Table I.
Yeast expression, substrate binding, and activity of the engineered forms of CYP73A1
C4H activity was measured at saturating substrate concentration (100 μm). CYP73A1 expression was evaluated from CO-reduced versus reduced difference spectra. Dissociation constants for the enzyme-substrate complex and εΔA(peak-trough) were calculated from the saturation curves of difference spectra recorded upon cinnamate binding to the oxidized yeast microsomes. P450 and C4H activity were not detected in W(R) transformed with a void plasmid.
| Protein | C4H Activity | Relative Expression | Cinnamate Binding | |
|---|---|---|---|---|
| KD | εΔ(390-420nm) | |||
| min-1 | μm | mm-1 cm-1 | ||
| CYP73A1 | 20 ± 3 | 100 ± 3.6 | 8.8 ± 0.3 | 125 |
| 73His | 22 ± 2.6 | 61 ± 3 | 4.2 ± 0.1 | 121 |
| PD73 | 7.9 ± 0.9 | 98 ± 3.5 | 6.4 ± 0.3 | 113 |
| PD73His | 13 ± 0.3 | 77 ± 4.8 | 2.7 ± 0.1 | 119 |