High Level QM/MM Modeling of the Formation of the Tetrahedral Intermediate in the Acylation of Wild Type and K73A Mutant TEM-1 Class A β-Lactamase

Abstract

The breakdown of beta-lactam antibiotics by beta-lactamases is the most important resistance mechanism of gram negative bacteria against these drugs. The reaction mechanism of class A beta-lactamases, the most widespread family of these enzymes, consists of two main steps: acylation of an active site serine by the antibiotic, followed by deacylation and release of the cleaved compound. We have investigated the first step in acylation (the formation of the tetrahedral intermediate) for the reaction of benzylpenicillin in the TEM-1 enzyme using high level combined quantum mechanics/molecular mechanics (QM/MM) methods. Structures were optimized at the B3LYP/6-31+G(d)/CHARMM27 level, with energies for key points calculated up to the ab initio SCS-MP2/aug-cc-pVTZ/CHARMM27 level. The results support a mechanism in which Glu166 removes a proton (via an intervening water molecule) from Ser70, which in turn attacks the beta-lactam of the antibiotic. Depending on the method used, the calculated barriers range from 3 to 12 kcal mol(-1) for this step, consistent with experimental data. We have also modeled this reaction step in a model of the K73A mutant enzyme. The barrier to reaction in this mutant model is found to be slightly higher: the results indicate that Lys73 stabilizes the transition state, in particular deprotonated Ser70, lowering the barrier by about 1.7 kcal mol(-1). This finding may help to explain the conservation of Lys73, in addition to the role we have previously found for it in the later stages of the reaction (Hermann et al. Org. Biomol. Chem. 2006, 4, 206-210).

Crystal Structure of a Preacylation Complex of the β-Lactamase Inhibitor Sulbactam Bound to a Sulfenamide Bond-Containing Thiol-β-lactamase

“…We anticipate that the C70–K73 sulfenamide is likely not formed in solution as both residues 70 and 73 have previously been shown to be important for acylation. 27 Future studies with additional substrates will provide insight into the general nature of these hypotheses.…”

Theoretical Aspects of Hydrolysis of Peptide Bonds by Zinc Metalloenzymes

“…Therefore, in a few recent QM or QM/MM studies, the authors tried to address this problem by improving the description of the electronic structure of the region of interest (i.e., the enzyme active site) by using more accurate (wave-function) methods. [23][24][25][26][27][28] It is generally accepted that for systems with a closedshell electronic structure a hierarchy of standard ab initio quantum chemical methods exists, starting from the Hartree-Fock methods through the popular second-order Møller-Plesset perturbation theory up to the coupled cluster methods (mentioning here only the methods that can be conveniently used for systems containing at least a few dozen atoms). Therefore, in many applications, such as the weak intermolecular interactions, CCSD(T) calculations using a larger basis set are considered as a good reference (benchmark, or the so-called ''golden standard'') for cheaper methods, including the approaches based on DFT.…”

Simulation with quantum mechanics/molecular mechanics for drug discovery

“…Unfortunately, resistant bacteria can cleave this ring through b-lactamase very efficiently which represents a serious menace to the antibacterial chemotherapy [58]. Hermann and coworkers have determined, through QM/MM computations, the cleavage mechanism of benzyl-penicillin with class A b-lactamase [59][60][61][62]. This mechanism, schematically presented in Figure 5, starts with the acylation of Ser70.…”