Table 2.

Binding affinities and kinetic constants of full-length PRORP1 to 5′-fluorescein-pre- tRNA^Asp

PRORP1 Variant	k obs (min−1)a	k obs relative to WT	K D (nM)b	K D relative to WT	ΔΔG°binding (kcal/mol)
Δ76 WT	2.62 ± 0.28	1	390 ± 30	1	0
Y133D	1.17 ± 0.22	0.45	11100 ± 1600	28.4	1.9
Y133Q	2.76 ± 0.34	1.05	3200 ± 600	8.2	1.2
R210A	1.59 ± 0.20	0.61	4200 ± 400	10.8	1.4
R210S	0.49 ± 0.09	0.19	10600 ± 1000	27.2	1.9
R212K	n.d.c	n.d.	≥ 50000d	≥ 130	≥ 2.9

^aMeasured using single turnover cleavage assays in 30 mM MOPS, pH 7.8, 330 mM NaCl, 1 mM TCEP and 20 mM CaCl₂ at 25 ± 1°C. The k_obs and standard error values are determined from nonlinear least squares regression of a single exponential equation to the data points from a single experimental trial using GraphPad Prism.

^bMeasured using fluorescence anisotropy in 30 mM MOPS, pH 7.8, 330 mM NaCl, 1 mM TCEP and 20 mM CaCl₂ at 25 ± 1°C. The K_D and standard error values are determined from non-linear least squares regression of a single binding isotherm to the data points from a single experimental trial using GraphPad Prism.

^cBased on the binding affinity data, we were unable to measure single turnover kinetics for the R212K mutant under saturating enzyme concentration.

^dThe lower limit for the K_D for R212K was estimated from the observation of <10% change in anisotropy at 20 μM R212K mutant, assuming that the total change in anisotropy upon binding Fl-pre-tRNA was similar to the that of the other PRORP1 variants.