Directed Evolution of Aminoglycoside Phosphotransferase (3′) Type IIIa Variants That Inactivate Amikacin but Impose Significant Fitness Costs
Figure 2
Michaelis-Menten plots of the wild-type and 4.1 APH(3′)-IIIa variants.
The plots show the dependence of initial velocity upon substrate concentration for the following reactions: a) wild-type APH(3′)-IIIa with kanamycin, b) artificially evolved 4.1 variant with kanamycin, c) wild-type APH(3′)-IIIa with amikacin, and d) 4.1 variant with amikacin. Substrate concentration is in units of micromolar and reaction velocity is in moles of substrate/moles of enzyme/second. Each series of reactions was conducted in triplicate. The average initial velocity values were fit to the Michaelis-Menten equation (a, c) or a simple substrate inhibition model (d) as described in the Methods; the derived kinetic parameters are presented in Table 4.