pK_a, MM, and QM studies of mechanisms of β‐lactamases and penicillin‐binding proteins: Acylation step

Abstract

The acylation step of the catalytic mechanism of beta-lactamases and penicillin-binding proteins (PBPs) has been studied with various approaches. The methods applied range from molecular dynamics (MD) simulations to multiple titration calculations using the Poisson-Boltzmann approach to quantum mechanical (QM) methods. The mechanism of class A beta-lactamases was investigated in the greatest detail. Most approaches support the critical role of Glu-166 and hydrolytic water in the acylation step of the enzymatic catalysis in class A beta-lactamases. The details of the catalytic mechanism have been revealed by the QM approach, which clearly pointed out the critical role of Glu-166 acting as a general base in the acylation step with preferred substrates. Lys-73 shuffles a proton abstracted by Glu-166 O(epsilon ) to the beta-lactam nitrogen through Ser-130 hydroxyl. This proton is transferred from O(gamma) of the catalytic Ser-70 through the bridging hydrolytic water to Glu-166 O(epsilon ). Then the hydrogen is simultaneously passed through S(N)2 inversion mechanism at Lys-73 N(zeta) to Ser-130 O(gamma), which loses its proton to the beta-lactam nitrogen. The protonation of beta-lactam nitrogen proceeds with an immediate ring opening and collapse of the first tetrahedral species into an acyl-enzyme intermediate. However, the studies that considered the effect of solvation lower the barrier for the pathway, which utilizes Lys-73 as a general base, thus creating a possibility of multiple mechanisms for the acylation step in the class A beta-lactamases. These findings help explain the exceptional efficiency of these enzymes. They emphasize an important role of Glu-166, Lys-73, and Ser-130 for enzymatic catalysis and shed light on details of the acylation step of class A beta-lactamase mechanism. The acylation step for class C beta-lactamases and six classes of PBPs were also considered with continuum solvent models and MD simulations.

The Importance of a Critical Protonation State and the Fate of the Catalytic Steps in Class A β-Lactamases and Penicillin-binding Proteins

“…We underscore that a deprotonated Glu-166 and a free-base Lys-73 would be unsuitable, as the lone electron pairs in the two side chains would repel each other, probably causing local detrimental conformational changes within the active site, consistent with the enzyme losing activity above pH of 9. This is supported by previous molecular dynamics simulations of TEM-1 with deprotonated Glu-166 and free-base Lys-73 that resulted in unstable trajectories (56).…”

Can Molecular Dynamics and QM/MM Solve the Penicillin Binding Protein Protonation Puzzle?

“…Moreover, Mobashery and co-workers 38 state that it would be unsuitable for Glu166 and Lys73 to both be deprotonated according to molecular dynamics studies performed on TEM-1 that resulted in unstable trajectories as reported by Massova and Kollman. 39 Hence, for class A β-lactamases they conclude that only one of these residues is protonated; however, that residue could vary based upon the acylation mechanism. Their work agrees with previous reports 27−32 stating that the native state of Lys73 in class A β-lactamases is protonated.…”

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

“…1 (c) to (d)). The acylation mechanism has been examined in many experimental [6][7][8][9][10][11][12][13][14] and theoretical works, 11,[15][16][17][18][19][20][21][22] and acylation is common to serine-b-lactamase and penicillin-binding protein (PBP). Deacylation, on the other hand, is only seen in serine-b-lactamase.…”