Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism. Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme. These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da. Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate. The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme. Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme. Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70. A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130. A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde. Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.

substituent thus offer the potential to test mechanistic proposals by differentiating between those inhibited forms that retain the oxazolidine ring of the clavulanate skeleton and those that do not. Forms of C-9 modified clavulanate-analogue-inhibited TEM-2 that retained the oxazolidine ring would be expected to exhibit a mass increment relative to unmodified TEM-2 enzyme diffferent from those detected in normal clavulanateinhibited enzyme. Forms which did not retain the oxazolidine ring might be expected to be indistinguishable from comparable forms of clavulanate-inhibited enzyme. FEB. 1997 ESI-MS spectra were acquired after incubation of TEM-2/Mactamase with r-butylammonium clavulanate 1, potassium 9-0-methylclavulanate 6 and potassium 9-AMsobutylaminodeoxyclavulanate 7. As previously reported in the case of clavulanate 17), three mass increments were observed in each case: A, B and a broad series X. A corresponds to unmodified TEM-2and was not visible in the case of the analogues 6 or 7. The broad series X was clearly present in each case and corresponds to an approximate mass of 28985 Da. High resolution studies in the case of 1 have demonstrated the broad series Xto be a superposition of three species with mass increments of 52, 70 and 88Da, corresponding to 4, 2 and 3, respectively.In contrast to the presence of series X in all three cases, differences existed between the largest apparent mass increments (B) resulting from the incubations of 1, 6 and 7 with the TEM-2/Mactamase. Incubation of TEM-2/Mactamase with 1 results in an observed mass of 29067+6Da, incubation with 6 in an observed mass of 29078±1Da, and incubation with 7 in an observed mass of 29117±7Da. These correspond to mass increments relative to unmodified enzyme of 155 for 1, 173 for 6, and 212Da for 7. The observed differences in mass increments between 1 and the analogues 6 and 7 may be accounted for by the difference in their C-9 substituents, resulting in the formation of 5, 8 and 9, respectively.Furthermore, as observed for 1, in the cases of 6 and 7 there was no evidence for the presence of any mass increment corresponding to the mass of the intact clavam inhibitor, implying rapid decarboxylation of the each of the three clavam inhibitors occurs after binding to the /Mactamase. These studies support a commonmechanistic scheme for the inhibition of /Mactamases by clavulanates 1, 6 and 7 in which acylation of Ser-70 by the /Mactam is followed by ring opening of the oxazolidine ring. Rapid decarboxylation of the resultant /Mteto acid 10 gives the imines 5a (R=OH), 8a (R=OMe) or 9a (R= N+H2CHMe2)which may be in equilibrium with E and/or Z enamines 5b, 8b or 9b, respectively. The imines 5a, 8a or 9a are then hydrolysed to give the aldehyde 2 and hydrated aldehyde 3 with concomitant loss of the elements of the oxazolidine ring and its C-9 substituent. Aldehyde 2/hydrated aldehyde 3 may undergo further slow hydrolysis to reform unmodified enzyme. Alternatively, nucleophilic attack of the alcohol of Ser-130 onto either the imine 5a, 8a or ...