Table 2.
Kinetic parameters for conformational and proteolytic activation of [Lys]Pg by SK constructsa
| SK construct | No 6-AHA
|
10 mM 6-AHA
|
||||
|---|---|---|---|---|---|---|
| KA (v1) (nM) | kcat/Km (v1) (nM−1s−1) | KA (v2) (nM) | kPg (v2) (nM−1s−1) | KA (v1) (nM) | kcat/Km (v1) (nM−1s−1) | |
| Native SK | 2 ± 1b | 13.2 ± 0.4 | 1.6 ± 0.2 | 1020 ± 40 | 40 ± 5 | 12.6 ± 0.3 |
| wild-type SK | 4 ± 1 | 13.4 ± 0.5 | 1.6 ± 0.2 | 1010 ± 40 | 33 ± 4 | 12.5 ± 0.3 |
| SKΔ(R253-L260) | 10 ± 2 | 15.5 ± 0.5 | NAc | NA | 78 ± 13 | 13.3 ± 0.7 |
| SK-His6 | 10 ± 4 | 12.5 ± 0.7 | 4 ± 1 | 900 ± 40 | 32 ± 3 | 11.8 ± 0.3 |
| SKΔ(R253-L260)ΔK414-His6 | 28 ± 5 | 12.0 ± 0.5 | NA | NA | 70 ± 7 | 11.8 ± 0.7 |
a
The apparent dissociation constant for conformational activation (KA (v1)) of 10 nM [Lys]Pg by the indicated SK constructs, and the bimolecular rate constant for VLK-pNA hydrolysis by the SK·[Lys]Pg* complex (kcat/Km (v1)) in the absence (No 6-AHA) and presence of 6-AHA (10 mM 6-AHA). Also listed are KA obtained from v2 (KA (v2)) and the bimolecular rate constant for Pm generation from [Lys]Pg (kPg). Parameters were determined by analysis of the data shown in Fig. 6 as described in Materials and methods.
b
Experimental error in the parameters represents the 95% confidence interval.
c
NA represents not analyzable