The binding of IMP to Ribonuclease A

Abstract

The binding of inosine 5′ phosphate (IMP) to ribonuclease A has been studied by kinetic and X‐ray crystallographic experiments at high (1.5 Å) resolution. IMP is a competitive inhibitor of the enzyme with respect to C>p and binds to the catalytic cleft by anchoring three IMP molecules in a novel binding mode. The three IMP molecules are connected to each other by hydrogen bond and van der Waals interactions and collectively occupy the B1R1P1B2P0P‐1 region of the ribonucleolytic active site. One of the IMP molecules binds with its nucleobase in the outskirts of the B2 subsite and interacts with Glu111 while its phosphoryl group binds in P1. Another IMP molecule binds by following the retro‐binding mode previously observed only for guanosines with its nucleobase at B1 and the phosphoryl group in P‐1. The third IMP molecule binds in a novel mode towards the C‐terminus. The RNase A–IMP complex provides structural evidence for the functional components of subsite P‐1 while it further supports the role inferred by other studies to Asn71 as the primary structural determinant for the adenine specificity of the B2 subsite. Comparative structural analysis of the IMP and AMP complexes highlights key aspects of the specificity of the base binding subsites of RNase A and provides a structural explanation for their potencies. The binding of IMP suggests ways to develop more potent inhibitors of the pancreatic RNase superfamily using this nucleotide as the starting point.

Influence of naturally‐occurring 5′‐pyrophosphate‐linked substituents on the binding of adenylic inhibitors to ribonuclease a: An X‐ray crystallographic study

“…The 67-fold drop in K i experienced when moving from 5′-AMP to 5′-ADP coincides with a radical change in nucleotide conformation (Class Ia/Ib to Class II; Table I). 22,26 We now present the crystal structure of the RNase A·5′-ATP complex. The electron density map indicates the position of the base, the β- and the γ-phosphate but there is significant disorder in between (Figure 2a).…”

The crystal structure of ribonuclease A in complex with thymidine‐3′‐monophosphate provides further insight into ligand binding

“…In their simplest mononucleotide product form, 5′‐purines and 3′‐pyrimidines [Fig. 1(B)] are also competitive inhibitors of the reaction (with micromolar affinities) and crystal structures of the natural RNA single nucleotides 5′‐AMP (PDB accession code: 1Z6S)21, 5′‐GMP (PDB accession code: 1RNC)22, 3′‐CMP (PDB accession code: 1RPF),23 and 3′‐UMP (PDB accession code: 1O0N)24 were previously determined. These structures offer insight into the ground state stabilization of these competitive inhibitors and offer basis for the design of new tight‐binding inhibitors to various members of the ribonuclease superfamily.…”

Recognition of Ribonuclease A by 3′–5′-Pyrophosphate-Linked Dinucleotide Inhibitors: A Molecular Dynamics/Continuum Electrostatics Analysis

“…Based on this expectation, structureassisted inhibitor design studies have mainly focused on RNase A. Structural and kinetic studies have examined the complexes between RNase A and several mono-or dinucleotide inhibitors containing adenine (10)(11)(12)(13)(14)(15)(16)(17) or inosine (18) at the 39 position of the scissile bond, and uracil (13)(14)(15)(16)(17) or cytosine (11,12) at the 59 position. The high-resolution structures of several complexes have provided a detailed picture of the inhibitor binding modes.…”