3-DIMENSIONAL STRUCTURES OF GLYCOLATE OXIDASE WITH BOUND ACTIVE-SITE INHIBITORS

A key step in plant photorespiration, the oxidation of glycolate to gl yoxylate, is carried out by the peroxisomal flavoprotein glycolate oxi dase (EC 1.1.3.15). The three-dimensional structure of this alp barrel protein has been refined to 2 Angstrom resolution (Lindqvist Y. 1989. J Mol Biol 209:151-166). FMN dependent glycolate oxidase is a member of the family of alpha-hydroxy acid oxidases. Here we describe the cry stallization and structure determination of two inhibitor complexes of the enzyme, TKP (3-Decyl-2,5-dioxo-3-hydroxy-3-pyrroline) and TACA Ca rboxy-5-(1-pentyl)hexylsulfanyl-1,2,3-triazole). The structure of the TACA complex has been refined to 2.6 Angstrom resolution and the TKP c omplex, solved with molecular replacement, to 2.2 Angstrom resolution. The R-free for the TACA and TKP complexes are 24.2 and 25.1%, respect ively. The overall structures are very similar to the unliganded holoe nzyme, but a closer examination of the active site reveals differences in the positioning of the flavin isoalloxazine ring and a displaced f lexible loop in the TKP complex. The two inhibitors differ in binding mode and hydrophobic interactions, and these differences are reflected by the very different K-i values for the inhibitors, 16 nM for TACA a nd 4.8 mu M for TKP. Implications of the structures of these enzyme-in hibitor complexes for the model for substrate binding and catalysis pr oposed from the hole-enzyme structure are discussed.