TABLE 2.
Kinetic parameters of wild-type SUMO-BDP and mutant ΔΝ83/F361X SUMO-BDP proteins with pNPP as substrate
SUMO-BDP and ΔΝ83/F361X SUMO-BDP proteins were expressed in growth medium supplemented with 1 mm MnCl2. The purified enzymes were exhaustively dialyzed against 50 mm HEPES, pH 7.5, 100 mm KCl, 5% glycerol, 5 mm EDTA, and 5 mm EGTA. The residual EDTA and EGTA were removed by a second dialysis step in the absence of the metal chelators. The phosphatase activity of SUMO-BDP (33 nm) and ΔΝ83/F361X SUMO-BDP (35 nm) were assayed spectrophotometrically at 30 °C in a Tris-HCl buffer using varying concentrations of pNPP (0.95 mm to 20 mm) as a substrate; increase in A410 nm was monitored. The kcat is equivalent to a specific activity of 14.8 μmol/min/mg for SUMO-BDP and 8.7 μmol/min/mg for ΔΝ83/F361X SUMO-BDP.
| SUMO-BDP | Metals | Substrate | kcat | Km (pNPP) | Km (metal) | kcat/ Km (pNPP) | kcat/ Km (metal) |
|---|---|---|---|---|---|---|---|
| s−1 | mm | mm | m−1s−1 | m−1s−1 | |||
| Wild type | MnCl2 | pNPP | 43.2 ± 2 | 4.4 ± 0.1 | 5.1 ± 0.2 | 9,818 ± 410 | 8,470 ± 430 |
| Wild type | MgCl2 | pNPP | 0 | ||||
| Wild type | CaCl2 | pNPP | 0 | ||||
| ΔN83/F361X | MnCl2 | pNPP | 25.5 ± 1 | 7.7 ± 0.6 | 8.9 ± 0.2 | 3,311 ± 120 | 2,865 ± 130 |
| ΔN83/F361X | MgCl2 | pNPP | 0 | ||||
| ΔN83/F361X | CaCl2 | pNPP | 0 |