Table 1. Attributes of Untreated and Esterified RNase 1 Variants.
| RNase 1 | Za | Carboxyl groups |
1 (equiv)b | Estersc |
kcat/KM (107 M–1s–1)d |
IC50 (μM)e | |
|---|---|---|---|---|---|---|---|
| HeLa Cells | H460 Cells | ||||||
| Wild-type | +5 | 13 | 0 | — | 2.5 ± 0.1 | >100 | >100 |
| Wild-type | +5 | 13 | 100 | 4 | 1.1 ± 0.5 | 10 ± 1 | 7 ± 1 |
| Wild-type | +5 | 13 | 200 | 6 | 1.6 ± 0.1 | NDf | 5.1 ± 0.5 |
| DDADD | 0 | 17 | 0 | — | 0.022 ± 0.002 | >100 | >100 |
| DDADD | 0 | 17 | 100 | 7 | 0.023 ± 0.003 | 6 ± 1 | 8.4 ± 0.5 |
| DDADD | 0 | 17 | 200 | 11 | 0.025 ± 0.004 | ND | 1.0 ± 0.2 |
| H12A/K41A/H119A | +4 | 13 | 0 | — | <1 × 10–5 | >100 | >100 |
| H12A/K41A/H119A | +4 | 13 | 100 | 4 | <1 × 10–5 | >100 | >100 |
| FLAG-labeled | +2 | 18 | 0 | — | 0.72 ± 0.07 | ND | ND |
| FLAG-labeled | +2 | 18 | 100 | 5 | 0.35 ± 0.04 | ND | ND |
a
Ref. 28.
b
Reaction conditions: 200 μL of enzyme (0.14 μmol) in 10 mM MES–HCl buffer (pH 5.5) plus 200 μL of diazo compound 1 (14 or 28 μmol) in acetonitrile; 37 °C for 4 h.
c
Values are the most prevalent species apparent in the mass spectra of Figures S3–S6.
d
Values are the mean ± SD for the cleavage of 6-FAM–dArU(dA)2–6-TAMRA in 100 mM Tris–HCl buffer (pH 7.5), containing NaCl (100 mM). Individual values are shown in Figure S7.
f
ND, not determined.