Table 2.
Kinetic parameters for miscellaneous ACCO mutants influencing enzyme activity and binding sites for ACC, Asc and bicarbonate. ACC oxidase activity was determined using the ACCO fusion protein and standard assay. ND, not determined.
| ACCO protein | ACC Km (µM) | Asc Ka (mM) | HCO3 Ka (mM) | ACCO activity (nmol min−1 mg−1) |
|---|---|---|---|---|
| Native | 51 | 2.3 | 4.9 | 3.48 |
| Glu294Phe | ND | ND | ND | 0.7 |
| Lys296Glu | ND | ND | ND | 4.16 |
| Glu297Leu | ND | ND | ND | 0.07 |
| Pro298Ala | 62 | 1.7 | 3.1 | 14.5 |
| Phe300Gln | 129 | 0.52 | 32.4 | 0.013 |
| Phe300Tyr | 36 | 1.0 | 6.2 | 0.47 |
| Glu301Leu | 31 | 1.5 | 5.5 | 0.19 |
| Glu301Asp | ND | ND | ND | 1.09 |
| Trp203Phe | 63 | 3.1 | 2.7 | 0.83 |
| Asn216Phe | 158 | 3.8 | 2.8 | 1.5 |
| Tyr251Phe | 62 | 1.5 | 1.2 | 1.09 |
| Trp203Phe | 63 | 3.1 | 2.7 | 2.0 |
| Phe187Tyr | 90 | 3.3 | 0.5 | 6.4 |
| Cys28Ala | 62 | 2.6 | 2.6 | 0.032 |
| Cys133ALA | ND | ND | ND | 0.22 |
| Cys133Pro | ND | ND | ND | 3.59 |
| Cys165Ala | ND | ND | ND | 0.13 |
| Gln188Ala | ND | ND | ND | 0.19 |
| Gln188Asn | 287 | 1.5 | 8.4 | 0.047 |
| Gln188Lys | ND | ND | ND | 0.12 |
| Lys199Glu | 78 | 0.54 | 3.1 | 0.032 |
| Lys230Glu | ND | 3.6 | 2.9 | 0.2 |
| Lys230Qln | ND | ND | 1.1 | 0.39 |
| Lys230Arg | ND | 3.6 | 1.4 | 1.16 |
| Lys172Glu | ND | 3.2 | ND | 1.75 |
| Lys144Glu | ND | 4.5 | ND | 2.48 |