8-HYDROXYPENILLIC ACID FROM 6-AMINOPENICILLANIC ACID - A NEW REACTIONCATALYZED BY A CLASS-C BETA-LACTAMASE

The hydrolysis of 6-aminopenicillanic acid (APA) at neutral pH, cataly zed by the class C beta-lactamase of Enterobacter cloacae P99, was obs erved from H-1 NMR spectra to yield an unexpected product, 8-hydroxype nillic acid, in bicarbonate-containing buffers. This product probably arises from turnover of the carbamate of APA by the enzyme. H-1 and C- 13 NMR spectra of APA in bicarbonate solutions clearly demonstrate the presence of the carbamate. Turnover of APA at saturating concentratio ns by the enzyme is accelerated by bicarbonate and by methanol. These results suggest that deacylation of the enzyme is rate determining and that the presence of the carbamate affects this step. A mechanism inv olving rate-determining intramolecular nucleophilic attack by the carb amate on the acyl enzyme is proposed to rationalize these observations and lead to formation of 8-hydroxypenillate. This reaction can be see n as an example of substrate-assisted enzymic catalysis. Quantitative analysis of the data indicated a dissociation constant of the APA-carb amate to APA and bicarbonate of 7 mM at pH 7.5. The P99 beta-lactamase also catalyzes the formation of an alternative product from 7-aminoce phalosporanic acid in the presence of bicarbonate. Ampicillin and ceph aloglycine which also possess an amine-bearing side chain [PhCH(NH2)CO -] react with bicarbonate to form carbamates at neutral pH but these d o not yield alternative products on turnover by the P99 beta-lactamase . Typical class A beta-lactamases do not catalyze 8-hydroxypenillate f ormation from APA and bicarbonate even when deacylation is rate determ ining. This difference is discussed in terms of beta-lactamase active site structure.