Cleavage of 3‘,5‘-Pyrophosphate-Linked Dinucleotides by Ribonuclease A and Angiogenin,

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Abstract
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 kcat/Km values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the Km values (10−16 μM) are lower than those reported for any earlier small substrates (e.g., 500−700 μM for CpA and UpA). The kcat/Km value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on Km 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 (t1/2 > 15 days for uncatalyzed degradation of UppA at pH 6 and 25 °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 ∼105-and 103.3-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 pKa 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 Å 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 π−π stacking with the six-membered ring of adenine.