Examples of the hydrolysis of d- and l-methylphenidate (d-MP and l-MP) by CES1A1, CES2, and CES3
d- and l-Methylphenidate (128 μM) were incubated with 100 μg/ml CES1A1, CES2, or CES3, respectively, in the total volume of 0.5 ml 20 mM Hepes buffer, pH 7.0, at 25°C for a 20 min or b 24 hours. This is a single experiment to demonstrate the enzymatic versus nonenzymatic hydrolysis of methylphenidate. Replicate kinetic analysis is shown in Table 3.
Enzyme | Total RA | Control RA | Enzymatic RA | |
|---|---|---|---|---|
| μM | μM | μM | ||
| CES1A1 (native)a | l-MP | 24.6 | 1.3 | 23.3 |
| d-MP | 6.8 | 1.2 | 5.6 | |
| CES1A1 (recombinant)a | l-MP | 21.9 | 1.3 | 20.6 |
| d-MP | 6.3 | 1.2 | 5.1 | |
| CES2 (native)b | l-MP | 43.9 | 43.2 | 0.7 |
| d-MP | 44.2 | 45.7 | -0.15 | |
| CES2 (recombinant)b | l-MP | 43.5 | 43.2 | 0.3 |
| d-MP | 44.3 | 45.7 | -1.4 | |
| CES3 (recombinant)b | l-MP | 54.4 | 54.1 | 0.3 |
|
| d-MP | 48.6 | 47.9 | 0.7 |