Table 1.
Apparent kinetic parameters for PmST1 and its mutants. Data from previous reports include PmST1 (aRef15, bRef45), cPd2,3ST,35 Pd2,6ST (dRef54, eRef20) and Psp2,6ST (fRef54, gRef62). ND, not determined. nd, not detected.
| Parent Enzyme |
Mutant | Product sialyl linkage |
Sialyltransferase | CMP-Neu5Ac Hydrolysis | Sialidase Activity | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| kcat (s−1) | KM (mM) |
kcat/KM (s−1 mM−1) |
Specificity (% 6′) |
kcat (s−1) | KM (mM) | kcat/KM (s−1 mM−1) |
kcat (s−1) |
KM (mM) |
kcat/KM (s−1 mM−1) |
|||
| Pd2,3ST | WT | α2–3 | c 24 | c 1.5 | c 16 | c < 0.6 | ND | ND | ND | ND | ND | ND |
| P7H | α2–6 | c 16 | c 3.8 | c 4.3 | c 94.7 | ND | ND | ND | ND | ND | ND | |
| P7H/M117A | α2–6 | c 18 | c 6.4 | c 2.8 | c > 99.6 | ND | ND | ND | ND | ND | ND | |
| PmST1 | WT | α2–3 | a 47 | a 1.4 | a 34 | 3.9 ± 0.8 | b 27 ± 1 | b 1.5 ± 0.2 | b 18 | a 230 | a 24 | a 9.5 |
| P34H | α2–6 | 8.6 ± 1.6 | 0.6 ± 0.4 | 13.5 | 94.0 ± 0.4 | 925.5 ± 66.7 | 1.2 ± 0.5 | 771.3 | 1.2 ± 0.1 | 13.7 ± 1.5 | 0.088 | |
| P34H/M144A | 9.0 ± 1.3 | 1.7 ± 0.5 | 5.3 | 97.9 ± 0.5 | 34.5 ± 0.3 | 2.3 ± 0.1 | 15.1 | 0.51 ± 0.03 | 15.1 ± 1.1 | 0.034 | ||
| P34H/ M144L | 20.0 ± 4.4 | 0.8 ± 0.2 | 26 | 98.7 ± 0.1 | 30.5 ± 0.8 | 3.4 ± 0.1 | 8.9 | 1.14 ± 0.1 | 6.4 ± 0.6 | 0.178 | ||
| P34H/M144V | 5.5 ± 0.4 | 1.4 ± 0.2 | 3.9 | 98.5 ± 0.1 | 62.4 ± 7.6 | 10.5 ± 2.7 | 6.0 | 0.27 ± 0.019 | 14.9 ± 1.3 | 0.018 | ||
| Pd2,6ST | WT | d 2.3 ± 0.1 | d 0.8 ± 0.1 | d 3 | nd | 6.7 ± 1.6 | 44.9 ± 14.2 | 0.15 | e 0.14 ± 0.01 | e 7.6 ± 0.5 | e 0.018 | |
| Psp2,6ST | WT | f 1.7 ± 0.2 | f 0.4 ± 0.1 | f 4.8 | nd | g 4.8 ± 0.5 | g 17 ± 4 | g 0.28 | (7.9 ± 0.3) × 10−3 | 4.7 ± 0.3 | 0.0017 | |