Table 1. Steady-State Kinetic Values and Acidity Constants (pKa1, pKa2) for Wild-Type and Mutant MtIGPSs and Ratios of kcat(mutant)/kcat(wild-type)a.
| MtIGPS | kcat (s–1) | KM (μM) | kcat(mutant)/kcat(wild-type) | pKa1 | pKa2 |
|---|---|---|---|---|---|
| wild-type | 0.022 ± 0.002 | 6.9 ± 1.4 | 1.0 | 6.3 ± 0.1 | 9.0 ± 0.1 |
| Glu57Ala | b | b | b | b | b |
| Glu57Asp | 0.0078 ± 0.0009 | 12 ± 4 | 0.35 | 6.5 ± 0.1 | 7.7 ± 0.1 |
| Glu57Gln | 1.10 × 10–4 ± 0.05 × 10–4 | 4.5 ± 0.8 | 0.0050 | ||
| Lys59Ala | b | b | b | b | b |
| Lys59Arg | 4.8 × 10–5 ± 0.2 ×10–5 | 19 ± 2 | 0.0022 | 6.4 ± 0.1 | 8.5 ± 0.1 |
| Lys119Ala | b | b | b | b | b |
| Lys119Arg | 8.1 × 10–6 ± 0.1 × 10–6 | 5.1 ± 0.7 | 0.00037 | 7.3 ± 0.1 | 8.7 ± 0.1 |
| Glu168Ala | b | b | b | b | b |
| Glu168Asp | 0.0110 ± 0.0003 | 26 ± 3 | 0.50 | 6.8 ± 0.2 | 9.0 ± 0.2 |
| Glu168Gln | 2.3 × 10–6 ± 0.1 × 10–6 | 2.4 ± 0.3 | 0.00010 | 7.0 ± 0.1 | 8.3 ± 0.1 |
| Glu219Ala | b | b | b | b | b |
| Glu219Asp | 2.70 × 10–4 ± 0.02 × 10–4 | 3.9 ± 0.3 | 0.012 | 6.1 ± 0.1 | 7.6 ± 0.2 |
| Glu219Asn | 0.0103 ± 0.0007 | 18 ± 3 | 0.47 | 6.0 ± 0.1 | 10.0 ± 0.2 |
| Glu219Gln | 0.23 ± 0.02 | 21 ± 4 | 10.5 | 6.2 ± 0.1 | 9.9 ± 0.1 |
a
Data are presented with standard errors (SE). Lys119Arg and Glu168Gln values are based on fluorescence measurements. All rate versus pH profiles showed bell-shaped curves, and pKa1 and pKa2 are shown with SEs. Note that the rate versus pH data for Glu57Gln MtIGPS did not yield a good fit with eq 1 (section 2.5) with a maximum normalized rate close to 1.0 and pKa values are not included for this mutant.
b
We observed no activity for the alanine variants.