Elucidation of the role of arginine-224 in the turnover processes of class A .beta.-lactamases

Abstract

The highly conserved arginine-244 of beta-lactamases has been postulated to play a role in their initial recognition of substrates, presumably through ion pairing interactions [Moews, P. C., Knox, J. R., Dideberg, O., Charlier, P., & Frère, J. M. (1990) Proteins: Struct., Funct., Genet. 7, 156-171]. However, in the Michaelis enzyme-substrate complex, no direct function has been attributed to this residue. Two mutants with substitutions of this residue in the TEM-1 beta-lactamase (lysine-244 and serine-244) have been prepared to explore whether the guanidinium group of arginine-244 plays a critical role in the turnover processes. The mutant enzymes are effective catalysts for the hydrolysis of both penicillins and cephalosporins, and the lysine mutant enzyme behaves virtually identically to the wild-type beta-lactamase. Comparative kinetic characterization of the serine mutant and wild-type enzymes attributed apparent binding energies of 1.3-2.3 kcal/mol for the penicillins and 0.3-1.0 kcal/mol for the cephalosporins to the transition-state species by arginine-244. Furthermore, it was shown that arginine-244 also contributes equally well to ground-state binding stabilization. These results were interpreted to indicate the involvement of a long hydrogen bond between arginine-244 and the substrate carboxylate, both in the ground and transition states. A reassessed picture for substrate anchoring involving interactions of the substrate carboxylate with the side chains of Ser-130, Ser-235, and Arg-244 is proposed to accommodate these observations.

Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase.

“…The side chain is well stabilized by multiple interactions with Ser-318 and Ser-343. This Arg-349 is unlikely to be the counterpart ofthe class A Arg-244 (27)(28)(29), however, because the two guanidinium groups are separated 4.5 A and the side chains differ greatly in solvent accessibility (2.5 A3 vs. 24.5 A3 for Arg-244). It is more likely that nearby Asn-346 (Fig.…”

Mechanistic Basis for the Emergence of Catalytic Competence against Carbapenem Antibiotics by the GES Family of β-Lactamases

“…Determination of Dissociation Constants-The substrate dissociation constant (K s ) for imipenem, with GES-1, -2, and -5, was determined by treating it as an inhibitor of nitrocefin hydrolysis as described previously (20). Reactions containing 50 mM NaP i (pH 7.0), 100 mM NaCl, 160 M (GES-1), 50 M (GES-2), or 120 M (GES-5) of nitrocefin, and varying concentrations of imipenem were initiated by the addition of the enzyme (200 pM final for GES-1 and GES-5 or 10 nM final for GES-2).…”

The 2.4-Å crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin

“…In class A and class D b-lactamases, the carboxylate interacts with the guanidinium group of an arginine that can occupy different positions in the primary structure of these enzymes. The presence of such a guanidinium group in b-lactamases is favorable to acylation by b-lactams [40]. Acylation of PBP5fm is very slow (second-order rate constant k 2 /K = 20 M -1 s -1 ) [12].…”