TABLE 2.
Enzyme | Km (mM) | kcat (s−1) | kcat/Km (mM−1 s−1) | Relative efficiency |
---|---|---|---|---|
Wild type | 115 | 12.2 | 106 | 5.3 |
Mutant | 275 | 5.6 | 20 | 1.0 |
For details, see reference 10. The substrate His-Lys-Ala-Arg-Val-Leu/p(NO2) Phe-Glu-Ala-Nle-Ser-NH2 is referred to as substrate 1 in reference 10. The assay conditions are 1 mM dithiothreitol–1 mM EDTA in 50 mM citrate (pH 4.5). Vials containing this buffer and various amounts of the substrate were preincubated at 37°C for 10 min before initiating reactions by the addition of wild-type or mutant HIV-1 protease. After sufficient time to allow about 15% hydrolysis of the substrate (15 to 30 min), trifluoroacetic acid was added to a final concentration of 5%. Products were analyzed by high-pressure liquid chromatography, and the data were fitted to the Michaelis-Menten equation to evaluate kcat and Km.