Paul Bash, Northwestern University Medical School

Research Objectives

The most successful defense bacteria have developed against antibiotics is the expression of enzymes known as b-lactamases, which hydrolyze the amide bond in the b-lactam ring of anti-biotic agents such as penicillin and cephalosporin. Despite intensive experimental scrutiny, basic information about how these enzymes achieve their catalytic efficiency is still lacking. This project uses computational techniques to define the specific chemical mechanisms employed by the enzyme TEM-1, a prototypical class A b-lactamase, and AmpC, a prototypical class C enzyme. This research will provide insights into the roles of the different active-site residues in substrate recognition and binding and in the catalytic process.

Computational Approach

	The enzyme beta-lactamase with the drug penicillin bound to its active site. A hydoxyl oxygen group (OH) of the enzyme is in position for the transfer of a proton (H) to a water molecule and for the nucleophilic attack of an oxygen (O) on the carbonyl carbon (C) of penicillin.

Our hybrid quantum/molecular mechanics method (QM/MM) begins with structural information obtained from x-ray crystallography and uses first-principles physical and chemical numerical methods, calibrated to characterize the basic chemical interactions found in the b-lactamase system, to describe the catalytic process at an atomic level. This hybrid method utilizes a semiempirical quantum mechanics description of atoms in the active site while representing the remaining atoms with a molecular mechanics model. We use the ab initio quantum mechanical code Gaussian 98, the CHARMM molecular mechanics code, and the semiempirical QM code MOPAC97.

Accomplishments

We have performed several molecular dynamics simulations of the TEM-1 enzyme with a penicillin substrate to gain insights into accessible conformations of the enzyme/substrate (Michaelis) complex. We used a molecular mechanics Hamiltonian, and we were interested in defining the roles of various active-site residues in binding and orienting the substrate into a conformation suitable for the catalytic reactions to proceed. We performed four separate simulations of the TEM-1 b-lactamase using different starting conformations to assess the sensitivity of our calculations to initial conditions. The results of the simulations are consistent with two of three proposed mechanisms for the acylation step of the reaction. These simulations provide insights into the roles of several residues in binding and orienting the substrate in the active site.

We have found that the interactions between Ser-130, Lys-234, Ser-235, and Arg-244 and the carboxylate oxygen atoms of the substrate are essential for securing the substrate in the active site. Additionally, the O8 carbonyl oxygen of the b-lactam ring interacts with the main chain nitrogen atoms from Ser-70 and Ala-237. The N14 nitrogen and O16 oxygen atoms of the carboxyamide group interact with a main chain carbonyl oxygen from Ala-237 and the side chain of Asn-132, respectively. Together, these interactions orient the substrate in the active site into a conformation suitable for the subsequent catalytic reactions to proceed.

Significance

Under intense evolutionary pressure, bacteria have developed several countermeasures to antibacterial agents, posing a serious risk to public health. The most successful of these defenses is expression of enzymes known as b-lactamases, which efficiently hydrolyze the amide bond in the b-lactam ring (red) of antibiotic agents such as benzylpenicillin and cephalosporins. The focus of our research is the development of a detailed understanding of the reaction mechanisms employed by these enzymes to hydrolyze b-lactam antibiotics. Understanding these mechanisms will yield insights that may prove useful for the development of new antibacterial agents.

Publications

M. A. Cunningham and P. A. Bash, "Systematic procedure for the development of accurate QM/MM model Hamiltonians," in Computer Simulations in Biomolecular Systems, edited by W. F. Van Gunsteren, P. K. Weiner, and A. J. Wilkinson (Kluwer, Dordrecht, Neth., 1997), pp. 177-95.

M. A. Cunningham, R. E. Gillilan, and P. A. Bash, "Computational enzymology," Can. Chem. News 49, 9 (1997).

M. A. Cunningham and P. A. Bash, "Computational enzymology," Biochimie. 79, 687 (1997).