TABLE 4.
Biotin- and biocytin-dependent oxaloacetate decarboxylation activities for site-directed mutants of ΔBCΔBCCP RePC compared with wild-type ΔBCΔBCCP RePC
Reaction conditions were as follows: 100 mm Tris-HCl (pH 7.8), 30 °C, 1 mm oxaloacetate, 0.24 mm NADH.
| 1 mm oxaloacetate, kcata | +5 mm biotin |
+5 mm biocytin |
|||
|---|---|---|---|---|---|
| kcat | kcat (+biotin)/kcat (−biotin) | kcat | kcat (+biocytin)/kcat (−biocytin) | ||
| min−1 | min−1 | min−1 | |||
| Wild-type CT | 0.46 ± 0.007 | 0.88 ± 0.01 | 1.9 | 2.56 ± 0.006 | 5.6 |
| Y628F | 0.38 ± 0.002 | 0.43 ± 0.01 | 1.1 | 0.41 ± 0.007 | 1.1 |
| Y628A | 0.33 ± 0.03 | 0.35 ± 0.006 | 1.1 | 0.36 ± 0.006 | 1.1 |
| D590A | 0.38 ± 0.01 | 1.16 ± 0.01 | 3.1 | 0.40 ± 0.006 | 1.1 |
a Specific activities were determined in triplicate, and the error reported is the S.D. value of the three determinations.