的磷酸结合亚位点）的突变体（K66 Q-RNase A）显示出更多的核酸内切模式。这表明了亚位点对键断裂的优先性所起的重要作用。分子模拟表明，在p（2）突变体的情况下，谷氨酰胺的酰胺基团可以与2 '，3'-环状末端磷酸形成氢键，而到3 '，5'-磷酸二酯键的距离太长，无法形成这样的氢键。这可以解释p（2）突变体对核酸外切的偏好。
The cleavage pattern of oligocytidylic acid substrates by bovine pancreatic ribonuclease A (RNase A) was studied by means of reversed-phase HPLC. Oligocytidylic acids, ranging from dinucleotides to heptanucleotides, were obtained by RNase A digestion of poly(C). They were identified by MALDI-TOF mass spectrometry; it was confirmed that all of them corresponded to the general structure (CP)(n)C >p, in which C >p indicates a 2',3'-cyclic phosphate. This is a confirmation of the proposed mechanism for RNase A, wherein the so-called hydrolytic (or second) step is in fact a special case of the reverse of transphosphorylation (first step). The patterns of cleavage for the oligonucleotide substrates show that the native enzyme has no special preference for endonucleolytic or exonucleolytic cleavage, whereas a mutant of the enzyme (K7Q/R10Q-RNase A) lacking p(2) (a phosphate binding subsite adjacent, on the 3' side, to the main phosphate binding site p(1)) shows a clear exonucleolytic pattern; a mutant (K66Q-RNase A) lacking p(0) (a phosphate binding subsite adjacent, on the 5' side, to the main phosphate binding site p(1)) shows a more endonucleolytic pattern. This indicates the important role played by the subsites on the preference for the bond cleaved. Molecular modeling shows that, in the case of the p(2) mutant, the amide group of glutamine can form a hydrogen bond with the 2',3'-cyclic terminal phosphate, whereas the distance to a 3',5'-phosphodiester bond is too long to form such a hydrogen bond. This could explain the preference for exonucleolytic cleavage shown by the p(2) mutant.