Kinetic Study of Laboratory Mutants of NDM-1 Metallo-β-Lactamase and the Importance of an Isoleucine at Position 35

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T) and NDM-1(I35S) enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. The k(cat), K-m, and k(cat)/K-m values calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, the k(cat)/K-m of NDM-1I(35S) for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T) and NDM-1(I35S) enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of alpha-helix content in the mutants.