Table 1.

Steady-State Kinetic Parameters for Cleavage of Ribonucleotides by Wild-Type and Mutant Ribonucleases^a

RNase A	substrateb	kcat (s–1)	Km (mM)	kcat/Km (M–1 s–1)	$\frac{{(k_{\mathrm{cat}}∕K_{\mathrm{m}})}^{\mathrm{mutant}}}{{(k_{\mathrm{cat}}∕K_{\mathrm{m}})}^{\text{wild-type}}}$
wild-type	poly(C)	(4.1 ± 0.1) × 102 c	0.034 ± 0.002c	(1.5 ± 0.1) × 107 c	1.0
H12A	poly(C)	0.073 ± 0.006	0.105 ± 0.025	(7.3 ± 0.2) × 102	(4.9 ± 0.9) × 10–5
H119A	poly(C)	0.24 ± 0.03	0.21 ± 0.03	(1.1 ± 0.1) × 103	(7.3 ± 0.8) × 10–5
wild-type	UpA	(1.40 ± 0.15) × 103 c	0.62 ± 0.09c	(2.3 ± 0.4) × 106 c	1.0
H12A	UpA	0.15 ± 0.02	0.86 ± 0.17	(1.7 ± 0.1) × 102	(7.4 ± 1.4) × 10–5
H119A	UpA	0.76 ± 0.10	0.80 ± 0.15	(9.5 ± 0.5) × 102	(4.1 ± 0.7) × 10–4
wild-type	UpOC6H4-p-NO2	18.8 ± 0.6	0.33 ± 0.05	(5.7 ± 0.6) × 104	1.0
H12A	UpOC6H4-p-NO2	0.0029 ± 0.0001	0.275 ± 0.041	(1.1 ± 0.1) × 101	(1.9 ± 0.3) × 10–4
H119A	UpOC6H4-p-NO2	27 ± 1	0.76 ± 0.08	(3.6 ± 0.1) × 104	0.63 ± 0.11

^aAll reactions were performed at 25 °C in 50 mM MES buffer, pH 6.0, containing 0.1 M NaCl. Steady-state kinetic parameters were determined by fitting the initial velocity data to a hyperbolic curve using the program HYPERO.²²

^bCleavage of poly(C) and UpOC₆H₄-p-NO₂ were monitored at 250 nm (Δε₂₅₀ = 2380 M^–1 cm^–1) and 330 nm (Δε₃₃₀ = 6200 M^–1 cm^–1), respectively; cleavage of UpA was monitored at 265 nm in the presence of excess adenosine deaminase²³ (Δε₂₆₅ = –6000 M^–1 cm^–1).

^cData from ref 8.