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.
Mazzella
L J
LJ
Department of Chemistry, Wesleyan University, Middletown, CT 06457.
Pazhanisamy
S
S
Pratt
R F
RF
D000588:Amines / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D009154:Mutation; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids; D013056:Spectrophotometry, Ultraviolet; D001618:beta-Lactamases / Q000235:genetics*
D000588:Amines; D010455:Peptides; D001618:beta-Lactamases