Table 3. Kinetic Parameters for the Pre-Steady-State Reaction of CpIMPDHa.
parameter | absorbance | fluorescence |
---|---|---|
kmax (s−1) | 41 ± 2 | 16 ± 1 |
Kapp (μM) | 510 ± 90 | 290 ± 60 |
kmax/Kapp (M−1 s−1) | 8.0 × 104 | 5.6 × 104 |
[NADH]/[E]total | 0.43 ± 0.01 | ndb |
Experimental conditions: 1 μM CpIMPDH, 500 μM IMP, 50 mM Tris-HCl, pH 8.0, 100 mM KCl, and 1 mM DTT at 25 °C. Progress curves exhibited a single exponential burst phase followed by a linear steady state and were fit by eq 6. Replots of kobs vs [NAD+] were fit by eq 8 to derive the above pre-steady-state parameters.
Not determined.