TABLE 1.
Comparison of the enzyme kinetic constants for wild-type and mutant TL-3-resistant HIV proteases
| HIV proteasea | Fluorogenic substrateb
|
MA-CA peptidec (% cleavage ± SD) | ||
|---|---|---|---|---|
| Mean Km ± SD (μM) | kcat (s−1) | kcat/Km (s−1 μM−1)d | ||
| Wild type | 45.4 ± 3.9 | 2.73 | 0.060 | 31.1 ± 3.3 |
| V82A (1x) | 73.5 ± 9.1 | 1.74 | 0.024 (40) | 18.2 ± 1.3 |
| M46I/F53L/V82A (3x) | 194.2 ± 19.5 | 5.45 | 0.028 (47) | 26.4 ± 3.8 |
| L24I/M46I/F53L/L63P/V77I/V82A (6x) | 187.2 ± 31.9 | 6.86 | 0.037 (60) | 32.2 ± 2.7 |
Indicated are the viral isolates from which the cloned protease was obtained. The term within the parentheses reflects the number of mutations present.
The kinetics of the proteases were assayed with the fluorogenic substrate Abz-T-I-Nle∗ (p-NO2)F-Q-R. The protease assay was performed in 0.1 M morpholine ethanesulfonate buffer (pH 5.6), 0.2 M NaCl, 1 mM dithiothreitol, and 5% glycerol.
For the HIV-1 MA-CA junction peptide SSQVSQNY*PIVQNLQG, the percent cleavage was assayed by HPLC and is expressed as the percent cleaved in the 30-min assay. The protease and substrate concentrations were 2 and 100 mM, respectively.
The values within the parentheses indicate the catalytic efficiency as a percentage of wild-type virus (R8).