INACTIVATION OF CLASS-A BETA-LACTAMASES BY CLAVULANIC ACID - THE ROLEOF ARGININE-244 IN A PROPOSED NONCONCERTED SEQUENCE OF EVENTS

From the refined 2 angstrom crystal structure of the Bacillus lichenif ormis 749/C beta-lactamase, energy-minimized models for active-site bi nding of the precatalytic (''Michaelis'') complex with the clinically utilized inactivator, clavulanic acid, for the acyl enzyme intermediat e, and for the ultimate acylated acyclic species that leads to inactiv ation of class A beta-lactamases by clavulanate have been generated. O n the basis of these models, the details of the chemistry of inactivat ion of clavulanate are reassessed. A nonconcerted process for the inac tivation chemistry of class A beta-lactamases by clavulanate is propos ed. These models reveal that the Arg-244 side chain and the Val-216 ca rbonyl anchor a structurally conserved water molecule, W673, which ser ves as the most likely source of a critical proton in a stepwise seque nce of events. Disruption of this ''electrostatic anchor'' for W673 by mutational replacement of Arg-244 with Ser in the TEM beta-lactamase would account for the resulting observed severe impairment of the effi ciency of inactivation of the mutant enzyme by clavulanate. The kineti c impact of the Arg-244-Ser mutation on interaction with clavulanate i s reflected by resistance to ampicillin plus clavulanate of a strain o f E. coli bearing the mutant enzyme. Molecular dynamics computations o n the acylated acyclic intermediate-the putative inactivating species- indicated that irreversible inactivation of the beta-lactamase may not occur as a consequence of a transimination reaction, in contrast to p revious suggestions. The most likely scenario for irreversible inactiv ation involves the capture of the beta-hydroxyl of conserved Ser-130 b y the iminium moiety of the acylated acyclic intermediate, followed by a deprotonation at C6 of clavulanate. The deprotonation is likely to be carried out by the conserved Glu-166 via the intervening crystallog raphic water W712. Deprotonation prior to nucleophile capture is propo sed as the mechanism of generation of the so-called transiently inhibi ted enamine species. For the wild-type TEM-1 beta-lactamase, both irre versible inactivation and the formation of the transiently inhibited s pecies proceed with comparable rates. In addition, a new function for the Ser-130 in the formation of the acyl-enzyme intermediate with both clavulanate and typical beta-lactamase substrates is proposed. It is suggested that the beta-hydroxyl of Ser-130 stabilizes the transition state for the expulsion of the incipient amine from the high-energy te trahedral species by hydrogen bonding to the oxazolidine amine in the course of Ser-70 acylation.