Human immunodeficiency virus-1 protease. 2. Use of pH rate studies and solvent kinetic isotope effects to elucidate details of chemical mechanism.
The pH dependence of the peptidolytic reaction of recombinant human immunodeficiency virus type 1 protease has been examined over a pH range of 3-7 for four oligopeptide substrates and two competitive inhibitors. The pK values obtained from the pKis vs pH profiles for the unprotonated and protonated active-site aspartyl groups, Asp-25 and Asp-25', in the monoprotonated enzyme form were 3.1 and 5.2, respectively. Profiles of log V/K vs pH for all four substrates were "bell-shaped" in which the pK values for the unprotonated and protonated aspartyl residues were 3.4-3.7 and 5.5-6.5, respectively. Profiles of log V vs pH for these substrates were "wave-shaped" in which V was shifted to a constant lower value upon protonation of a residue of pK = 4.2-5.2. These results indicate that substrates bind only to a form of HIV-1 protease in which one of the two catalytic aspartyl residues is protonated. Solvent kinetic isotope effects were measured over a pH (D) range of 3-7 for two oligopeptide substrates, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 and Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2. The pH-independent value for DV/K was 1.0 for both substrates, and DV = 1.5-1.7 and 2.2-3.2 at low and high pH (D), respectively. The attentuation of both V and DV at low pH (D) is consistent with a change in rate-limiting step from a chemical one at high pH (D) to one in which a product release step or an enzyme isomerization step becomes partly rate-limiting at low pH (D). Proton inventory data is in accord with the concerted transfer of two protons in the transition state of a rate-limiting chemical step in which the enzyme-bound amide hydrate adduct collapses to form the carboxylic acid and amine products.
Hyland
L J
LJ
Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Tomaszek
T A
TA
Meek
T D
TD
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D016333:HIV Protease / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010103:Oxygen Isotopes; D012997:Solvents; D013379:Substrate Specificity
D009842:Oligopeptides; D010103:Oxygen Isotopes; D012997:Solvents; D016333:HIV Protease