Catalytic Mechanism of Cruzain from Trypanosoma cruzi As Determined from Solvent Kinetic Isotope Effects of Steady-State and Pre-Steady-State Kinetics

Cruzain, an important drug target for Chagas disease, is a member of clan CA of the cysteine proteases. Understanding the catalytic mechanism of cruzain is vital to the design of new inhibitors. To this end, we have determined pH–rate profiles for substrates and affinity agents and solvent kinetic isotope effects in pre-steady-state and steady-state modes using three substrates: Cbz-Phe-Arg-AMC, Cbz-Arg-Arg-AMC, and Cbz-Arg-Ala-AMC. The pH–rate profile of kcat/Km for Cbz-Arg-Arg-AMC indicated pK1 = 6.6 (unprotonated) and pK2 ∼ 9.6 (protonated) groups were required for catalysis. The temperature dependence of the pK = 6.2–6.6 group exhibited a ΔHion value of 8.4 kcal/mol, typical of histidine. The pH–rate profile of inactivation by iodoacetamide confirmed that the catalytic cysteine possesses a pKa of 9.8. Normal solvent kinetic isotope effects were observed for both D2Okcat (1.6–2.1) and D2Okcat/Km (1.1–1.4) for all three substrates. Pre-steady-state kinetics revealed exponential bursts of AMC production ...