Computational Enzymology

Research Objectives

Advances in protein crystallography have enabled scientists to determine the three-dimensional structures of enzymes and explore the mechanisms by which enzymes achieve both strict specificity in recognizing their substrates and extraordinary efficiency in catalyzing chemical transformations. Some aspects of the reaction mechanism, however, lie outside the reach of experimental methods, due in large part to the short-lived nature of the key steps along the reaction pathway.

In our program, we are attempting to develop numerical tools, based on first-principles physical and chemical models, that can provide information complementary to that obtained from experiment. Our goal is a basic understanding of the reaction mechanism in enzyme systems. Our methods are designed to fill in some of the details missing from present experimental studies of enzyme systems and complete the picture of how enzymes achieve such remarkable catalytic efficiencies and substrate specificities.

Computational Approach

Our principal computational tool is a hybrid, quantum mechanical/molecular mechanical (QM/MM) method developed to simulate the behavior of biological molecules in the condensed phase. Relatively few atoms in the active site of the enzyme are treated quantum mechanically; the bulk of the atoms are represented by a molecular mechanics hamiltonian. The method enables us to investigate the critical events of bond formation and cleavage catalyzed by the enzyme and yet retain the principal effects of the protein environment. To calibrate the quantum hamiltonian parameters, we rely on large quantum chemistry calculations conducted on the J90 cluster using the code GAUSSIAN-94. Our molecular dynamics simulations are performed with the code CHARMM on the T3E, using the parallel architecture to routinely simulate systems involving twenty thousand atoms.

Accomplishments

We have investigated the reaction mechanism in the class of enzymes known as beta-lactamases, the primary source of antibiotic resistance in bacteria. These enzymes hydrolyze antibiotic agents such as penicillin with great alacrity. We have performed a number of molecular dynamics simulations aimed at defining the so-called Michaelis complex: the state in which the enzyme has captured the substrate but prior to any bond reorganization. We have found two possible mechanisms for the initial attack of the enzyme on its penicillin substrate to be consistent with the results of our simulations. Our next efforts will be directed to understanding the relative energetics of these two possible pathways.

Significance

Recent experiments have indicated that once bacteria develop a resistance to antibiotics, that resistance is not lost even after many thousands of generations of bacteria cultured in the absence of antibiotics. Additionally, bacteria seem able to find mechanisms to develop resistance to new antibiotics. An understanding of how the beta-lactamase enzymes function at an atomic level may provide new insights into the development of new antibiotics or enzyme inhibitors.

Publications

Bash, P. A., L. L. Ho, A. D. MacKerell, Jr., D. Levine, and P. Hallstrom.1996. Progress toward chemical accuracy in the computer simulation of condensed phase reactions. Proc. Natl. Acad. Sci. USA 93:3698-3703.

Cunningham, M A., L. L. Ho, D. T. Nguyen, R. E. Gillilan and P. A. Bash. Simulation of the enzyme reaction mechanism of malate dehydrogenase. 1997. Biochemistry 36:4800- 4816.

Ho, L. L., A. J. MacKerell, Jr., and P. A. Bash. 1996. Proton and hydride transfers in solution: hybrid QM/MM free energy perturbation study. J. Phys. Chem. 100:4466-4475.

CPK rendering of the active site of beta-lactamase. The catalytic serine residue (in blue) is positioned to transfer a proton (H) to the water molecule (in cyan). This transfer will activate the serine in preparation for its oxygen atom (O) to attack the carbon atom (C) of penicillin substrate (in yellow).

Ribbon diagram of the enzyme beta-lactamase from TEM-1. Helices are rendered in green and beta sheets in red. Connecting loops are indicated in blue. The penicillin substrate is drawn as a stick figure with carbon atoms in yellow, nitrogen in blue, oxygen in red, hydrogen in white, and sulfur in orange. The penicillin molecule is located in the active site of the enzyme.

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