Table 1. Dependence of Activity of Human HMGCL Single Cys→Ser Mutants on Exogenous Thiol.
| Activity Recovered Following Reduction of Dialyzed Enzyme (U/mg) | Activity Following Dialysis (U/mg) | Stimulation of Activity by Exogenous Thiol | Intersubunit Dimer Formationa | |
|---|---|---|---|---|
| Enzyme | Reducing Assay Conditions | Non-Reducing Assay Conditions | Non-Reducing Assay Conditions | |
| WT HMGCL | 123 (± 7) | 11.5 (± 1.4) | 10.7 fold | Moderate |
| C141S HL | 42.0 (± 3.7) | 8.83 (± 0.23) | 4.76 fold | Strong |
| C170S HL | 53.1 (± 8.3) | 0.084 (± 0.012) | 636 fold | None |
| C174S HL | 42.1 (± 5.9) | 2.66 (± 0.30) | 15.8 fold | Moderate |
| C197S HL | 21.2 (± 0.6) | 0.141 (± 0.012) | 150 fold | Weak |
| C234S HL | 128 (± 6) | 0.742 (± 0.062) | 172 fold | Weak |
| C266S HL | 0.224 (± 0.010) | 0.137 (± 0.005) | 1.63 fold | None |
| C307S HL | 132 (± 5) | 13.8 (± 0.1) | 9.56 fold | Moderate |
| C323S HL | 132 (± 2) | 13.8 (± 1.6) | 9.57 fold | None |
a
Strong indicates a SDS PAGE dimer band (Fig. 4B) which is >65% of the total intensity in that lane; moderate, 34-65%; weak, <34%.