BETA-LACTAMASE-CATALYZED HYDROLYSIS OF CEPHALEXIN - EVOLUTION OF THE CEPHALOSPOROATE INTERMEDIATE

Hydrolysis of cephalexin catalysed by P99 beta-lactamase from Enteroba cter cloacae has been studied at pD 6.4 and 8.0. The enzymatic hydroly sis product is the cephalosporoate intermediate, which in aqueous medi a undergoes tautomerization of the double bond in the dihydrothiazine ring from position 3-4 to 4-5, with the consequent uptake of a proton at C(3). This latter process is governed by steric constraints. NMR re sults show that the cephalosporoate compound bears the aromatic group, present in the side chain at C(7), folded over the dihydrothiazine ri ng, Under these conditions the proton is chiefly taken up by the a fac e of the dihydrothiazine ring. Subsequently, the six-membered ring can be subjected to several reactions according to the pH.