TABLE 3.
Affect of amino acid substitutions on P1 specificity of OpdB using Cbz-Arg-AMCa
| Mutant | Kinetics
|
||
|---|---|---|---|
| Km (μM) | kcat (s−1) | kcat/Km (s−1 μM−1) | |
| Wild type | 4.4 | 9 | 2 |
| D460T | 4.6 | 10 | 2 |
| D462N | 4.7 | 10 | 2 |
| D460T-D462N | 5.2 | 7 | 1 |
| E494H | 4.6 | 9 | 2 |
| D567H | 5.0 | 9 | 2 |
| E576A | 33.4 | 2 | 0.06 |
| E578A | 16.4 | 4 | 0.2 |
| E576A-E578A | ND | ND | ND |
| E576W-E578T | ND | ND | ND |
| E576R-E578R | ND | ND | ND |
| D599H | 4.5 | 9 | 2 |
| E624H | 5.4 | 10 | 2 |
| D638Q | 4.0 | 11 | 3 |
a
No activity was observed against H-Arg-AMC, H-Leu-AMC, H-Lys-AMC, Cbz-Leu-Leu-Glu-βNA, Cbz-Ala-Ala-Phe-AMC, Cbz-Gly-Gly-Leu-pNA, Suc-Ile-Ala-AMC, Suc-Gly-Pro-AMC, Ac-Tyr-Val-Ala-Asp-pNA, and Suc-Ala-Ala-Pro-Phe-AMC. ND, no detectable activity. The standard errors of the Km and kcat values were within 10% of their respective means.