CRYSTAL-STRUCTURE OF 4-OXALOCROTONATE TAUTOMERASE INACTIVATED BY 2-OXO-3-PENTYNOATE AT 2.4 ANGSTROM RESOLUTION - ANALYSIS AND IMPLICATIONS FOR THE MECHANISM OF INACTIVATION AND CATALYSIS

The crystal structure of 4-oxalocrotonate tautomerase (4-OT) inactivat ed by the active site-directed irreversible inhibitor 2-oxo-3-pentynoa te (2-OF) has been determined to 2.4 Angstrom resolution. The structur e 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 s urrounding Pro-1. Both structures can be described as a trimer of dime rs where each dimer consists of a four-stranded beta-sheet with two an tiparallel alpha-helices on one side. Examination of the structure als o reveals noncovalent interactions between the adduct and two residues in the active site, The epsilon and eta 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 ad duct while the side chain of Arg-61' from the same dimer as the modifi ed Pro-1 interacts with the C-1 carboxylate group in a bidentate fashi on. An additional interaction to the 2-oxo group of the adduct is prov ided by one of the two ordered water molecules within the active site region. These interactions coupled with the observation that 2-oxo-3-b utynoate is a more potent irreversible inhibitor of 4-oxalocrotonate t automerase than is 2-OF suggest that Arg-39 '' and the ordered water m olecule polarize the carbonyl group of 2-OF which facilitates a Michae l reaction between Pro-1 and the acetylene compound. On the basis of t he crystal structure, a mechanism for the enzyme-catalyzed reaction is proposed.