Theoretical Study on Catalysis by Protein Enzymes, Ribosome, and Molecular Motors

Principal Investigator: Martin Karplus, Harvard University

Research Objectives

This project's goal is to develop a greater understanding of the mechanisms involved in enzyme catalysis and related protein functions. We are studying the protein enzymes chorismate mutase, flavoxireductase, and aminopeptidase, and a nucleic acid enzyme, the hammerhead ribosome. We are also studying another class of enzymes known as molecular motors, which play important roles in bioenergy transduction and gene replication.

Computational Approach

For active-site models in the gas phase, Gaussian98 and NWChem are used forab initio or density functional calculations. To determine the catalytic mechanism in the presence of the enzyme environment, the CHARMM program (developed by the Karplus group) is used for a combined quantum and molecular mechanics (QM/MM) approach.

Accomplishments

We studied the role of tunneling for two proton transfer steps in reactions catalyzed by triosephosphate isomerase (TIM). The effect of tunneling on the reaction rate is less than a factor of 10 at room temperature; the tunneling became more important at lower temperatures. The imaginary frequency mode and modes having large contributions to the reaction path curvature were localized on the atoms in the active site, within 4 Å of the substrate. This suggests that only a small number of atoms close to the substrate and their motions directly determine the magnitude of tunneling.

In horse liver alcohol dehydrogenase (LADH) proton and hydride transfers, proton transfers proceeded in a virtually concerted fashion before hydride transfers. The catalytic efficiency of LADH was low for a pH below 5.5, and the hydride transfer was hardly affected for a pH between 5.5 and 8.1. Perturbation analysis of the QM/MM energies suggests a number of charged residues close to the active site as well as the phosphate groups in NAD⁺ make important contributions to the energetics of proton and hydride transfer reactions.

Chorismate mutase (CM) acts at the first branch-point of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Two nonreactive conformers of chorismate were found to be more stable than the reactive pseudo-diaxial chair conformer in solution. When these inactive conformers were bound to the active site, they rapidly converted to the reactive chair conformer. This suggests that the enzyme binds the more prevalent nonreactive conformers and transforms them into the active form in a step prior to the chemical reaction.

Significance

Despite the growing availability of enzyme crystal structures, details of the chemical mechanisms employed by enzymes to achieve their catalytic efficiency remain elusive. This is mainly because the chemical events of bond formation and cleavage that define the reaction are exceedingly short and currently inaccessible to direct experimental measurement. It is also very difficult to probe directly the coupling between chemical events and conformational transitions with atomic details, which remains a major obstacle for understanding the working mechanism of molecular motors. Theoretical studies, therefore, are of great value for providing insights into these mechanisms.

Publications

H. Guo, Q. Cui, W. N. Lipscomb, and M. Karplus, "Substrate conformational transitions in the active site of chorismate mutase: Their role in the catalytic mechanism," Proc. Natl. Acad. Sci. USA98, 9032 (2001).

Q. Cui and M. Karplus, "Triosephosphate isomerase (TIM): A theoretical comparison of alternative pathways," J. Am. Chem. Soc.123, 2284 (2001).

P. D. Lyne and M. Karplus, "Determination of the pK_a of the 2'-hydroxyl group of a phosphorylated ribose: Implications for the mechanism of hammerhead ribozyme catalysis," J. Am. Chem. Soc. 122, 166 (2000).