Evidence from a mutant beta-lactamase for the mechanism of beta-lactamase-catalysed depsipeptide aminolysis.

The Ser-70----Gly mutant of the TEM-1 beta-lactamase, where the active-site serine hydroxy group has been lost, does not catalyse the hydrolysis of either benzylpenicillin or N-(phenylacetyl)glycyl depsipeptides. This is as would be expected for a double-displacement mechanism where the Ser-70 becomes acylated at an intermediate stage. Further, however, the mutant enzyme, unlike the wild-type, does not catalyse aminolysis of depsipeptides by D-phenylalanine. If the active site is not structurally disrupted by the mutation, this result shows that Ser-70 is necessary for the aminolysis reaction and implies that this reaction, like the hydrolysis, proceeds by way of an acyl-(serine)-enzyme intermediate. Although physical evidence suggests that the mutant enzyme does not have a structure in solution identical with that of the wild-type, the mutant does still bind beta-lactam substrates. The latter result suggests sufficient conservation of the active-site structure for the major conclusion above to hold.