1c9x
From Proteopedia
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H119A VARIANT OF RIBONUCLEASE A
Overview
His12 and His119 are critical for catalysis of RNA cleavage by, ribonuclease A (RNase A). Substitution of either residue with an alanine, decreases the value of k(cat)/K(M) by more than 10(4)-fold. His12 and, His119 are proximal to the scissile phosphoryl group of an RNA substrate, in enzyme-substrate complexes. Here, the role of these active site, histidines in RNA binding was investigated by monitoring the effect of, mutagenesis and pH on the stability of enzyme-nucleic acid complexes., X-ray diffraction analysis of the H12A and H119A variants at a resolution, of 1.7 and 1.8 A, respectively, shows that the amino acid substitutions do, not perturb the overall structure of the variants. Isothermal titration, calorimetric studies on the complexation of wild-type RNase A and the, variants with 3'-UMP at pH 6.0 show that His12 and His119 contribute 1.4, and 1.1 kcal/mol to complex stability, respectively. Determination of the, stability of the complex of wild-type RNase A and 6-carboxyfluorescein, approximately d(AUAA) at varying pHs by fluorescence anisotropy shows that, the stability increases by 2.4 kcal/mol as the pH decreases from 8.0 to, 4.0. At pH 4.0, replacing His12 with an alanine residue decreases the, stability of the complex with 6-carboxyfluorescein approximately d(AUAA), by 2.3 kcal/mol. Together, these structural and thermodynamic data provide, the first thorough analysis of the contribution of histidine residues to, nucleic acid binding.
About this Structure
1C9X is a Single protein structure of sequence from Bos taurus with CL as ligand. Full crystallographic information is available from OCA.
Reference
Contribution of the active site histidine residues of ribonuclease A to nucleic acid binding., Park C, Schultz LW, Raines RT, Biochemistry. 2001 Apr 24;40(16):4949-56. PMID:11305910
Page seeded by OCA on Tue Nov 20 12:17:54 2007
