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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 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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-[[Image:1jn4.gif|left|200px]]<br /><applet load="1jn4" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1jn4.gif|left|200px]]<br /><applet load="1jn4" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1jn4, resolution 1.80&Aring;" />
 '''The Crystal Structure of Ribonuclease A in complex with 2'-deoxyuridine 3'-pyrophosphate (P'-5') adenosine'''<br />
 ==Overview==
-Recently, 3',5'-pyrophosphate-linked 2'-deoxyribodinucleotides were shown, to be &gt;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'--&gt;5') adenosine (CppA) and, uridine 3'-pyrophosphate (P'--&gt;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) &gt; 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'--&gt;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.
+Recently, 3',5'-pyrophosphate-linked 2'-deoxyribodinucleotides were shown to be &gt;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'--&gt;5') adenosine (CppA) and uridine 3'-pyrophosphate (P'--&gt;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) &gt; 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'--&gt;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 Structure==
-JN4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with 139 as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Pancreatic_ribonuclease Pancreatic ribonuclease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.27.5 3.1.27.5] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1JN4 OCA].
+JN4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with <scene name='pdbligand=139:'>139</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Pancreatic_ribonuclease Pancreatic ribonuclease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.27.5 3.1.27.5] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1JN4 OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Pancreatic ribonuclease]]
 [[Category: Single protein]]
-[[Category: Acharya, K.R.]]
+[[Category: Acharya, K R.]]
-[[Category: Jardine, A.M.]]
+[[Category: Jardine, A M.]]
-[[Category: Jenkins, J.L.]]
+[[Category: Jenkins, J L.]]
-[[Category: Leonidas, D.D.]]
+[[Category: Leonidas, D D.]]
 [[Category: Park, C.]]
-[[Category: Raines, R.T.]]
+[[Category: Raines, R T.]]
 [[Category: Shapiro, R.]]
 [[Category: 139]]
@@ Line 27: / Line 27: @@
 [[Category: ribonuclease]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 18:23:51 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:24:28 2008''