Crystal Structure of 4-Oxalocrotonate Tautomerase Inactivated by 2-Oxo-3-pentynoate at 2.4 Å Resolution:  Analysis and Implications for the Mechanism of Inactivation and Catalysis

The crystal structure of 4-oxalocrotonate tautomerase (4-OT) inactivated by the active site-directed irreversible inhibitor 2-oxo-3-pentynoate (2-OP) has been determined to 2.4 Å resolution. The structure of the enzyme covalently modified at Pro-1 by the resulting 2-oxo-3-pentenoate adduct is nearly superimposable on that of the free enzyme and confirms that the active site is located in a hydrophobic region surrounding Pro-1. Both structures can be described as a trimer of dimers where each dimer consists of a four-stranded β-sheet with two antiparallel α-helices on one side. Examination of the structure also reveals noncovalent interactions between the adduct and two residues in the active site. The ε and η nitrogens of the guanidinium side chain of Arg-39‘ ‘ from a neighboring dimer interact respectively with the C-2 carbonyl oxygen and one C-1 carboxylate oxygen of the adduct while the side chain of Arg-61‘ from the same dimer as the modified Pro-1 interacts with the C-1 carboxylate group in a bidentate fashion. An additional interaction to the 2-oxo group of the adduct is provided by one of the two ordered water molecules within the active site region. These interactions coupled with the observation that 2-oxo-3-butynoate is a more potent irreversible inhibitor of 4-oxalocrotonate tautomerase than is 2-OP suggest that Arg-39‘ ‘ and the ordered water molecule polarize the carbonyl group of 2-OP which facilitates a Michael reaction between Pro-1 and the acetylene compound. On the basis of the crystal structure, a mechanism for the enzyme-catalyzed reaction is proposed.