TABLE 3.
Catalytic efficiencies and substrate binding constants of MBP-Δ29NST and single point mutants in the hydrolysis and sialyltransfer reactions
| Hydrolysis of CMP-Neu5Ac in the absence of acceptor substrates | |||||
| Kinetic parameters | MBP-29NST | R282A | E124A | H280A | D258Na |
| kcat (min−1) | 0.59 | 0.32 | 0.55 | 0.05 | – |
| Km (μm) | 20 | 55 | 29 | 12.7 | – |
| kcat/Km (mm−1min−1) | 29 | 5.76 | 18.7 | 3.9 | – |
| Transfer of CMP-Neu5Ac to acceptor substrates | |||||
| Kinetic parameters | MBP-29NST | R282A | E124A | H280A | D258Na |
| kcat (min−1) | 5.4 | 0.62 | 4.4 | 0.108 | – |
| Km (μm) | 43 | 62 | 194 | 54 | – |
| kcat/Km (mm−1min−1) | 124 | 10 | 23 | 2 | – |
a –, D258N mutant had no activity in either enzyme-catalyzed hydrolysis of CMPNeuAc or enzyme-catalyzed transfer of CMPNeuAc.