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

Zhai, Xiang, Meek, Thomas D.
Biochemistry 2018 v.57 no.22 pp. 3176-3190
Chagas disease, Trypanosoma cruzi, catalytic activity, cruzipain, cysteine, drugs, histidine, isotopes, solvents, temperature
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 for Cbz-Phe-Arg-AMC and Cbz-Arg-Arg-AMC, but not for Cbz-Arg-Ala-AMC. The overall solvent isotope effect on kcₐₜ can be attributed to the solvent isotope effect on the deacylation step. Our results suggest that cruzain is unique among papain-like cysteine proteases in that the catalytic cysteine and histidine have neutral charges in the free enzyme. The generation of the active thiolate of the catalytic cysteine is likely preceded (and possibly triggered) by a ligand-induced conformational change, which could bring the catalytic dyad into the proximity to effect proton transfer.