Table 1.

Single-turnover kinetic parameters measuring pre-tRNA cleavage by wild-type and mutant T. maritima RNase P

Holoenzyme (protein/RNA)	Amino acid conservation (%)a	kcat / KM (106 M−1 min−1)b	n	Relative reactivityc
WT / WT		10.8 ± 1.70	6	1
R14A / WT	9.7 (13.5)	3.90 ± 0.06	1	0.4
R15A / WT	16.6 (34.6)	3.25 ± 0.15	2	0.3
F17A / WT	84.3 (97.7)	0.50 ± 0.25	2	0.05
F21A / WT	58.1 (88.6)	1.7 ± 0.10	2	0.2
K51A / WT	2.0 (2.65)	4.2 ± 1.3	3	0.4
R52A / WT	9.0 (98.2)	4.0 ± 1.0	2	0.4
K53A / WT	93.9 (99.0)	2.4 ± 0.8	2	0.2
K56A / WT	26.4 (48.3)	10.9 ± 1.9	2	1.1
R59A / WT	3.3 (7.7)	11.9 ± 1.3	2	1.2
R60A / WT	99.8 (100)	7.2 ± 2.6	2	0.7
K62A / WT	10.2 (79.2)	10.2 ± 1.2	2	1
R65A / WT	100	15.7 ± 3.6	2	1.5
R89A / WT	70.8 (95.3)	0.60 ± 0.30	3	0.06
K90A / WT	43.5 (48.8)	2.65 ± 1.50	2	0.3
WT / U52C		0.8 ± 0.4	2	0.08
R89A / U52C		0.050 ± 0.011	3	0.005
F17 / U52C		0.006 ± 0.003	2	0.0006
- / WT (hs)d		0.50 ± 0.03	1	0.05
- / WT (salt)e		0.030 ± 0.028	1	0.003

^aFrequency of occurrence of amino acid in a large group of bacterial RNase P proteins (n = 491). In parenthesis is shown the frequency of occurrence of R + K for either R or K, and F + Y for F. This reflects better the overall conservation of this amino acid.

^bk_cat/K_M were determined from the observed reaction rates (k_obs) at various concentrations and assuming Michaelis–Menten kinetics. The enzyme activity was obtained from the average of n complete time course reaction assays performed. For n > 1, the estimated error represents the propagated standard error from all time-dependent kinetic measurements. For n = 1, the estimated error was propagated from individual time-dependent kinetic measurements as determined by Kaleidagraph 4 (Synergy Software).

^cApproximate relative enzyme activity using the ratio: k_cat/K_M^{(modified RNase P)}/k_cat/K_M^{(wild-type RNase P).}

^dRibozyme (P RNA alone) under high salt (hs) conditions (100 mM MgCl₂, 1.0 M NH₄OAc).

^eRibozyme (P RNA alone) under elevated salt conditions (50 mM MgCl₂, 0.6 M NH₄OAc).