Martin Karplus, Ioan Andricioaei, Guo Hong, Fabrice Leclerc, Paul Lyne,
and Cui Qiang, Harvard University
Paul Bash, Northwestern University Medical School

Research Objectives

The goal of this project is to develop a greater understanding of the mechanisms involved in enzyme catalysis and related protein functions. We are studying two types of enzymes: nucleic acids (specifically hammerhead ribozyme) and proteins.

Computational Approach

Our methods are based on the physical and chemical principles of statistical mechanics and quantum mechanics, implemented in computational form using techniques from computational chemistry. For example, we use the structural data as inputs for molecular dynamics simulations to determine the structural and dynamics properties of enzyme systems. Given the reactive conformation, the chemical steps involved in the reaction mechanism are studied using ab initio QM on model systems in the gas phase and the hybrid semiempirical and ab initio quantum-classical methods (QM/MM) implemented in the CHARMM program.

Accomplishments

	X-ray structure of the hammerhead ribozyme. The RNA backbone is represented by a white ribbon. The active site region, treated by quantum mechanics, is included in a yellow circle, and the phosphodiester bond cleaved during the reaction is indicated by a purple arrow. The metal cations (Mg2+) used as cofactors are represented by CPK spheres.

Simulations of the hammerhead ribozyme several nanoseconds in length have been performed for a system consisting of the hammerhead plus counterions to neutralize the backbone phosphate charges, all solvated with explicit water molecules. The simulation system is stable but some conformational changes do occur. In particular, the two arms which are constrained in the crystal structure undergo relative displacements of about two angstroms. As a result of the motion, the parts of the ribozyme involved in the catalysis undergo a conformational change. The details of the changes are being examined to provide an appropriate system for initiating the quantum and molecular mechanics (QM/MM) on the ribozyme.

Ab initio studies of the reaction path of phosphate ester hydrolysis of a model for the hammerhead ribozyme have also been completed, and the effects of thio-substitution at the pro-R site and at the leaving group have been studied. A theoretical estimate of the acid-base ionization constant (pKa) of the 2'-OH at the active site of hammerhead ribozymes was made based on quantum chemistry calculations of a phosphorylated ribose. The large value found (pKa = 14.9) has implications for the mechanism of hammerhead ribozyme catalysis and more specifically for the role played by metal ions in the reaction.

Significance

The dynamic properties of proteins and nucleic acids are difficult to investigate experimentally, but they are essential for an understanding of their function. Computer simulations can provide the necessary insights, at an atomic level of detail, for a complete understanding of the relationship between biomolecular dynamics/structure and function.

Publications

F. Leclerc and M. Karplus, "MCSS based predictions of RNA binding sites," Theoretical Chemistry Accounts 101, 131 (1999).

P. D. Lyne, M. Hodoscek, and M. Karplus, "A hybrid QM/MM potential employing Hartree-Fock or density functional methods in the quantum region," J. Phys. Chem. A 103, 3462 (1999).

P. D. Lyne and M. Karplus, "Determination of the pKa of the 2'-hydroxyl group at the active site of hammerhead ribozyme from ab initio calculations with solvation corrections," J. Am. Chem. Soc. (submitted).