TABLE 5

Kinetic parameters of purified His-tagged PykA and PykFa

SubstratePykAPykF
Km (mM)kcat (s−1)kcat/Km (M−1 s−1)Km (mM)kcat (s−1)kcat/Km (M−1 s−1)
ADP0.060 ± 0.009129 ± 14(2.17 ± 0.26) × 1060.210 ± 0.06680 ± 11(3.99 ± 0.80) × 105
UDP0.089 ± 0.019164 ± 20(1.88 ± 0.22) × 1060.648 ± 0.085166 ± 29(2.55 ± 0.19) × 105
GDP0.345 ± 0.036209 ± 17(6.07 ± 0.20) × 105NDNDND
CDP0.560 ± 0.02843 ± 2(7.71 ± 0.32) × 104NDNDND
  • a ND, not detectable. E. coli BL21(DE3) and pET30a vector were used to express the His-tagged pyruvate kinases. The pykA gene was amplified from genomic DNA of BW25113 with primers pykA-NdeI-1 and pykAChis-XhoI-2 (Table S2) and cloned into pET30a vector through NdeI and XhoI sites, while the pykF gene was amplified from genomic DNA of BW25113 with primers pykF-NdeI-1 and pykFChis-HindIII-2 (Table S2) and cloned into pET30a vector through NdeI and HindIII sites. The Michaelis-Menten equation was used to calculate the kinetic parameters. Three repeats were performed, and the error data represent standard deviations.