Table 3. Kinetic Analysis of Arabidopsis PPA-AT Wild Type and Mutants
Enzymeskcat (s−1)Km (μM)kcat/Km (mM−1 s−1)
Prephenate (Asp amino donor)
Wild type21.3 ± 1.0312 ± 3069.0 ± 4.2
T84V4.5 ± 1.2**676 ± 96*6.5 ± 0.9**
K169S5.5 ± 1.5**791 ± 97*6.7 ± 1.1**
T84VK169SN.D.N.D.N.D.
4-Hydroxyphenylpyruvate (Trp amino donor)
T84VK169S5.0 ± 0.3288 ± 1517.2 ± 0.7
  • Kinetic parameters were obtained for prephenate using aspartate amino donor (20 mM) in 10-min reactions using 0.5 and 2 μg/mL recombinant enzymes for wild type and mutants of Arabidopsis PPA-AT enzymes, respectively. For the T84VK169S double mutant, PPA-AT activity was not detectable (N.D.); instead, kinetic parameters were obtained for 4-hydroxyphenylpyruvate using Trp amino donor (20 mM) in 3-min reactions using 4 μg/mL recombinant enzyme. PLP (200 μM) was used as a cofactor. Data are means ± se (n ≥ 3). Significant difference from the corresponding wild type value is indicated (*P < 0.05, **P < 0.01, Student’s t test).