Metal content and steady state kinetics of recombinant yDbr1. (A) Molar equivalents of metals ions for recombinant and apo yDbr1 measured with ICP-MS. Zn and Fe were present at 2.7 and 0.7 molar equivalents, while Ni, Cu, Mn, and Co are <0.02 equivalents. Apo-yDbr1 has <0.02 equivalents of all metals. (B) Schematic of the fluorescent AK88 bRNA substrate. When intact, the “black-hole” quencher DABCYL prevents the fluorescein (FAM) from emitting light at 488 nm. Following Dbr1 cleavage, the quencher is no longer proximal to the FAM which becomes highly fluorescent. (C) Steady-state kinetic analysis of recombinant yDbr1. The AK88 bRNA was prepared at concentrations from 0.08–5 µM and initial rates of product formation were measured. The resulting data were fit with the Michaelis–Menten equation to obtain estimates of kcat = 5.6 sec−1, Km = 0.7 µM. No metal ions were added to any component of these reactions; the only metal ions present are those that copurified with the enzyme or those present as trace metal contaminants.
