Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases

Abstract

The pH dependence of the pK(a) values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 beta-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates. By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction intermediate, we found that acid-catalyzed protonation of the beta-lactam nitrogen is energetically favored as the initiating event, followed by base-catalyzed nucleophilic attack on the carbonyl carbon of the beta-lactam group. N-protonation is catalyzed through a hydrogen-bonded cluster involving the 2-carboxylate group of the substrate, the side chains of S130 and K234, and a solvent molecule. Nucleophilic attack on the carbonyl carbon is carried out by the side chain of S70 with proton abstraction catalyzed by a water molecule hydrogen-bonded to the side chain of E166. Stabilization of ion pairs in the active site through interactions with distant clusters of charged residues in the enzyme was concluded to be an important driving force of the catalytic mechanism.

Figure 1

Hydrogen-bonding and electrostatic interactions in the active site of TEM-1 β-lactamase. The hydrogen-bonded serine-lysine side chains (S70·K73) and (S130·K234) are highlighted because of their functional importance. The numbered designations of active-site water molecules are taken from x-ray studies of the free enzyme (27).

Figure 2

Comparison of mechanisms for hydrolysis of β-lactam antibiotics catalyzed by TEM-1 β-lactamase to illustrate as the initiating event (i) general-base catalyzed nucleophilic attack by S70 and (ii) general-acid catalyzed protonation of the β-lactam N(1). The two schemes differ mechanistically up to formation of the first tetrahedral adduct of the reaction T1. Structural formulae are, therefore, illustrated only for the acylation portion of each reaction cycle. Formation of the T2 tetrahedral adduct (not shown) occurs through nucleophilic attack on the penicilloyl moiety in EY by a water molecule sequestered in the active site. The structures of the tetrahedral adducts T1 and T2 were deduced by close examination of sterically allowed interactions that an incoming nucleophilic S70 side chain, correspondingly, water molecule, may experience in the ES and EY intermediates, respectively. We discuss results for only sequential reaction mechanisms. The corresponding concerted reaction mechanisms were found to be less favorable energetically. T1a and T1b represent transient species in formation and breakdown of T1. T1a was modeled by applying the electronic configurations of active-site atoms in T1 to nuclear positions in ES. T1b was modeled by applying the electronic configurations of EY to the nuclear coordinates of T1. This sequence of changes in electronic structure and nuclear coordinates follows from first-principle considerations of the Born-Oppenheimer approximation and the Franck-Condon rule. A similar procedure was applied to evaluate electrostatic interactions governing deacylation.

Figure 3

Contour maps of the electrostatic potential in the active site of TEM-1 β-lactamase for E, ES, T1, and EY (top to bottom). The contour levels are calculated at intervals of 0.5 kcal/mol⋅e (≈0.83 RT/e). Solid red contours indicate negative isopotential curves; broken blue contours show positive isopotential curves while broken green contours represent curves of 0 potential. The contours are deleted at distances less than 2.5 Å from hydrogen-bound atoms. The maps are drawn for x = 30–50 Å and y = 28–42 Å with respect to the crystal axis system of the E166N acylenzyme (4). The contour maps are projected along the z axis from z = 29.5 Å to z = 32.5 Å. [The level of z = 31 Å corresponds to the position of N^ɛ(K73).] The circles represent substrate or acyl atoms viewed in projection within (filled) and outside (unfilled) of the 3-Å slab projected here and identify catalytically important regions where steep gradients are visible.

Figure 4

Histogram comparison of the electrostatic potential Φ_el,i at the positions of catalytically important atoms for N-protonation. The value of Φ_el,i was calculated in each case at grid points coinciding with the position of the catalytically important atom of the residue, i.e., N(1), C(7), O(8), O^γ(S70) (=S70′), N(S70) (=S70′′), N^ɛ(K73), O^γ(S130), O^ɛ1,2(E166), N^ɛ(K234), N(A237), and O(WAT297). The value for O^ɛ1,2(E166) is given at the grid point midway between the two carboxylate oxygens. Although the N(1), C(7), and O(8) atoms do not occur in the free enzyme E, the values of Φ_el,i are given at the grid points that are occupied by these atoms in the ES complex.