Steady-state kinetics of recombinant wild-type and mutant P5′N-1 as a function of UMP. (A) Steady-state kinetics of recombinant wild-type and mutant P5′N-1 using UMP as substrate. (B) Semilog plot of the data in panel A to highlight the difference of K_m of mutants with respect to the wild-type enzyme. Enzyme activity was assayed at 37°C, pH 7.5, as described in “Materials and methods.” • indicates wild type; □, D87V; ▴, L131P; ♦, N179S; ▿, G230R. The solid line differentiates the wild type from mutants (dashed line).