Journal of Computer-Aided Molecular Design, 11 (1997) 153–162 153 *To whom correspondence should be addressed. ESCOM J-CAMD 388 Role of the tautomerism of 2-azaadenine and 2-azahypoxanthine in substrate recognition by xanthine oxidase Begoña Hernández a , Modesto Orozco a and Francisco J. Luque b, * a Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University of Barcelona, Martí i Franquès 1, E-08028 Barcelona, Spain b Department of Pharmacy, Section Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Avinguda Diagonal s/n, E-08028 Barcelona, Spain Received 30 August 1996 Accepted 6 December 1996 Keywords: Xanthine oxidase; Molecular recognition; Tautomerism; Quantum mechanics; Xanthine Summary The tautomerism of 2-azaadenine and 2-hypoxanthine has been examined in the gas phase and in aqueous solution. The tautomerism in the gas phase has been studied by means of semiempirical and ab initio quantum-mechanical computations, as well as density-functional calculations. The influence of the aqueous solvent on the relative stability between tautomers has been estimated from self-consist- ent reaction field calculations performedwith different high-level continuum models. The results provide a detailed picture of the tautomeric preference for these purine bases. The importance of tautomerism in the substrate recognition by xanthine oxidase is discussed. Finally, the rate of oxidation of 2-aza- adenine and 2-hypoxanthine by xanthine oxidase is discussed in terms of the recognition model at the enzyme active site. Introduction The degradation metabolism of purines is mainly con- trolled by two enzymes: adenosine deaminase (EC 3.5.4.4) and xanthine oxidase (xanthine:O 2 oxidoreductase; EC 1.2.3.2) [1]. The former enzyme deaminates adenosine yielding inosine, which is subsequently decomposed into the ribose and the purine base, hypoxanthine (1,7-dihydro- 6H-purin-6-one). Oxidation at position 2 of hypoxanthine by xanthine oxidase yields xanthine (1,7-dihydro-6H- purin-2,6-dione), which is also an intermediate product in the catabolism of guanine. Indeed, this enzyme catalyzes oxidation at position 8 of xanthine leading to uric acid, the final degradation product of purines in humans. Xanthine oxidase is a very important pharmacological target, since a malfunctioning of this enzyme increases the content of uric acid and eventually leads to the deposition of sodium hydrogen urate monohydrate crystals in joints. This gives rise to a painful disease known as gout, which is clinically treated by the antihyperuricemic drug allo- purinol [2] (pyrazolo[3,4-d]pyrimidin-6-one), a drug struc- turally related to hypoxanthine. Allopurinol is converted by xanthine oxidase into alloxanthine (pyrazolo[3,4-d]- pyrimidin-2,6-one). This latter compound inactivates the enzyme [3], thus inhibiting the formation of uric acid. The treatment of gout with allopurinol leads to the excretion of purines mainly as hypoxanthine and xanthine. Owing to the biochemical and pharmacological rel- evance of xanthine oxidase, a great deal of research has focused on the structural and kinetic aspects of the enzy- matic mechanism [4]. Xanthine oxidase and the related xanthine dehydrogenase (xanthine:NAD + oxidoreductase; EC 1.2.1.37) both comprise two equivalent, independent subunits [4], each containing one atom of molybdenum, one molecule of flavin adenine dinucleotide and two distinct iron-sulfur clusters. There are two spatially separ- ated binding sites for the reducing (hypoxanthine, xan- thine) and oxidizing (O 2 , NAD + ) substrates. The enzyme follows a two-site ping-pong mechanism [4,5], in which all the prosthetic groups participate in catalysis forming an electron transport chain connecting the two binding sites. The basic catalytic unit consists of a MoOS moiety [4c,d]. 0920-654X/$ 6.00 + 1.00 © 1997 ESCOM Science Publishers B.V.