Table 2.
Kinetic Data for BtGH97a with DNP-Glc, pNP-Glc and Maltose, BtGH97b with pNP-Gal and Melibiose, and BtGH97a Mutants with pNP-Glc and DNP-Glc
| Enzyme/Mutant | Substrate | KM (mM) | kcat (s−1) | kcat/KM (s−1 mM−1) |
|---|---|---|---|---|
| BtGH97a | DNP-Glc | 0.14 ± 0.01 | 280 ± 7 | 1997 |
| pNP-Glc | 0.085 ± 0.009 | 53 ± 2 | 620 | |
| Maltosea | 2.0 ± 0.4 | 0.015 ± 0.002 | 0.0072 | |
| BtGH97b | pNP-Gal | 0.23 ± 0.01 | 91 ± 2 | 391 |
| Melibiose | 1.5 ± 0.17 | 0.015 ± 0.0004 | 0.011 | |
| BtGH97a | ||||
| E194A | pNP-Glc | 0.31 ± 0.02 | 2.8 ± 0.07 | 9.2 |
| E532A | pNP-Glc | 0.14 ± 0.02 | 1.1 ± 0.04 | 7.7 |
| Wild-type | DNP-Glc | — | — | 1807 ± 68 |
| E194A | DNP-Glc | — | — | 255 ± 3 |
| E439A | DNP-Glc | — | — | 0.007 ± 0.001 |
| E508A | DNP-Glc | — | — | ND |
| E526A | DNP-Glc | — | — | 48 ± 4 |
| E532A | DNP-Glc | — | — | 402 ± 29 |
Kinetic data for BtGH97a with DNP-Glc, pNP-Glc and maltose, BtGH97b with pNP-Gal and melibiose, and BtGH97a mutants with pNP-Glc were determined using full Michaelis-Menten kinetics, and those for BtGH97a mutants with DNP-Glc were determined using substrate depletion methods. Where no data are reported for BtGH97a mutants with pNP-Glc, no activity could be measured. ND, none detected.
a
Substrate inhibition was observed. The Ki value for maltose was 17.2 mM.