PDBsum entry 1jn4

Hydrolase

PDB id: 1jn4

Contents

Protein chains

124 a.a. *

Ligands

139

Waters ×265

* Residue conservation analysis

PDB id:

1jn4

Name:

Hydrolase

Title:

The crystal structure of ribonuclease a in complex with 2'- deoxyuridine 3'-pyrophosphate (p'-5') adenosine

Structure:

Pancreatic ribonuclease a. Chain: a, b. Synonym: rnase 1. Rnase a. Ec: 3.1.27.5

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Organ: pankreas

Resolution:

1.80Å R-factor: 0.225 R-free: 0.295

Authors:

A.M.Jardine,D.D.Leonidas,J.L.Jenkins,C.Park,R.T.Raines,K.R.Acharya, R.Shapiro

Key ref:

A.M.Jardine et al. (2001). Cleavage of 3',5'-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin. Biochemistry, 40, 10262-10272. PubMed id: 11513604 DOI: 10.1021/bi010888j

Date:

23-Jul-01 Release date: 03-Jun-03

Protein chains

P61823  (RNAS1_BOVIN) -  Ribonuclease pancreatic from Bos taurus

Seq: 150 a.a.

Struc: 124 a.a.

Enzyme reactions

• Enzyme class: E.C.4.6.1.18 - pancreatic ribonuclease.
• Reaction:
  1. an [RNA] containing cytidine + H2O = an [RNA]-3'-cytidine- 3'-phosphate + a 5'-hydroxy-ribonucleotide-3'-[RNA]
  2. an [RNA] containing uridine + H2O = an [RNA]-3'-uridine-3'-phosphate + a 5'-hydroxy-ribonucleotide-3'-[RNA]

DOI no: 10.1021/bi010888j

Biochemistry 40:10262-10272 (2001)

PubMed id: 11513604

Cleavage of 3',5'-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin.

A.M.Jardine, D.D.Leonidas, J.L.Jenkins, C.Park, R.T.Raines, K.R.Acharya, R.Shapiro.

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 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.