Criterion | Implementation | Remarks |
---|---|---|
Empty vector controls | Purify, assay MBP | De Rigueur, but unconvincing |
Renaturation from inclusion bodies | Tagged Urzymes purified from pellet | WT Enzymes do not segregate with inclusion bodies |
MBP fusions release cryptic activity on TEV cleavage. | Assay fusion proteins ± TEV cleavage | Inhibition in fusion proteins is widespread, not universal. |
Active-site titrations Urzyme preparations have significant bursts. | Single turnover time-courses | A key criterion, this is also essential for comparing kcat/KM. |
Mutations, modular alterations induce predictable changes in activity. | Determine effect of active-site mutations, genetic manipulations | Active-site mutations generally affect Urzyme activities differently and can actually enhance activity because mechanisms are different. |
Urzymes, WT enzymes have different Steady-state KM values. | Measure: kcat, KM, kcat/KM | Contamination by WT enzyme would saturate at WT KM. |
Amino acid specificity is different from full-length | Compare: (kcat/KM)W/(kcat/KM)Y | Urzymes are generally low specificity, high kcat catalysts. |