TETRAHEDRON: ASYMMETRY Tetrahedron: Asymmetry 13 (2002) 475–489 Pergamon Computational studies on the enantioselectivity of -chymotrypsin towards -carbomethoxy--lactams F. Felluga, a M. Fermeglia, b M. Ferrone, b G. Pitacco, a S. Pricl b, * and E. Valentin a a Department of Chemical Sciences, University of Trieste, Via Giorgeri 1, 34127 Trieste, Italy b Computer -aided Systems Laboratory, Department of Chemical, Environmental and Raw Materials Engineering -DICAMP, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy and ICS -UNIDO, Area Science Park, Padriciano 99, 34100 Trieste, Italy Received 31 January 2001; accepted 15 March 2002 Abstract—In our previous work, racemic -carbomethoxy--lactams were subjected to enzymatic hydrolysis in the presence of -chymotrypsin. The hydrolysis of three N-substituted lactams proved to be highly enantioselective, whereas an unsubstituted lactam was recovered in racemic form. Thus, in this paper we applied several molecular modeling protocols to explain the substrate specificity and the enantioselectivity of this enzyme. The adopted procedures involved accurate docking experiments of both enantiomers of each lactam to the protein active site, whose 3-D structure was obtained from X-ray crystallographic data, followed by extensive conformational and energetic analysis of the computer-generated complexes. The results obtained fully account for the experimental evidences on the enantioselective hydrolysis of these interesting, potential drugs by -chymotrypsin. © 2002 Elsevier Science Ltd. All rights reserved. 1. Introduction Enzymes play a major role in organic synthesis as environmentally friendly catalysts for a wide range of chemical transformations. 1,2 Among this plethora of biomacromolecules, hydrolytic enzymes are particularly useful as they exhibit broad substrate specificities and can induce high enantioselectivities, are commercially available, and do not necessitate the use of expensive and/or unstable coenzyme systems. To date, esterolytic enzymes, such as lipases and proteases, appear to be the most widely employed in organic processes. For an enzyme to gain extensive application, the factors gov- erning its specificity, particularly the relationships between its selectivity and the structural features of the substrate it processes, must be understood. Accord- ingly, several simple and easy-to-use active site models have been proposed for a number of different esterases, 3,4 and some well defined SARs (structure– activity relationships) are emerging. Rather recently, straightforward models for enzyme selectivity, known as rules, have been proposed in the literature for some popular lipases such as Pseudomonas cepacia (PCL), 5,6 Candida rugosa (CRL), 7,8 and Rhizo - mucor miehei (RML). 9 Nonetheless, these types of models are limited to isosteres of known substrates, and can only predict which the fastest reacting enantiomer is. Therefore, the goals of more accurate modeling in this field should be at least threefold: (i) to explain, on a molecular level, the known behavior of an enzyme; (ii) to suggest how to change the selectivity of a reac- tion by modification of substrate, enzyme or reaction conditions, and (iii) to predict quantitatively the degree of stereoselectivity of an enzyme-catalyzed reaction. In the last decades, X-ray data of a great number of enzymes have become available to the scientific commu- nity by means of public data-base depositories, such as the Protein Data Bank (PDB). 10 These records provide the three-dimensional structure of the relevant biomacromolecule active site. Further, the progressively increasing development of sophisticated molecular modeling suites allows the researcher to explore and characterize the interactions between a given substrate and the amino acids making up the catalytic site. In this context, the reaction mechanism of -chymotrypsin was deduced, in part, from its three-dimensional structure determined by X-ray crystallography 11 (Fig. 1). The enzyme contains three subunits, the A, B and C chains, * Corresponding author. Tel.: +39-040-6763750; fax: +39-040-569823; e-mail: sabrinap@dicamp.units.it 0957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S0957-4166(02)00144-1