Table 2.
Steady state kinetic parameters for various PhnY constructs and WT GAPDH. See also Supplemental Figure S6.
| KM,Pn (µM) | KM,NAD+ (µM) | kcat (s−1) | |
|---|---|---|---|
| PhnY constructs with PnAA | |||
| Wild type | 3.2 ± 0.7 | 58 ± 9 | 2.2 ± 0.1 |
| Arg108Ala | 9.7 ± 0.8 | 18 ± 2 | 0.051 ± 0.001 |
| Asn158Ala | 29 ± 9 | 40 ± 6 | 0.010 ± 0.001 |
| Glu254Ala | n.d.1 | n.d. | n.d. |
| Arg290Ala | 5.1 ± 0.6 | 75 ± 8 | 0.12 ± 0.003 |
| Cys291Ala | n.d. | n.d. | n.d. |
| Glu385Ala2 | 19 ± 3 | 370 ± 10 | 0.19 ± 0.03 |
| Arg447Ala | 150 ± 20 | 54 ± 1 | 0.076 ± 0.003 |
| PhnY constructs with G3P | |||
| Wild type | 97 ± 7 | 530 ± 50 | 0.098 ± 0.003 |
| Cys291Ala | n.d. | n.d. | n.d. |
| PhnY constructs with 3-OPP | |||
| Wild type | 3300 ± 100 | n.d. | 1.5 ± 0.1 |
| Cys291Ala | n.d. | n.d. | n.d. |
| GAPDH with various substrates | |||
| PnAA | n.d. | n.d. | n.d. |
| 3-OPP | 1500 ± 300 | 330 ± 20 | 0.038 ± 0.022 |
| G3P (racemic) | 25 ± 7 | 22 ± 1 | 22 ± 2 |
1
n.d.: Not determined. Low levels of detectable activity precluded kinetic parameter determination.
2
: This mutant displayed substrate inhibition (see Figure S6); the Ki,PnAA was 420 ± 70 µM.