TABLE 1.

Steady-state kinetic parameters of the purified VIM-7 in comparison with those of the other variants, VIM-1 and VIM-2a

SubstrateKm (μM)kcat (s−1)kcat/Km (μM−1·s−1)
VIM-7VIM-1bVIM-2bVIM-7VIM-1bVIM-2bVIM-7VIM-1bVIM-2b
Penicillins
    Benzylpenicillin17 ± 2c84070430 ± 230280250.0364
    Ampicillin15 ± 2c92090190 ± 1735125130.0381.4
    Carbenicillin84 ± 10c752051,200 ± 74170185142.30.9
    Piperacillin26 ± 3c3,500125140 ± 131,9003005.40.542.4
    Azlocillin66 ± 1612020078 ± 41,5002001.212.51.0
    Cloxacillin860 ± 310ND250 ± 472,500 ± 400ND350 ± 182.9ND1.4
Cephalosporins
    Cephalothin45 ± 55511180 ± 62801304.05.112
    Cephaloridine250 ± 213050180 ± 63151400.7210.52.8
    Cefoxitin68 ± 71301310 ± 0.326150.150.21.2
    Cefuroxime29 ± 4422016 ± 0.532580.557.70.4
    Cefotaxime22 ± 22501256 ± 2170702.60.685.8
    Ceftazidime120 ± 25800721.4 ± 0.1603.60.0120.0750.05
    Cefepime580 ± 61150>4005.3 ± 0.2550>400.00913.70.1
    Nitrocefin58 ± 317181,500 ± 2995770265.642.8
    Moxalactam75 ± 1545055230 ± 1343903.10.0961.6
Carbapenems
    Imipenem27 ± 21.59100 ± 20.2343.70.133.8
    Meropenem38 ± 450242 ± 0.81351.10.262.5
    Ertapenem28 ± 3ND9 ± 0.68 ± 0.2ND0.2 ± 0.010.29ND0.022
Monobactam (aztreonam)2,700 ± 630c>1,000>1,000NH<0.01<0.01ND<1.0 × 102<1.0 × 102
Inhibitors
    Tazobactam3,500 ± 36034087568 ± 35.3280.0190.0160.032
    Sulbactam740 ± 90200320110 ± 610230.150.050.072
    Clavulanic acid940 ± 320cND>5,000c2.3 ± 0.4dND5.4 ± 2.1d0.0025ND0.0011
  • a ND, data not determined; NH, no measurable hydrolysis.

  • b Kinetic constants for VIM-1 are from Franceschini et al. (3), and those for VIM-2 are from Docquier et al. (2) except for those for cloxacillin, ertapenem, and clavulanic acid (this study).

  • c Km values were measured as inhibition constants (Ki) in a competitive model using nitrocefin as the reporter substrate.

  • d kcat values were calculated from fits of initial velocity against the clavulanate concentration assuming the Km equals the Ki (from the experiment).