Jardine A M, Leonidas D D, Jenkins J L, Park C, Raines R T, Acharya K R, Shapiro R
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 2001 Aug 28;40(34):10262-72. doi: 10.1021/bi010888j.
Recently, 3',5'-pyrophosphate-linked 2'-deoxyribodinucleotides were shown to be >100-fold more effective inhibitors of RNase A superfamily enzymes than were the corresponding monophosphate-linked (i.e., standard) dinucleotides. Here, we have investigated two ribo analogues of these compounds, cytidine 3'-pyrophosphate (P'-->5') adenosine (CppA) and uridine 3'-pyrophosphate (P'-->5') adenosine (UppA), as potential substrates for RNase A and angiogenin. CppA and UppA are cleaved efficiently by RNase A, yielding as products 5'-AMP and cytidine or uridine cyclic 2',3'-phosphate. The k(cat)/K(m) values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the K(m) values (10-16 microM) are lower than those reported for any earlier small substrates (e.g., 500-700 microM for CpA and UpA). The k(cat)/K(m) value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on K(m) is more modest. Ribonucleotide 3',5'-pyrophosphate linkages were proposed previously to exist in nature as chemically labile intermediates in the pathway for the generation of cyclic 2',3'-phosphate termini in various RNAs. We demonstrate that in fact they are relatively stable (t(1/2) > 15 days for uncatalyzed degradation of UppA at pH 6 and 25 degrees C) and that cleavage in vivo is most likely enzymatic. Replacements of the RNase A catalytic residues His12 and His119 by alanine reduce activity toward UppA by approximately 10(5)-and 10(3.3)-fold, respectively. Thus, both residues play important roles. His12 probably acts as a base catalyst in cleavage of UppA (as with RNA). However, the major function of His119 in RNA cleavage, protonation of the 5'-O leaving group, is not required for UppA cleavage because the pK(a) of the leaving group is much lower than that for RNA substrates. A crystal structure of the complex of RNase A with 2'-deoxyuridine 3'-pyrophosphate (P'-->5') adenosine (dUppA), determined at 1.7 A resolution, together with models of the UppA complex based on this structure suggest that His119 contributes to UppA cleavage through a hydrogen bond with a nonbridging oxygen atom in the pyrophosphate and through pi-pi stacking with the six-membered ring of adenine.
最近研究表明，3',5'-焦磷酸连接的2'-脱氧核糖二核苷酸对核糖核酸酶A超家族酶的抑制作用比相应的单磷酸连接（即标准）二核苷酸强100倍以上。在此，我们研究了这些化合物的两种核糖类似物，胞苷3'-焦磷酸（P'→5'）腺苷（CppA）和尿苷3'-焦磷酸（P'→5'）腺苷（UppA），作为核糖核酸酶A和血管生成素的潜在底物。CppA和UppA能被核糖核酸酶A有效切割，产生5'-AMP和胞苷或尿苷环状2',3'-磷酸作为产物。其催化常数与米氏常数的比值（k(cat)/K(m)）仅比标准二核苷酸CpA和UpA小4倍，且米氏常数（10 - 16μM）低于此前报道的任何小分子底物（例如，CpA和UpA为500 - 700μM）。CppA与血管生成素的k(cat)/K(m)值也仅比CpA小几倍，但核苷酸间连接延长对米氏常数的影响较小。此前有人提出核糖核苷酸3',5'-焦磷酸连接在自然界中作为各种RNA生成环状2',3'-磷酸末端途径中的化学不稳定中间体存在。我们证明实际上它们相对稳定（在pH 6和25℃下，UppA未催化降解的半衰期t(1/2) > 15天），且体内切割很可能是酶促反应。将核糖核酸酶A催化残基His12和His119替换为丙氨酸后，对UppA的活性分别降低约10^5倍和10^3.3倍。因此，这两个残基都起重要作用。His12可能在UppA切割中起碱催化剂作用（与RNA切割时一样）。然而，His119在RNA切割中的主要功能，即5'-O离去基团的质子化，在UppA切割中并非必需，因为离去基团的pK(a)远低于RNA底物。以1.7 Å分辨率测定的核糖核酸酶A与2'-脱氧尿苷3'-焦磷酸（P'→5'）腺苷（dUppA）复合物的晶体结构，以及基于该结构的UppA复合物模型表明，His119通过与焦磷酸中非桥连氧原子形成氢键以及与腺嘌呤六元环形成π-π堆积，对UppA切割起作用。
