Table 1.
Steady-state kinetic parameters for AfChoAp and AfBadApa
| Protein | Substrate | Ligand | kcat, s−1 | KA, mM | kcat/KA, M−1 s−1 | KO2, mM | kcat/KO2, M−1 s−1 | KNAD(P)+, mM | kcat/KNAD(P)+, M−1 s−1 | Kia, mM |
|---|---|---|---|---|---|---|---|---|---|---|
| AfChoAp | Choline | 40.0 ± 0.2 | 1.8 ± 0.1 | 22,200 ± 700 | 0.69 ± 0.01 | 58,000 ± 1,000 | 1.4 ± 0.1 | |||
| Betaine-aldehyde | 18.2 ± 0.4 | 1.6 ± 0.1 | 11,400 ± 800 | 0.36 ± 0.02 | 52,000 ± 3,200 | 3.8 ± 0.3 | ||||
| Choline | NAD+ | 23.0 ± 1.0 | 0.62 ± 0.03 | 37,400 ± 2,400 | 2.4 ± 0.1 | |||||
| AfBadAp | NAD+ | 10.2 ± 0.4 | 0.025 ± 0.001 | 408,000 ± 20,000 | 0.037 ± 0.004 | 276,000 ± 11,000 | 50 ± 1 | |||
| NADP+ | 1.4 ± 0.1 | 0.11 ± 0.01 | 13,000 ± 1,300 | 1.7 ± 0.1 | 820 ± 70 | 150 ± 13 |
Parameters are for AfChoAp with oxygen and choline or betaine aldehyde as substrates and for AfBadAp with betaine aldehyde and NAD(P)+ as substrates. Values are sample means and standard errors. KA is the Km for either choline or betaine aldehyde. Data for reactions with no ligand were fitted to the equation v/e = kcatAB/(KaB + KbA + AB + KiaKb) (Equation 1) and those for AfChoAp with choline and NAD+ ligand were fitted to the equation v/e = kcatAB/(KbA + AB + Kia) (Equation 2), where Ka and Kb are the Michaelis constants for choline and betaine aldehyde (Ka) and oxygen or NAD(P)+ (Kb), respectively, and kcat is the turnover enzyme (e) saturated with both substrates. The choline oxidase activity was measured in 20 mM Tris-HCl (pH 8.0) at 25°C and the betaine aldehyde dehydrogenase activity in 100 mM HEPES-KOH (pH 8.0) at 25°C.