Papain-Like Cysteine Proteinases 171 Applied Biochemistry and Biotechnology Vol. 118, 2004 Copyright © 2004 by Humana Press Inc. All rights of any nature whatsoever reserved. 0273-2289/04/118/0171/$25.00 171 *Author to whom all correspondence and reprint requests should be addressed. Insight Into Catalytic Mechanism of Papain-Like Cysteine Proteinases The Case of D 158 E. M. PAPAMICHAEL,* ,1 L. G. THEODOROU, 1 AND J. G. BIETH 2 1 University of Ioannina, Department of Chemistry, Ioannina 45110, Greece, E-mail: epapamic@cc.uoi.gr; and 2 Université Louis Pasteur Strasbourg, Faculté de Pharmacie, I.N.S.E.R.M. Unité 392, Laboratoire d’Enzymologie, 74 Route du Rhin, Illkirch, F-67400, France Received May 21, 2003; Revised August 20, 2003; Accepted August 28, 2003 Abstract We studied the role of D 158 in papain-like cysteine proteinases by using subtilisin Carlsberg, and its chemically modified analog thiolsubtilisin, by applying the proton inventory (PI) method and also by taking into account the pH profiles of the k cat /K m parameter. In the case of thiolsubtilisin, we estimated large inverse solvent isotope effects for k cat /K m , as in papain, whereas for subtilisin we found “dome-shaped” PI, suggesting a completely different mechanism. Finally, the kinetic behavior of thiolsubtilisin presented similarities as well as differences, compared to papain, suggesting a possible role for D 158 as part of a catalytic triad in papain-like cysteine proteinases. Index Entries: Cysteine proteinases; proton inventory; catalytic mecha- nism; aspartate 158 ; thiolsubtilisin. Introduction Serine proteinases hydrolyze amide or ester substrates by means of a charge-relay system formed by D 102 , H 57 , and S 195 (chymotrypsin number- ing) (1), whereas papain-like cysteine proteinases carry out catalysis by an ion pair (C 25 -S – /H 159 -Im + H; papain numbering). Although in both cases the minimal mechanism could be presented by Scheme 1 (2), the ambiguous role of D 158 in papain-like cysteine proteinases has been argued (3–6), and, therefore, it should be elucidated: