Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2′-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/ base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the 18 O KIEs on the 2′O nucleophile, 5′O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium-or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5′O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/ Lys41), together with proton transfer from His119, render departure of the 5′O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2′-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure. E nzymes achieve powerful rate enhancements by providing multiple catalytic modes to stabilize reaction transition states, including electrostatic interactions and proton transfer (1). For RNA 2′-O-transphosphorylation reactions, interactions with acid, base, or metal ion catalysts in solution can influence transition state structure (2). Understanding biological catalysis therefore requires knowledge of chemical mechanisms, transition state structure, and transition state interactions for both enzymatic and nonenzymatic RNA cleavage reactions. Comparisons of enzymatic and nonenzymatic catalysis can provide information on which catalytic modes are used by enzymes and whether unique features of the active site environment may alter the transition state charge distribution (3).RNA undergoes two competing transesterification reactions in solution: isomerization to a 2′,5′-phosphodiester and 2′-Otransphosphorylation to yield a cyclic 2′,3′-phosphodiester with concomitant release of the 5′O-nucleoside (4, 5). Reactions catalyzed by acid and by buffers yield both isomerization and cleavage products and proceed via a pentacoordinated phosphorane intermediate formed by an attack of the 2′OH at the adja...

We introduce a new hybrid molecular orbital/density-functional modified divide-and-conquer (mDC) approach that allows the linear-scaling calculation of very large quantum systems. The method provides a powerful framework from which linear-scaling force fields for molecular simulations can be developed. The method is variational in the energy, and has simple, analytic gradients and essentially no break-even point with respect to the corresponding full electronic structure calculation. Furthermore, the new approach allows intermolecular forces to be properly balanced such that non-bonded interactions can be treated, in some cases, to much higher accuracy than the full calculation. The approach is illustrated using the second-order self-consistent charge density-functional tight-binding model (DFTB2). Using this model as a base Hamiltonian, the new mDC approach is applied to a series of water systems, where results show that geometries and interaction energies between water molecules are greatly improved relative to full DFTB2. In order to achieve substantial improvement in the accuracy of intermolecular binding energies and hydrogen bonded cluster geometries, it was necessary to extend the DFTB2 model to higher-order atom-centered multipoles for the second-order self-consistent intermolecular electrostatic term. Using generalized, linear-scaling electrostatic methods, timings demonstrate that the method is able to calculate a water system of 3000 atoms in less than half of a second, and systems of up to one million atoms in only a few minutes using a conventional desktop workstation.