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

Cysteine proteinases are relevant to several aspects of the parasite life c ycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of c ruzipain, 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-al pha -benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degreesC and 40 .0 degreesC and between pH 4.5 and 8.5. Values of K-m were independent of t emperature and pH, whereas values of V-max V-max/K-m, and K-i were temperat ure-dependent and pH-dependent. Over the whole pH range explored, values of logV(max), log(V-max/K-m), and log(Ki) increased linearly with respect to T-1. Values of V-max and V-max/K-m were affected by the acid-base equilibri um of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degreesC). Moreover, values of K-i were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(un l)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degreesC) upon Z-Phe-A rg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were tempe rature-independent. Conversely, values of logK(unl)" were linearly dependen t on T-1. As a whole, total substrate inhibition of cruzipain decreased wit h increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain eithe r productively or following a binding mode which results in enzyme inhibiti on. However, allosteric effect(s) cannot be excluded.