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 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 Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). 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.
Salvati
L
L
Dipartimento di Biologia, Università 'Roma Tre', Roma, Italy.
Mattu
M
M
Polticelli
F
F
Tiberi
F
F
Gradoni
L
L
Venturini
G
G
Bolognesi
M
M
Ascenzi
P
P
D000818:Animals; D002364:Caseins / Q000737:chemistry; D002384:Catalysis; D003374:Coumarins / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D004151:Dipeptides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D015800:Protozoan Proteins; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D014349:Trypanosoma cruzi / Q000201:enzymology*
D002364:Caseins; D003374:Coumarins; D004151:Dipeptides; D015800:Protozoan Proteins; C026018:azocasein; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; D003546:Cysteine Endopeptidases; C063214:cruzipain