Probing the role of Asn 152 in the class C Â²-lactamase AmpC
College of Liberal Arts and Sciences
Physical Sciences and Mathematics
AmpC, a class C Â²-lactamase, is a main cause of antibiotic resistance to cephalosporins in many species of bacteria. In the hydrolytic cleavage of antibiotics by AmpC, the current proposed mechanism involves an acyl-intermediate, where the enzyme becomes covalently attached to the drug at serine-64, before an activated water molecule hydrolyzes the bond and regenerates the enzyme. Although this mechanism is generally accepted, the exact roles that the other active site residues play in recognition and breakdown of the substrate are not fully understood. Here, we investigated the role of the active site residue asparagine-152 (Asn152) in E. coli AmpC by mutating it to a glycine, serine, or threonine residue and examining the effect that these mutations have on kinetic and structural properties with four different Â²-lactam drugs: cefotaxime, cefoxitin, oxacillin, and a derivative of cephalothin (CENTA). We discovered that although the mutations cause higher Km values with all substrates, they result in 50 to 150 times higher kcat values against cefotaxime. In addition, the N152 mutations provided the enzyme the ability to break down oxacillin, which is not a viable substrate for the wild type AmpC.
Enzyme Mechanisms Conference
Wallar, Bradley J.; Docter, Brianne; Anwar, Mujahid; Leonard, David; and Powers, Rachel, "Probing the role of Asn 152 in the class C Â²-lactamase AmpC" (2013). Faculty Scholarly Dissemination Grants. 1016.
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