Transition states of native and drug-resistant HIV-1 protease are the same

HIV-1 protease is an important target for the treatment of HIV/AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary ¹⁴C and ¹⁵N KIEs and secondary ³H and ¹⁸O KIEs for native and multidrug-resistant HIV-1 protease (I84V). We observed ¹⁴C KIEs ( ¹⁴V/K) of 1.029 ± 0.003 and 1.025 ± 0.005, ¹⁵N KIEs ( ¹⁵V/K) of 0.987 ± 0.004 and 0.989 ± 0.003, ¹⁸O KIEs ( ¹⁸V/K) of 0.999 ± 0.003 and 0.993 ± 0.003, and ³H KIEs ( ³V/K) KIEs of 0.968 ± 0.001 and 0.976 ± 0.001 for the native and I84V enzyme, respectively. The chemical reaction involves nucleophilic water attack at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32 Å) with partial hydrogen bond formation to the accepting nitrogen (1.20 Å) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52 Å). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.