TABLE 2.

Kinetic analysis of the HIV-1 protease mutant (M46I, G48V, I50V, I84L)^a

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

^aFor 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 k_cat and K_m.