Table 2.
Dimer to monomer dissociation constants for various SARS-CoV 3CLpro constructs and mutants determined by different methods.
| 13CLpro Construct | 2 WT or mutant | 3 Relative Activity | D↔M Dissociation Constant (μM) | 4 Method | Reference |
|---|---|---|---|---|---|
| Nat | WT | 100% | 0.25 –1.0 | enzymatic | This work |
| N-H6-U | WT | <5% | > 10.0 | enzymatic | |
| N-H6-X | WT | 0.015 | enzymatic& AGE | (Kuo et al., 2004) | |
| N-H6-X | WT | 0.00035 | AUC-SE | (Hsu et al., 2005) | |
| N-H6-X | 10aa-C145A | 0.0172 | |||
| C-H6-X | C145A-10aa | 0.0056 | |||
| C-H6-U | WT | 0.81 | enzymatic | (Chen et al., 2006) | |
| C-H6-U | WT | 100 | AGE | (Fan et al., 2004) | |
| C-H6-U | WT | 100% | 0.28 | AUC-SV | (Hsu et al., 2005) |
| Δ(1–3) | 76% | 3.4 | |||
| Δ(1–4) | 1.3% | 57.5 | |||
| C-H6-U | WT | 14.0 | AUC-SE | (Wei et al., 2006) | |
| R4E | |||||
| C-H6-U | WT | 0.19 | AUC-SV | (Chou et al., 2004) | |
| N-H6-U | WT | 100% | 227 | ITC | (Chen et al., 2005) |
| Δ(1–7) | <1% | 262 | |||
| N-GSTX | 5 WT(G278D) | 100% | <100 | 6 DLS | (Shi and Song, 2006) |
| Δ(1–5) | <1% | >100 |
Different constructs used in studies were: (Nat) Native construct with n-terminal methionine; (N-H6U) n-terminal (His)6-tag uncleaved; (N-H6X) n-terminal (His)6-tag cleaved; (C-H6U) c-terminal (His)6-tag uncleaved; (C-H6X) c-terminal (His)6-tag cleaved; (N-GSTX) n-terminal GST-tag cleaved
The wild type enzyme is defined as SARS 3CLpro purified from the particular construct. Delta (Δ) refers to the amino acid number(s) that were removed from the construct.
If a relative activity number is reported, it is based on the comparison to the wild type enzyme from the same construct.
The analytic method used to determine the dimer dissociation constant: (AUC) Analytical Ultracentrifugation; (SV) Sedimentation Velocity; (SE) Sedimentation Equilibrium; (ITC) Isothermal Titration Calorimetry; (AGE) Analytical Gel Exclusion chromatography; (DLS) Dynamic Light Scattering
WT(G278D) In these studies, the authors had a spurious mutation of G278D which they allowed to carry through all experiments so wild type is actually the G278D mutant (Shi and Song, 2006; Shi et al., 2004). The other indicated mutants are based on G278D.
DLS experiments were performed at 100 μM enzyme. The dissociation constants are based on either all dimer (Kd <100 μM) or all monomer (>100 μM).