Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH

Abstract

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite±host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters K m , V max , V max /K m , and K i for the cruzipain-catalysed hydrolysis of N-a-benzyloxycarbonyl-l-phenylalanyl-larginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 8C and 40.0 8C and between pH 4.5 and 8.5. Values of K m were independent of temperature and pH, whereas values of . As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.Keywords: cruzipain; effect of pH; effect of temperature; papain-like cysteine proteinase; total substrate inhibition.The haemoflagellate protozoan parasite Trypanosoma cruzi, the causative agent of the Chagas' disease, afflicts more than 20 million people in Central and South America. A much larger population is considered at risk worldwide [1±3].T. cruzi has a complex life cycle, involving a triatomine bug as haematophagous vector and a mammalian host. The major forms of T. cruzi present in the haematophagous insect vector are a replicative stage, the epimastigote, and a nonreplicative form, the infective metacyclic trypomastigote. In the mammalian host, T. cruzi has an obligate intracellular replicative stage, the amastigote, and a nonreplicative form, the bloodstream trypomastigote [1±3]. The T. cruzi life cycle is affected by changes in temperature and pH, both in the insect vector and in the mammalian host. In the triatomine bug, the optimal temperature range for parasite replication is 23 8C to 27 8C, being higher in mammalian hosts (< 37 8C). Note that the replication rate of T. cruzi increases with temperature. Moreover, T. cruzi replication is facilitated at the pH of the mammalian host cytoplasm (i.e. pH 7.4) [4±8].Cysteine proteinases are relevant to several aspects of the parasite life cycle and of the parasite±host relationship. In particular, cruzipain, the major papain-like cysteine proteinase from T. cruzi, is expressed in all stages of the parasite life cycle, being more abundant in replicating forms and particularly in the insect epimastigote stage. Among others, cruzipain participates in the penetration of T. cruzi trypomastigotes into host cells, in the nutrition of the parasite at the expense of the host, and in the escape mechanisms of the parasite from the immune system of the host [1,2,9]. Moreover, cruzipain is a promising target for antiparasite chemotherapy. Notably, cysteine proteinase inhibitors block replication and differentiation of T. cruzi both in vitro a...

Computational Study of the Michaelis Complex Formation and the Effect on the Reaction Mechanism of Cruzain Cysteine Protease

“…The covalent inhibitor Bz–Tyr–Ala–CH 2 F was replaced by the peptide Ac–Ala–Ala–Ala–Gly–Ala–OCH 3 . Taking into account the low substrate specificity of this protein, − this peptide was chosen because it was already used in our previous study of the catalytic mechanism from the ion pair dyad, thus facilitating the comparison between mechanisms . In addition, it presents computational advantages due to its small size.…”

Proteome of the Triatomine Digestive Tract: From Catalytic to Immune Pathways; Focusing on Annexin Expression

“…Regarding blood digestion, hemoglobin (Hb) as well as peptides, amino acids, and heme proteins, are also released into the TDT (Francis et al, 1997). Besides the digestion process, the volume of blood consumed by triatomines determines the limit of their nymphal development (Kollien et al, 2001;Salvati et al, 2001Salvati et al, , 2002 as well as the metacyclogenesis of T. cruzi (Garcia et al, 1995;Lander et al, 2020). In its free state, the heme molecule acts as a potent pro-oxidant and cytotoxic agent, leading to the lysis of many cells and ROS generation through the catalytic decomposition of organic hydroperoxides (Van der Zee et al, 1996;Graça-Souza et al, 2006).…”

Does Inhibition of Trypanosoma cruzi Key Enzymes Affect Parasite Life Cycle and Geographic Distribution?

“…Remarkably, the catalytic activity of T. cruzi key enzymes is modulated by pHand temperature-dependent substrate inhibition. This modulation mechanism appears to be substrate-independent (10)(11)(12). Modulation of the catalytic activity of T. cruzi key enzymes occurs over pH and temperature ranges overlapping those affecting parasite life cycle and distribution (1,3,13).…”

“…Interestingly, also T. cruzi oligopeptidase B (10) and malate dehydrogenase (11) action is modulated by the pH-and temperature-dependent substrate inhibition mechanism. In particular, the remarkable substrate inhibition processes distinguish T. cruzi oligopeptidase B and cruzain from human proteases, facilitating drug design to counteract cell invasion by trypanosomes (10,12).…”

“…This may represent an adaptation to life in homeothermal animals, but it may result in a higher sensitivity of the parasite to climate changes along latitude gradients. The pH-and temperaturedependent substrate inhibition mechanism, until now described in three key enzymes participating to the life cycle of T. cruzi (10)(11)(12) may therefore represent a general mechanism to modulate the parasite life cycle and its geographic range. Last but not least, this molecular mechanism might also apply to T. brucei brucei (the parasite responsible for the sleeping disease) (1), expressing a soluble 80-kDa endopeptidase, which is modulated by a pronounced pH-and temperature-dependent substrate inhibition (15).…”