TABLE 2.
Kinetic constants for CCT dimers
CCT activity was assayed in the presence of 0.2 mm PG/PC (1:1) SUVs as described under “Experimental Procedures.” Plots of activity versus [phosphocholine] or versus [CTP] were fit to the Michaelis-Menten equation (V = Vmax[S]/(Km + [S]), using GraphPad Prism 4 software, to generate the kinetic parameters kcat and Km. The r2 values for the fit of the data to this equation were ≥0.96 for each trial. Km and kcat are derived from the best fit to this equation ±S.E. of the fit. n is the number of independent determinations. The S.E. values associated with the kcat values were obtained by error propagation of the S.E. of the kcat values for individual substrates (CTP and phosphocholine (PCho)).
|
CCT dimer
|
kcat (n)
|
Km (n)
|
|||
|---|---|---|---|---|---|
| PCho | CTP | ||||
| min-1 | mm | ||||
| 1 | 367/His236(K122A) | 454 ± 11a (4) | 0.48 ± 0.07 (2) | 1.82 ± 0.16 (2) | |
| 2 | 367/His367(K122A) | 474 ± 10a (5) | 0.28 ± 0.03 (3) | 0.61 ± 0.08 (2) | |
| 3 | 367/367 (denatured/renatured) | 861 ± 32 (4) | 0.41 ± 0.10 (2) | 0.72 ± 0.14 (2) | |
| 4 | 367/367 (untreated) | 885 ± 35 (2) | 0.46 ± 0.12 (2) | 0.64 ± 0.10 (2) | |
For data sets with n > 2, t test analyses showed that the probability of being the same as the CCT367/367 dimer on row 3 is <0.005.