Contribution of the active site histidine residues of ribonuclease A to nucleic acid binding.
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.
Park
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Department of Biochemistry and Department of Chemistry, University of Wisconsin-Madison, 53706, USA.
Schultz
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LW
Raines
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RT
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D018360:Crystallography, X-Ray; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; C024098:6-carboxyfluorescein; D006639:Histidine; C031034:3'-uridylic acid; D004247:DNA; D014542:Uridine Monophosphate; D012259:Ribonuclease, Pancreatic; D000409:Alanine