TABLE 2

In vitro kinetic data for enzyme catalysis of PED6 substrate via ubiquitin activation

EnzymeaMonoubiquitin activationK63-linked Ub2 activation
Kact (M)bkcat (s−1)ckcat/Kact (M−1 s−1)Kact (M)kcat (s−1)kcat/Kact (M−1 s−1)
PAU(2.6 ± 0.85) × 10−54.7 ± 0.461.8 × 105(3.9 ± 0.98) × 10−83.7 ± 0.189.5 × 107
PYU(9.2 ± 5.60) × 10−60.5 ± 0.035.4 × 104(4.8 ± 0.17) × 10−80.5 ± 0.031.0 × 107
BTU(2.1 ± 0.55) × 10−50.9 ± 0.064.2 × 104(4.6 ± 2.82) × 10−70.5 ± 0.081.1 × 106
PFU(1.5 ± 0.37) × 10−41.5 ± 0.161.0 × 104(5.2 ± 0.19) × 10−72.8 ± 0.284.8 × 106
  • a No enzyme activity was detectable in the absence of ubiquitin.

  • b Activation constant, or the concentration of ubiquitin activator required to reach half-maximal enzyme initial rate of PED6 cleavage, obtained from a one-site binding model nonlinear regression analysis of a theoretical curve generated using GraphPad Prism 5.0 (GraphPad Software Inc.) software (n = 3).

  • c Catalytic constants obtained from nonlinear regression analysis of a theoretical curve generated by GraphPad Prism 5.0 using the conversion of 59,293 relative fluorescence units, equal to 1 nmol of PED6.