The Rodin-Ohno hypothesis that two enzyme superfamilies descended from one ancestral gene: an unlikely scenario for the origins of translation that will not be dismissed

Table 2 Criteria for the authenticity of Urzyme catalytic activities

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 k_cat/K_M.
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 K_M values.	Measure: k_cat, K_M, k_cat/K_M	Contamination by WT enzyme would saturate at WT K_M.
Amino acid specificity is different from full-length	Compare: (k_cat/K_M)_W/(k_cat/K_M)_Y	Urzymes are generally low specificity, high k_cat catalysts.

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