Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis

• Published in: Journal of molecular biology Vol. 317; no. 1; pp. 21 - 40
• Main Authors: Campbell, Frank E, Cassano, Adam G, Anderson, Vernon E, Harris, Michael E
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 15.03.2002
• Subjects: Base Sequence; Catalysis; Cations, Divalent - metabolism; endonuclease; Endoribonucleases - chemistry; Endoribonucleases - metabolism; enzyme kinetics; Escherichia coli; Escherichia coli - enzymology; Escherichia coli Proteins; Fluorescence; Hydrogen-Ion Concentration; Kinetics; Nucleic Acid Conformation; phosphodiesterase; Protein Binding; Protein Conformation; ribonuclease II; Ribonuclease III; RNA - chemistry; RNA - genetics; RNA - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Solvents; stopped-flow fluorescence; Thermodynamics; endonuclease; ribonuclease III; stopped-flow fluorescence; dsRBD; phosphodiesterase; RRM; enzyme kinetics
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2002.5413

To better understand substrate recognition and catalysis by RNase III, we examined steady-state and pre-steady-state reaction kinetics, and changes in intrinsic enzyme fluorescence. The multiple turnover cleavage of a model RNA substrate shows a pre-steady-state burst of product formation followed by a slower phase, indicating that the steady-state reaction rate is not limited by substrate cleavage. RNase III catalyzed hydrolysis is slower at low pH, permitting the use of pre-steady-state kinetics to measure the dissociation constant for formation of the enzyme-substrate complex ( K d=5.4(±0.6) nM), and the rate constant for phosphodiester bond cleavage ( k c=1.160(±0.001) min −1, pH 5.4). Isotope incorporation analysis shows that a single solvent oxygen atom is incorporated into the 5′ phosphate of the RNA product, which demonstrates that the cleavage step is irreversible. Analysis of the pH dependence of the single turnover rate constant, k c, fits best to a model for two or more titratable groups with p K a of ca 5.6, suggesting a role for conserved acidic residues in catalysis. Additionally, we find that k c is dependent on the p K a value of the hydrated divalent metal ion included in the reaction, providing evidence for participation of a metal ion hydroxide in catalysis, potentially in developing the nucleophile for the hydrolysis reaction. In order to assess whether conformational changes also contribute to the enzyme mechanism, we monitored intrinsic tryptophan fluorescence. During a single round of binding and cleavage by the enzyme we detect a biphasic change in fluorescence. The rate of the initial increase in fluorescence was dependent on substrate concentration yielding a second-order rate constant of 1.0(±0.1)×10 8 M −1 s −1, while the rate constant of the second phase was concentration independent (6.4(±0.8) s −1; pH 7.3). These data, together with the unique dependence of each phase on divalent metal ion identity and pH, support the hypothesis that the two fluorescence transitions, which we attribute to conformational changes, correlate with substrate binding and catalysis.