The activity of these two mutants was measured with imipenem, benzylpenicillin, nitrocefin, cephaloridine, and cefotaxime as substrates in the absence of added zinc, i.e. The activity of the two mutants was measured in the absence of added Zn 2+ (<0.4 μ m) with various substrates representing the three major families of β-lactam antibiotics, namely carbapenems, penicillins, and cephalosporins ( Table IV). At a free zinc concentration of 100 μ m, inductively coupled plasma mass spectroscopy results show that the WT enzyme and the N116H/N220G double mutant bind 2 zinc ions per molecule, whereas the N220G mutant binds 1.7 zinc ions per molecule. Under these conditions, the N220G and N116H/N220G proteins contained one zinc ion per molecule as reported for the WT β-lactamase ( Table III). The enzymes were stored in 15 m m sodium cacodylate, pH 6.5, and in the presence of a free zinc concentration lower than 0.4 μ m. ![]() The N116H/N220G mutant is a little less stable than the WT enzyme and the N220G mutant, because the transition already occurred between 2 and 3 m urea. ![]() Transitions between native and denaturated states occurred near 3 m urea. The stability of the N220G mutant with urea as denaturating agent is similar to that of the WT enzyme. Glycobiology and Extracellular Matrices.
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