Table 2.
Steady-state kinetic parameters and product binding kinetics of native (l-DHFR) and heavy (h-DHFR) hsDHFR at pH 7.65 and 25 °Ca,b
| l-DHFR | h-DHFR | h-KIE | |
|---|---|---|---|
| kcatDHF, (s−1) | 14.03 ± 0.02 | 9.77 ± 0.12 | 1.44 ± 0.02 |
| KmDHF, (μM) | 0.29 ± 0.05 | 0.27 ± 0.09 | 1.07 ± 0.40 |
| (kcat/Km)DHF × 107 (M−1s−1) | 4.84 ± 0.83 | 3.62 ± 1.20 | 1.34 ± 0.50 |
| konTHF × 10−6(M−1s−1) | 38.7 ± 1.8 | 38.5 ± 0.5 | 1.01 ± 0.05 |
| koffTHF, (s−1) | 110 ± 10 | 80 ± 5 | 1.38 ± 0.13 |
|
| |||
| kcatNADPH, (s−1) | 14.10 ± 0.01 | 9.75 ± 0.11 | 1.45 ± 0.02 |
| KmNADPH, (μM) | 0.36 ± 0.05 | 0.34 ± 0.07 | 1.06 ± 0.26 |
| (kcat/Km)NADPH × 107 (M−1s−1) | 3.92 ± 0.54 | 2.87 ± 0.59 | 1.37 ± 0.34 |
| konNADP × 10−6 (M−1s−1) | 1.19 ± 0.05 | 1.26 ± 0.05 | 0.94 ± 0.06 |
| koffNADP, (s−1) | 140 ± 1 | 140 ± 1 | 1.00 ± 0.01 |
a
kcatDHF and KmDHF are the turnover number and Michaelis constant with DHF as substrate measured in the presence of 100 μM NADPH and varying concentrations of DHF
b
kcatNADPH and KmNADPH are the turnover number and Michaelis constant with NADPH as substrate, measured in the presence of 100 μM DHF and varying concentrations of NADPH.
c
h-KIE is the heavy enzyme KIE, calculated by taking the ratio between the kinetic parameter of l-DHFR and the same parameter of h-DHFR.