Ab Initio QM/MM Study of Class beta-Lactamase Acylation: Dual Participation of Glu166 and Lys73 in a Concerted Base Promotion of Ser70

摘要

beta-Lactamase acquisition is the most prevalent basis for Gram-negative bacteria resistance to the beta-lactam antibiotics.The mechanism used by the most common class A Gram-negative beta-lactamases is serine acylation followed by hydrolytic deacylation,destroying the beta-lactam.The ab initio quantum mechanical/molecular mechanical (QM/MM) calculations,augmented by extensive molecular dynamics simulations reported herein,describe the serine acylation mechanism for the class A TEM-1 beta-lactamase with penicillanic acid as substrate.Potential energy surfaces (based on approximately 350 MP2/6-31+G calculations) reveal the proton movements that govern Ser70 tetrahedral formation and then collapse to the acyl-enzyme.A remarkable duality of mechanism for tetrahedral formation is implicated.Following substrate binding,the pathway initiates by a low energy barrier (5 kcal mol~(-1)) and an energetically favorable transfer of a proton from Lys73 to Glu166,through the catalytic water molecule and Ser70.This gives unprotonated Lys73 and protonated Glu166.Tetrahedral formation ensues in a concerted general base process,with Lys73 promoting Ser70 addition to the beta-lactam carbonyl.Moreover,the three-dimensional potential energy surface also shows that the previously proposed pathway,involving Glu166 as the general base promoting Ser70 through a conserved water molecule,exists in competition with the Lys73 process.The existence of two routes to the tetrahedral species is fully consistent with experimental data for mutant variants of the TEM beta-lactamase.