title: The pH-dependence of the Escherichia coli RNase HII-catalysed reaction suggests that an active site carboxylate group participates directly in catalysis.
abstract: RNase HII specifically catalyses the hydrolysis of phosphate diester linkages contained within the RNA portion of DNA/RNA hybrids. The catalytic parameters of the enzyme derived from Escherichia coli BL21 have been measured using 5'-fluorescent oligodeoxynucleotide substrates containing embedded ribonucleotides. The products of the reaction and the chemistry of phosphate diester hydrolysis were assigned unequivocally using mass spectrometry. The pH-dependence of the catalytic parameters was measured under conditions of optimal magnesium ion concentration. The logarithm of the turnover number of the enzyme increases steeply with pH until a pH-independent region is reached close to neutrality. The slope of the pH-dependent region is 2, indicating that the catalytically proficient form of RNase HII is di-anionic. The pH-dependence of log 1/K(M) is a sigmoidal curve reaching a maximal value at higher pH, suggesting deprotonation of a residue stabilises substrate binding. Possible mechanisms for the RNase HII-catalysed reaction consistent with the pH-dependent behaviour of the enzyme are discussed. The active sites of RNase H enzymes contain a cluster of four strictly conserved carboxylate groups. Together, the data suggest a requirement for ionisation of an active site carboxylic acid for metal ion binding or correct positioning of metal ion(s) in the enzyme-substrate complex and a role for a second active site carboxylate in general base catalysis.
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, University of Sheffield, Sheffield, S3 7HF, UK.
authors.forename: James A
authors.initials: JA
authors.lastname: Bastock
authors.forename: Michelle
authors.initials: M
authors.lastname: Webb
authors.forename: Jane A
authors.initials: JA
authors.lastname: Grasby
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002264:Carboxylic Acids / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016914:Ribonuclease H / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002264:Carboxylic Acids; C091139:ribonuclease HII; D016914:Ribonuclease H