Larger increases in sensitivity to paracatalytic inactivation than in catalytic competence during experimental evolution of the second beta-galactosidase of Escherichia coli

Second-order rate constants (M-1.s(-1)) at 25 degrees C and pH 7.5 for inactivation of first-generation (ebg(a) and ebg(b)), second-generation (ebg(ab) and ebg(abcd)) and third-generation (ebg(abcde)) experimental evolvants of the title enzyme by 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside are 0.042, 0.30, 10, 24 and 57 respectively. Only partial inactivation is observed, except for ebg(abcde). At a single high inactivator concentration, inactivation of the wild-type ebg(o) is also seen. The changes in sensitivity to the paracatalytic inactivator (over a range of 10(3.3)) are larger than changes in k(cat)/K-m for lactose (over a range of 10(2.7)) or nitrophenyl galactosides (over a range of only 10(1.3)), or changes in degalactosylation rate (over a range of 10(1.7)). These data raise the possibility that evolution in the reverse sense, towards insensitivity to a paracatalytic inactivator with a proportionally lower effect on transformation of substrate, may become a mechanism for the development of bacterial resistance to antibiotics that act by paracatalytic enzyme inactivation.