TABLE 4.
Km and substrate selectivity coefficient values for CTP variants
The substrate selectivity coefficient is defined as Ki/Km. Ki values (mm) were determined as described under “Experimental Procedures” and are presented in Table 3. Standard errors were propagated from the standard errors in Km and Ki. For single-Cys CTP variant-inhibitor combinations where either the percent inhibition was less than 3% or the standard error of the measurement was greater than the calculated Ki value itself, then a minimal estimate for the selectivity coefficient was determined as follows: % inhibition + % S.E. is used to calculate a Ki, then Ki/Km is used to calculate a minimal selectivity coefficient. Thus, poorly inhibiting anions display different thresholds of selectivity based on inherent differences in the Km value of the variant. In these instances, the selectivity coefficient value presented represents a minimal estimate. Those cases where Ki + S.E. ≤ 0 (i.e. no observable inhibition) are denoted as ND as the selectivity coefficient value is not determinable. α-KG is α-ketoglutarate; PEP is phosphoenolpyruvate.
| CTP variant | Km valuesa | Selectivity coefficient values |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Isocitrate | α-KG | Malate | Succinate | Malonate | PEP | ADP | Phosphate | Pyruvate | Glutamate | ||
| Cys-less | 0.62 ± 0.04 | 10.3 ± 0.8 | 61.7 ± 10.2 | 81.0 ± 15.4 | 70.3 ± 8.4 | 61.7 ± 22.5 | 30.0 ± 2.8 | 75.3 ± 27.0 | 156.0 ± 44.7 | >370 | >756 |
| K83C | 28.2 ± 7.2 | 0.9 ± 0.3 | 0.6 ± 0.2 | 0.5 ± 0.2 | 0.7 ± 0.2 | 1.1 ± 0.4 | 0.6 ± 0.2 | 1.0 ± 0.3 | 1.0 ± 0.4 | 27.7 ± 16.3 | 2.6 ± 1.6 |
| R87C | 31.1 ± 4.7 | 0.5 ± 0.1 | 0.5 ± 0.2 | 0.5 ± 0.1 | 0.7 ± 0.2 | 0.9 ± 0.2 | 0.4 ± 0.1 | 1.0 ± 0.3 | 1.4 ± 0.5 | ND | 2.8 ± 1.3 |
| G119C | 25.4 ± 5.1 | 0.7 ± 0.2 | 1.3 ± 0.5 | 2.0 ± 0.8 | 3.2 ± 1.2 | 2.0 ± 0.7 | 0.9 ± 0.2 | 2.0 ± 0.5 | 2.3 ± 0.9 | 14.8 ± 17.8 | 4.0 ± 1.2 |
| R189C | 14.5 ± 1.7 | 0.6 ± 0.1 | 0.5 ± 0.1 | 0.5 ± 0.1 | 0.4 ± 0.1 | 0.8 ± 0.1 | 2.2 ± 0.3 | 3.9 ± 1.0 | 2.3 ± 0.5 | 1.5 ± 0.3 | 4.1 ± 1.2 |
| K37C | 2.3 ± 0.6 | 100.6 ± 40.6 | >750 | 118.7 ± 50.7 | >144 | 100.6 ± 83.8 | 100.6 ± 55.0 | 55.6 ± 37.6 | 76.6 ± 54.0 | >77 | >750 |
| R181C | 25.8 ± 5.2 | 5.3 ± 1.8 | 1.8 ± 0.4 | 1.5 ± 0.5 | 1.6 ± 0.6 | 1.7 ± 0.5 | 2.5 ± 1.0 | 7.5 ± 3.6 | 1.8 ± 0.6 | >6 | ND |
| K239C | 30.9 ± 10.9 | 4.8 ± 2.4 | >7 | 5.2 ± 3.1 | >38 | 6.3 ± 5.1 | 3.3 ± 1.7 | >5 | 3.8 ± 2.5 | 4.8 ± 3.1 | >78 |
| R276C | 16.4 ± 3.3 | 1.4 ± 0.4 | 2.0 ± 0.5 | 1.8 ± 0.4 | 1.8 ± 0.4 | 2.7 ± 0.7 | 0.6 ± 0.2 | 1.2 ± 0.3 | 2.7 ± 0.8 | 27.3 ± 27.3 | 2.2 ± 0.7 |
| R279C | 19.5 ± 5.4 | 1.4 ± 0.5 | 0.7 ± 0.3 | 1.1 ± 0.4 | 1.3 ± 0.4 | 0.9 ± 0.4 | 0.7 ± 0.3 | 1.6 ± 0.7 | 1.0 ± 0.4 | 16.2 ± 11.4 | 2.6 ± 0.9 |
a Km values (mm) were previously published (24) except for the G119C mutant data.