TABLE 4.
Affect of amino acid substitutions on P2 specificity of OpdBa
| Mutant | Amino acid substitution and kinetics
|
|||||
|---|---|---|---|---|---|---|
| Cbz-Arg-Arg-AMC
|
Cbz-Phe-Arg-AMC
|
|||||
| Km (μM) | kcat (s−1) | kcat/Km (s−1 μM−1) | Km (μM) | kcat (s−1) | kcat/Km (s−1 μM−1) | |
| Wild type | 0.8 | 29 | 36 | 2.1 | 21 | 10 |
| D460T | 2.1 | 22 | 10 | 2.9 | 18 | 6 |
| D462N | 2.3 | 23 | 10 | 3.0 | 19 | 6 |
| D460T-D462N | 2.9 | 18 | 6 | 2.9 | 17 | 6 |
| E494H | 1.0 | 33 | 33 | 2.5 | 18 | 7 |
| D567H | 0.8 | 29 | 36 | 2.0 | 22 | 10 |
| E576A | 4.5 | 9 | 2 | 12.0 | 7 | 0.6 |
| E578A | 3.1 | 9 | 3 | 5.9 | 9 | 2 |
| D599H | 1.0 | 30 | 30 | 2.1 | 20 | 10 |
| E624H | 1.0 | 33 | 33 | 2.6 | 22 | 8 |
| D638Q | 1.1 | 31 | 28 | 2.8 | 20 | 7 |
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, Suc-Ile-Ala-AMC, Suc-Gly-Pro-AMC, Ac-Tyr-Val-Ala-Asp-pNA, and Suc-Ala-Ala-Pro-Phe-AMC. The standard errors of the values for Km and kcat were within 10% of their respective means.