Table 3

KPC-2 R220 variant substrate kinetics

β-LactamKPC-2 variantKm (μM) (± SD)kcat (s−1) (± SD)kcat/Km (μM−1 s−1) (± SD)
PiperacillinWT12 ± 165 ± 75.4 ± 0.5
R220H13 ± 168 ± 75.2 ± 0.5
R220A164 ± 16193 ± 201.2 ± 0.1
R220M35 ± 485 ± 92.4 ± 0.2
R220K23 ± 240 ± 41.7 ± 0.2
E276A12 ± 159 ± 64.8 ± 0.5
E276D5.2 ± 0.538 ± 47.2 ± 0.7
CephalothinWT38 ± 4223 ± 205.9 ± 0.6
R220H108 ± 11206 ± 201.9 ± 0.2
R220A207 ± 21379 ± 381.8 ± 0.2
R220M98 ± 10156 ± 161.6 ± 0.2
R220K45 ± 588 ± 92.0 ± 0.2
E276A24 ± 2157 ± 166.5 ± 0.6
E276D12 ± 1139 ± 1411 ± 1
NitrocefinWT8 ± 1130 ± 1316 ± 2
R220H16 ± 2260 ± 2616 ± 2
R220A54 ± 5327 ± 336.1 ± 0.6
R220M94 ± 9724 ± 727.7 ± 0.8
R220K10 ± 1120 ± 1212 ± 1
E276A4.9 ± 0.497 ± 1019 ± 2
E276D4.1 ± 0.477 ± 819 ± 2
CefotaximeWT190 ± 19140 ± 140.7 ± 0.1
R220HN/DaN/DN/D
R220AN/DN/DN/D
R220MN/DN/DN/D
R220KN/DN/DN/D
E276A278 ± 28118 ± 120.4 ± 0.1
E276D253 ± 25100 ± 100.5 ± 0.1
ImipenemWT21 ± 221 ± 21.0 ± 0.1
R220H11 ± 19 ± 10.8 ± 0.1
R220A18 ± 24 ± 10.2 ± 0.1
R220M28 ± 310 ± 10.4 ± 0.1
R220K5 ± 17 ± 11.4 ± 0.1
E276A12 ± 111 ± 10.9 ± 0.1
E276D10 ± 113 ± 11.3 ± 0.1
  • a N/D, values could not be determined; variants did not hydrolyze cefotaxime at detectable levels. In addition, attempts to obtain a Ki for cefotaxime were not successful even when using excess (up to 10 mM) cefotaxime, revealing that binding of cefotaxime was also compromised in these variants.