1jn4
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The Crystal Structure of Ribonuclease A in complex with 2'-deoxyuridine 3'-pyrophosphate (P'-5') adenosine
OverviewOverview
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.
About this StructureAbout this Structure
1JN4 is a Single protein structure of sequence from Bos taurus with 139 as ligand. Active as Pancreatic ribonuclease, with EC number 3.1.27.5 Full crystallographic information is available from OCA.
ReferenceReference
Cleavage of 3',5'-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin., Jardine AM, Leonidas DD, Jenkins JL, Park C, Raines RT, Acharya KR, Shapiro R, Biochemistry. 2001 Aug 28;40(34):10262-72. PMID:11513604
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