Biochemistry
Biochemistry
5 years ago

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

Catalytic Mechanism of Cruzain from Trypanosoma cruzi As Determined from Solvent Kinetic Isotope Effects of Steady-State and Pre-Steady-State Kinetics
Thomas D. Meek, Xiang Zhai
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 performed pH-rate profiles for substrates and affinity agents, and have determined 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 groups of pK1 = 6.6 (unprotonated) and pK2 ~ 9.6 (protonated) required for catalysis. The temperature dependence of the group of pK = 6.2 – 6.6 exhibited a values of Hion = 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 k¬cat is attributable 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 are neutral in charge in the free enzyme. The generation of the active thiolate of the catalytic cysteine is likely proceeded (and possibly triggered) by a ligand-induced conformational change, which could bring the catalytic dyad to close proximity in order to effect proton transfer.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b01250

DOI: 10.1021/acs.biochem.7b01250

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