Table 1.
Amide exchange rates kOH− (M−1·s−1) for P. furiosus rubredoxin at 28°C
| Position | Rate | Position | Rate |
|---|---|---|---|
| Ala-1 | 2.2 × 108 | Glu-30 | 6.4 × 105 |
| Lys-2 | 2.9 × 106 | Glu-31 | 850 |
| Trp-3 | 90 | Leu-32 | 350 |
| Val-4 | 110 | Asp-34 | 2.0 × 108 |
| Cys-5 | 80 | Asp-35 | 2.6 × 107 |
| Lys-6 | 470 | Trp-36 | 2,300 |
| Ile-7 | 40 | Val-37 | 120 |
| Cys-8 | 20 | Cys-38 | 40 |
| Gly-9 | 850 | Ile-40 | ∼20 |
| Tyr-10 | 40 | Cys-41 | ∼20 |
| Ile-11 | 370 | Gly-42 | 220 |
| Tyr-12 | 90 | Ala-43 | 90 |
| Asp-13 | 5,900 | Lys-45 | 2.5 × 104 |
| Glu-14 | 1.5 × 105 | Ser-46 | 1.3 × 107 |
| Asp-15 | 7,200 | Glu-47 | 1,100 |
| Ala-16 | 6,000 | Phe-48 | 210 |
| Gly-17 | 8,200 | Glu-49 | 230 |
| Asp-18 | 8,000 | Lys-50 | 2.1 × 105 |
| Asp-20 | 4.3 × 107 | Leu-51 | 1.6 × 104 |
| Asn-21 | 3.1 × 104 | Glu-52 | 1.6 × 107 |
| Gly-22 | 7.8 × 106 | Asp-53 | 3.8 × 106 |
| Ile-23 | 4,600 | ||
| Ser-24 | 5.0 × 107 | Side chain | |
| Gly-26 | 1.6 × 104 | Trp-3 ring | 3.5 × 104 |
| Thr-27 | 5400 | Trp-36 ring | 2,800 |
| Lys-28 | 4.1 × 104 | Asn-21 Nδ1 | 1.0 × 105 |
| Phe-29 | 8,400 | Asn-21 Nδ2 | 2.6 × 105 |
The amide 1HN exchange values for Lys-28, Phe-29, and Trp-36 as well as for indole 1HN of Trp-3 and Trp-36 were fitted for kex data below pH 11. All positions having kOH− values between 150 s−1 and 5 × 105 s−1 predict an optimal slope for log kex vs. pH between 0.85 and 1.2. The pH (7.17, 10.38, 10.95, 11.49, 12.02, and 12.51) and temperature (3°C, 28°C, and 53°C) data observed for each amide then were fitted to a slope of log kex vs. pH set to 1.0 and an activation energy of 17 kcal/mol. The pH and the temperature-dependence analyses were satisfactorily fitted with a 25% standard error in the observed rates. The rates for all other amides (kOH− > 5 × 105 s−1 or kOH− < 150 s−1) were estimated assuming EX2 kinetics and a 17-kcal/mol activation energy.