Table 1.
Steady-State Kinetic Parameters for the Cleavage of Poly(C) by Wild-Type, Mutant, and Semisynthetic Ribonucleases
| residue 41 | side chain | kcat (s–1) | Km (mM) | kcat/Km (M–1 s–1) | ΔΔG‡a | pKa of side chain NHb |
|---|---|---|---|---|---|---|
| cysteine | ~CH2–SH | 0.026 ± 0.004 | 0.36 ± 0.12 | 73 ± 15 | 6.8 | — |
| lysine (wild-type) | ~CH2CH2CH2CH2NH3+ | 604 ± 47 | 0.091 ± 0.022 | (6.5 ± 1.2) × 106 | 0.0 | 10.6 |
| S-(aminoethyl)cysteine | ~CH2–S–CH2CH2NH3+ | 43 ± 3 | 0.075 ± 0.016 | (5.2 ± 1.0) × 105 | 1.5 | 10.6 |
| S-acetamidinocysteine | ~CH2–S–CH2C(NH2)NH2+ | 11.0 ± 0.4 | 0.041 ± 0.006 | (2.6 ± 0.3) × 105 | 1.9 | 12.5 |
| S-(carbamoylmethyl)cysteine | ~CH2–S–CH2C(O)NH2 | 0.074 ± 0.007 | 0.25 ± 0.05 | 301 ± 28 | 5.9 | 15.2 |
| S-((trimethylamino)ethyl)cysteine | ~CH2–S–CH2CH2N(CH3)3+ | nd | nd | <230c | >6.1c | — |
| S-(aminopropyl)cysteine | ~CH2–S–CH2CH2CH2NH3+ | 12.9 ± 0.4 | 0.12 ± 0.01 | (1.1 ± 0.1) × 105 | 2.4 | 10.6 |
| arginine | ~CH2CH2CH2NHC(NH2)NH2+ | 4.4 ± 0.3 | 0.091 ± 0.016 | (4.8 ± 0.6) × 104 | 2.9 | 13.7 |
a
ΔΔG‡ = RTln[(kcat/Km)wild-type/(kcat/Km)].
b
Based on aqueous solutions of the model compounds: lysine, S-(aminoethyl)cysteine, S-(aminopropyl)cysteine, CH3CH2CH2CH2NH3+ (Hall, H. K., Jr. J. Am. Chem. Soc. 1957, 79, 5441-5444); S-acetamidinocysteine, CH3C(NH2)NH2+ and arginine, H2NC(NH2)NH2+ (Albert, A. R.; Goldacre, R.; Phillips, J. J. Chem. Soc. 1948, 3, 2240-2249); S-(carbamoylmethyl)cysteine, CH3C(O)NH2 (Bordwell, F. G. Acc. Chem. Res. 1988, 21, 456-463).
c
Assuming Km ≥ 0.1 mM.