Docking of 4-Oxalocrotonate Tautomerase Substrates: Implications for the Catalytic Mechanism T. A. Soares 1,2 D. S. Goodsell 1 J. M. Briggs 3 R. Ferreira 2,3 A. J. Olson 1 1 Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 2 Departamento de Quimica Fundamental, Universidade Federal de Pernambuco, Recife, PE 50670-901, Brazil 3 Department of Pharmacology, University of California, San Diego, La Jolla, CA 92037, USA Received 21 October 1998; accepted 22 February 1999 Abstract: The enzyme 4-oxalocrotonate tautomerase catalyzes the ketonization of dienols, which after further processing become intermediates in the Krebs cycle. The enzyme uses a general acid– base mechanism for proton transfer: the amino-terminal proline has been shown to function as the catalytic base and Arg39 has been implicated as the catalytic acid. We report the results of molecular docking simulations of 4-oxalocrotonate tautomerase with two substrates, 2-hydroxymu- conate and 5-carboxymethyl-2-hydroxymuconate. pK a calculations are also performed for the free enzyme. The predicted binding mode of 2-hydroxymuconate is in agreement with experimental data. A model for the binding mode of 5-carboxymethyl-2-hydroxymuconate is proposed which explains the lower catalytic efficiency of the enzyme toward this substrate. The pK a predictions and docking simulations support residue Arg39 as the general acid for the enzyme catalysis. © 1999 John Wiley & Sons, Inc. Biopoly 50: 319 –328, 1999 Keywords: 4-oxalocrotonate tautomerase; molecular docking; pK a predictions; catalytic mechanism INTRODUCTION Microbial degradation of aromatic carbon sources oc- curs by a meta-fission pathway through the ketoniza- tion of dienols. 1,2 In Pseudomonas putida mt-2 the entire pathway is encoded by the TOL plasmid, en- abling strains of soil bacteria to use simple aromatic hydrocarbons as a unique source of carbon and en- ergy. A key enzyme of this metabolic pathway is 4-oxalocrotonate tautomerase (4OT). This enzyme promotes the ketonization of 2-hydroxymuconate (HM) to an ,-unsaturated ketone, which undergoes Correspondence to: A. J. Olson Contract grant sponsor: Conselho Nacional de Desenvolvi- mento Cientifico e Technologico and NIH; contract grant number: P01 GM48870 (NIH) Biopolymers, Vol. 50, 319 –328 (1999) © 1999 John Wiley & Sons, Inc. CCC 0006-3525/99/030319-10 319