TABLE 1.
Kinetic parameters determined for conformational and proteolytic [Lys]Pg and mini-Pg activation
Values of KA(v1) and kcat/Km(v1) (for VLK-pNA hydrolysis by the SK·Pg* complexes) derived from analysis of v1, and kPg(v2) from analysis of v2 are listed for wtSK, native SK, SKΔK414, the indicated SK deletion mutant, and Ala substitution mutants. All of the mutants were made on the background of SKΔK414. The parameters determined for [Lys]Pg and mini-Pg activation are listed for reactions in the absence of 6-AHA (No 6-AHA) and presence of 10 mm 6-AHA (10 mm 6-AHA). Experimental error represents ±2 S.D. NM, not measurable. Kinetics experiments were performed, and the progress curves were analyzed as described under “Experimental Procedures.”
| No 6-AHA |
10 mm 6-AHA |
||||
|---|---|---|---|---|---|
| KA (v1) | kcat/Km (v1) | kPg (v2) | KA (v1) | kcat/Km (v1) | |
| nm | nm−1s−1 | nm−1s−1 | nm | nm−1s−1 | |
| [Lys]Pg | |||||
| Wild-type SK | 2 ± 2 | 12.6 ± 0.3 | 780 ± 20 | 39 ± 6 | 12.5 ± 0.5 |
| Native SK | 1.4 ± 0.8 | 12.8 ± 0.4 | 880 ± 60 | 40 ± 5 | 12.2 ± 0.3 |
| SKΔK414 | 22 ± 7 | 13 ± 1 | 920 ± 60 | 38 ± 6 | 12.2 ± 0.5 |
| SKΔK414Δ(R253-L260) | 22 ± 5 | 13.6 ± 0.4 | NM | 54 ± 13 | 11.8 ± 0.4 |
| SKΔK414(E249A) | 21 ± 8 | 12.2 ± 0.9 | 1030 ± 90 | 58 ± 15 | 11.5 ± 0.8 |
| SKΔK414(R253A) | 27 ± 7 | 12.7 ± 0.7 | 370 ± 30 | 27 ± 7 | 11.3 ± 0.6 |
| SKΔK414(K256A) | 29 ± 5 | 12.3 ± 0.5 | 180 ± 10 | 39 ± 4 | 12.0 ± 0.3 |
| SKΔK414(K257A) | 32 ± 7 | 12.2 ± 0.6 | 390 ± 20 | 53 ± 7 | 11.5 ± 0.4 |
| SKΔK414(K256A/K257A) | 28 ± 5 | 13.8 ± 0.5 | NM | 54 ± 6 | 12.9 ± 0.4 |
| SKΔK414(E262A) | 22 ± 8 | 17.9 ± 1.5 | 1570 ± 70 | 37 ± 7 | 16.8 ± 0.9 |
| SKΔK414(E262A/E263A) | 23 ± 11 | 13.7 ± 1.3 | 1370 ± 50 | 58 ± 6 | 12.9 ± 0.3 |
| Mini-Pg | |||||
| Wild-type SK | 40 ± 13 | 16 ± 1 | 1110 ± 80 | 38 ± 4 | 14.5 ± 0.4 |
| Native SK | 52 ± 21 | 16 ± 1 | 1220 ± 160 | 47 ± 3 | 13.8 ± 0.3 |
| SKΔK414 | 36 ± 12 | 15 ± 1 | 1070 ± 30 | 33 ± 2 | 14.6 ± 0.2 |
| SKΔK414Δ(R253-L260) | 42 ± 4 | 13.8 ± 0.4 | NM | 38 ± 7 | 12.4 ± 0.6 |
| SKΔK414(E249A) | 50 ± 19 | 13 ± 1 | 960 ± 40 | 55 ± 26 | 10 ± 1 |
| SKΔK414(R253A) | 29 ± 7 | 13.1 ± 0.7 | 280 ± 10 | 27 ± 3 | 14.1 ± 0.3 |
| SKΔK414(K256A) | 24 ± 5 | 15.3 ± 0.8 | 160 ± 10 | 32 ± 5 | 16.3 ± 0.7 |
| SKΔK414(K257A) | 45 ± 17 | 15 ± 1 | 260 ± 10 | 47 ± 10 | 14.9 ± 0.9 |
| SKΔK414(K256A/K257A) | 39 ± 9 | 16 ± 1 | NM | 36 ± 5 | 15.2 ± 0.6 |
| SKΔK414(E262A) | 27 ± 9 | 15 ± 2 | 1570 ± 50 | 37 ± 5 | 14.7 ± 0.7 |
| SKΔK414(E262A/E263A) | 30 ± 10 | 13 ± 1 | 1030 ± 40 | 32 ± 4 | 14.6 ± 0.4 |