Amino acid sequence, active-site residue, and effect of suicide inhibitors on cephalosporinase of Citrobacter freundii GN346.

The structural gene for a cephalosporinase of Citrobacter freundii GN346 was sequenced. From the nucleotide sequence, the entire amino acid sequence of the mature enzyme with 361 amino acids and a molecular weight of 39,867 was determined. The active-site serine was directly confirmed to be serine 64 by studies in which the enzyme was labeled with dansylpenicillin. In investigations comparing the inhibitory effect of sulbactam (penicillanic acid sulfone) and cloxacillin sulfone on the cephalosporinase and on TEM-2-type penicillinase, sulbactam was found to be an effective progressive inhibitor but a poor competitive inhibitor for the cephalosporinase. The cephalosporinase and the inhibitor formed a long-lived complex with a half-life of 550 minutes. Cloxacillin sulfone could not inactivate the cephalosporinase progressively but irreversibly inactivated the penicillinase.