TABLE 1.
Substrate selectivity for the MhpE-catalyzed forward and reverse reactionsa
| Substrate(s) (concn) | Assay method (retention time)b | vrelc |
|---|---|---|
| 4-Hydroxy-2-keto-pentanoic acid (200 μM) | LDH/NADH | 1.00 |
| Acetaldehyde + pyruvic acid (both 100 mg/ml) | HPLC (34 min) | 0.13 |
| Propionaldehyde + pyruvic acid (both 100 mg/ml) | HPLC | — |
| Acetaldehyde + 2-keto-butyric acid (both 100 mg/ml) | HPLC (37 min) | 0.12 |
| Acetaldehyde + phenylpyruvic acid (both 100 mg/ml) | HPLC (33 min) | 0.13 |
a
Assay methods for forward and reverse reactions are described in the text and are illustrated in Fig. 1. Assays were conducted at 20°C in 50 mM potassium phosphate buffer, pH 7.0.
b
Retention time for α-keto-γ-butyrolactone derivatives observed on organic acids HPLC (Bio-Rad HPX-87H column, eluent 0.005 mM H2SO4, flow rate 0.6 ml/min). LDH, lactate dehydrogenase.
c
Reaction rate relative to HKP forward reaction. —, no reaction observed.