A SURVEY OF A FUNCTIONAL AMINO-ACID OF CLASS C-BETA-LACTAMASE CORRESPONDING TO GLU166 OF CLASS A-BETA-LACTAMASES

The class C beta-lactamase of Citrobacter freundii GN346 is a typical cephalosporinase comprising 361 amino acids. The aspartic acid at posi tion 217 and glutamic acid at position 219 in this beta-lactamase were , respectively, previously shown not to be the counterpart of Glu166 ( ABL166) in class A beta-lactamases, even though sequence alignment of class A and C enzymes strongly suggested this possibility [(1990) FEBS Lett. 264,211-214; (1990) J. Bacteriol. 172, 4348-4351]. We tried aga in to assign candidates for the counterpart of Glu166 through sequence alignment based on other criteria, the glutamic acids at positions 19 5 and 205 in the class C beta-lactamase being selected. To investigate this possibility, these two glutamic acids were changed to glutamine, lysine or alanine, respectively. All the mutant enzymes showed more t han 50% of the activity of the wild-type enzyme, indicating that the p ossibility was ruled out. These results strongly suggested the possibi lity that the class C beta-lactamase lacks a functional acidic residue corresponding to Glu166 in class A enzymes.