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

Abstract

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.

Structure of the wild-type TEM-1 β-lactamase at 1.55 Å and the mutant enzyme Ser70Ala at 2.1 Å suggest the mode of noncovalent catalysis for the mutant enzyme

“…The activity of the S70G -lactamases is higher than expected, suggesting the possibility of wild-type contamination. It has been suggested that in the resulting S70G mutant with single nucleotide change from the original Ser codon (AGC) to the glycine codon (GGC) that mistranslation of the glycine codon back to AGC leads to wild-type contamination (Mazzella et al, 1991). The same study has shown that the Table 3 Selected active-site distances in the wild-type and the mutant S70G -lactamase (Å ).…”

“…glycine codon GGG having a double-nucleotide mismatch prevents wild-type contamination and reduces the activity of the S70G mutant -lactamase 50-fold (Mazzella et al, 1991). For this study, a second construct of the S70G mutation was made using the preferred glycine codon for E. coli, GGG, having a double nucleotide change.…”

“…albus -lactamase Ser70 has been systematically mutated to alanine and cysteine and the kinetic properties of the S70A and S70C enzymes showed similar trends (Jacob et al, 1991). In TEM-1 -lactamase, the same mutants were evaluated kinetically (S70C and S70G) and the results were not fully consistent (Mazzella et al, 1991;Sigal et al, 1984;Toth et al, 1988). The S70G mutant in TEM-1 -lactamase has been constructed using two different glycine codons, GGC and GGA (Toth et al, 1988).…”

“…In this work, Ser70 in TEM-1 -lactamase has been changed to a glycine to yield mutant enzyme S70G. Although the S70A, S70G and S70C enzymes have been studied from -lactamases from several organisms, their kinetic properties have been quite different, which can be attributed to the different experimental conditions (Chen et al, 1996;Jacob et al, 1991;Mazzella et al, 1991;Sigal et al, 1982Sigal et al, , 1984Toth et al, 1988;Knap & Pratt, 1991). This study was designed to provide independent evaluation of the active-site mutants and to shed additional light onto the mode of non-covalent catalysis.…”

Steady-State Kinetics of the Binding of .beta.-Lactams and Penicilloates to the Second Binding Site of the Enterobacter cloacae P99 .beta.-Lactamase

“…Jamin et al (1993) have concluded that depsipeptides may also bind to a second site on acyl enzymes formed on reaction between the same depsipeptide and the Streptomyces R6 1 DD-peptidase. This is likely true for the P99 P-lactamase also (Mazzella et al, 1991).…”