Integration of kinetic isotope effect analyses to elucidate ribonuclease mechanism.
The well-studied mechanism of ribonuclease A is believed to involve concerted general acid-base catalysis by two histidine residues, His12 and His119. The basic features of this mechanism are often cited to explain rate enhancement by both protein and RNA enzymes that catalyze RNA 2'-O-transphosphorylation. Recent kinetic isotope effect analyses and computational studies are providing a more chemically detailed description of the mechanism of RNase A and the rate limiting transition state. Overall, the results support an asynchronous mechanism for both solution and ribonuclease catalyzed reactions in which breakdown of a transient dianoinic phosphorane intermediate by 5'OP bond cleavage is rate limiting. Relative to non-enzymatic reactions catalyzed by specific base, a smaller KIE on the 5'O leaving group and a less negative βLG are observed for RNase A catalysis. Quantum mechanical calculations consistent with these data support a model in which electrostatic and H-bonding interactions with the non-bridging oxygens and proton transfer from His119 render departure of the 5'O less advanced and stabilize charge buildup in the transition state. Both experiment and computation indicate advanced 2'OP bond formation in the rate limiting transition state. However, this feature makes it difficult to resolve the chemical steps involved in 2'O activation. Thus, modeling the transition state for RNase A catalysis underscores those elements of its chemical mechanism that are well resolved, as well as highlighting those where ambiguity remains. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment.
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44118, United States. Electronic address: meh2@cwru.edu.
Michael E
ME
Harris
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, United States; Department of Chemistry, University of Chicago, Chicago, IL 60637, United States.
Joseph A
JA
Piccirilli
Center for Integrative Proteomics Research, BioMaPS Institute for Quantitative Biology, Rutgers University, Piscataway, NJ 08854, United States; Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, United States.
Darrin M
DM
York
D055162:Biocatalysis; D020134:Catalytic Domain; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010103:Oxygen Isotopes / Q000737:chemistry; D010766:Phosphorylation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism
D010103:Oxygen Isotopes; D012313:RNA; D012259:Ribonuclease, Pancreatic
Enzyme catalysis; Kinetic isotope effect; Ribonuclease; Transition state