STRUCTURAL BASIS FOR THE INACTIVATION OF THE P54 MUTANT OF BETA-LACTAMASE FROM STAPHYLOCOCCUS-AUREUS PC1

The crystal structure of a mutant protein of a class A beta-lactamase from Staphylococcus aureus PC1, in which Asp179 is replaced by an asparagine (P54), has been determined and refined at 2.3-angstrom resolution (1 angstrom = 0.1 nm). The resulting crystallographic R factor (R = SIGMA-h parallel-to F(o)\ - \F(c) parallel-to/SIGMA-h\F(o)\, where \F(o)\ and \F(o)\ and \F(c)\ are the observed and calculated structure factor amplitudes) is 0.181 for 12 289 reflections with I greater-than-or-equal-to sigma(I) within the 6.0-2.3-angstrom resolution range. The mutated residue is located at the C-terminus of an extensive loop (the OMEGA-loop), remote from the active site, and results in a drastically reduced activity. Examination of the native and P54 structures reveals that the overall fold is similar, except that there is substantial disorder of the OMEGA-loop of P54. This is a consequence of the elimination of a salt bridge between Asp179 and Arg164 that links the two ends of the OMEGA-loop in native beta-lactamase. It is associated with a difference in side-chain conformation between Asn179 in P54 and Asp179 in the native structure. An alternate interaction occurs in P54 between Asn179 and Ala69, adjacent to the catalytic Ser70. This disorder affects catalysis since some of the disordered residues, in particular Glu166, form part of the active site. Stopped-flow kinetic measurments of native and P54 beta-lactamase with nicrofin confirm the prediction that disordering of the catalytic Glu166 in P54 makes deacetylation the rate-limiting step in hydrolysis: whereas the ratio of the acylation rate to the deacylation rate is about 1 in native beta-lactamase, in P54 this value is approximately 600, mainly due to the reduction in the deacetylation rate. The acylation rate is reduced about 10-fold. Thus, the kinetics of P54 support the proposal that the acylation and deacylation steps in beta-lactamase are functionally separate and are assisted by different amino acid residues.