TABLE 1.

Kinetic parameters (V_max(app), K_m(app), and V_max(app)/K_m(app)) in the RNase P cleavage of ptRNA^Ser or pr39 in the presence of different EGSs

Multiple turnover kinetic analyses to determine the values of V_max(app) and K_m(app) were carried out in buffer A (50 mm Tris, pH 7.4, 100 mm NH₄Cl, and 10 mm MgCl₂) at 37 °C, as described previously (18, 23, 45). To determine the binding affinity (K_d) between substrate pr39 and EGSs, binding assays were carried out in the absence of human RNase P in buffer B (50 mm Tris, pH 7.5, 100 mm NH₄Cl, 10 mm MgCl₂, 3% glycerol, 0.1% xylene cyanol, 0.1% bromophenol blue), using a protocol modified from Pyle et al. (24). The values shown are the average derived from triplicate experiments.

Substrate	Km	Vmax(app)	Vmax(app)/Km(app)	Kd
	μm	pmol min−1	pmol μm−1min−1	μm
ptRNASer	0.015 ± 0.003	0.045 ± 0.015	3.0 ± 0.6
PR mRNA(pr39)
+PR-SER	0.55 ± 0.10	0.030 ± 0.010	0.055 ± 0.020	1.3 ± 0.3
+PR-SER-C	NDa	ND	<0.001	1.2 ± 0.3
+PR-C125	0.40 ± 0.09	0.80 ± 0.20	2.0 ± 0.4	0.030 ± 0.005
+PR-C125-C	ND	ND	<0.001	0.032 ± 0.006

^a ND, not determined.