Catalytic properties of cysteine proteinases from Trypanosoma cruzi and Leishmania infantum: a pre-steady-state and steady-state study q Paolo Ascenzi, a,b, * Alessio Bocedi, a,b,c Paolo Visca, a,d Giovanni Antonini, a,b and Luigi Gradoni e a Dipartimento di Biologia, Universit  a ‘Roma Tre’, Viale Guglielmo Marconi 446, I-00146 Rome, Italy b Centro Interdipartimentale per la Microscopia Elettronica, Universit  a ‘Roma Tre’, Via della Vasca Navale 79, I-00146 Rome, Italy c Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universit  a di L’Aquila, Via Vetoio, Coppito, I-67100 L’Aquila, Italy d Istituto Nazionale per le Malattie Infettive IRCSS ‘Lazzaro Spallanzani’, Via Portuense 292, I-00149 Rome, Italy e Laboratorio di Parassitologia, Istituto Superiore di Sanit  a, Viale Regina Elena 299, I-00161 Rome, Italy Received 31 July 2003 This paper is dedicated to the memory of Prof. Eraldo Antonini on the occasion of the 20th anniversary of his death. Abstract Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite–host relationship. Moreover, they appear as promising targets for antiparasite chemotherapy. Here, the first quantitative investigation on the steady-state and pre- steady-state kinetics of the papain-like cysteine proteinases from epimastigotes of Trypanosoma cruzi (cruzipain), the agent of Chagas’ disease, and from promastigotes of Leishmania infantum, an agent of visceral and cutaneous leishmaniases, is reported. The results indicate that kinetics for the parasite proteinase catalyzed hydrolysis of N-a-benzyloxycarbonyl-L-phenylalanyl-L -arginine- (7-amino-4-methylcoumarin) may be consistently fitted to the minimum three-step mechanism involving the acylenzyme inter- mediate EP: At neutral pH, the k þ3 step (deacylation process) is rate limiting in enzyme catalysis, whereas, at pH < 6, the k þ2 step (acylation process) becomes rate limiting. This illustrates the potential danger in interpreting both k cat versus pH profile, given that the ac- ylation or the deacylation step is rate limiting throughout the whole pH range explored, and K m as the true affinity constant for the E:S complex formation. Comparison with the steady-state and pre-steady-state kinetics of homologous plant enzymes suggests that the parasite cysteine proteinase catalytic behavior appears to be of general significance. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Parasite cysteine proteinase; Trypanosoma cruzi; Leishmania infantum; Pre-steady-state kinetics; Steady-state kinetics Trypanosomatids are parasitic Protozoa, a number of them being agents of endemic diseases prevalent mainly in developing countries. In particular, Trypanosoma cruzi is the agent of the American trypanosomiasis (Chagas’ disease), affecting at least 20 million people. Furthermore, Leishmania are parasitic trypanosomatids Biochemical and Biophysical Research Communications 309 (2003) 659–665 www.elsevier.com/locate/ybbrc BBRC q Abbreviations: Z-Phe-Arg-AMC, N-a-benzyloxycarbonyl-L-phe- nylalanyl-L-arginine-(7-amino-4-methylcoumarin); AMC, 7-amino-4- methylcoumarin. * Corresponding author. Fax: +39-06-5517-6321. E-mail address: ascenzi@bio.uniroma3.it (P. Ascenzi). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.08.015