| SHV-1 beta-lactamase is the prototype of a growing family of SHV variants, some of which have developed resistance to the most common beta-lactamase inhibitors. The increasing number of such enzymes is creating great difficulties for clinicians in the treatment of infections with antibiotics. Understanding the mechanisms and structural basis for the inactivation of these enzymes is necessary to rationally design new inhibitors and antibiotics. In the present work, SHV-1 and a SHV-1 variant in which residue Serine 130 is replaced by Glycine (Ser130Gly) were inhibited with tazobactam and with clavulanic acid. The uninhibited and inhibited enzymes were analyzed by liquid chromatography—electrospray ionization mass spectrometry (LC-ESI/MS) to determine the mass increase after the inhibition reactions. The uninhibited and the inactivated SHV-1 and the variant enzymes were also digested with trypsin, and the digestion products were analyzed using LC-ESI/MS and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for characterizing the inhibition site and products.; The obtained results indicate that in both SHV-1 and its Ser130Gly variant, the residue Serine 70 is modified by the inhibition reactions with tazobactam and clavulanic acid. Inhibition mechanisms rationalizing the experimental observations are proposed. |
