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^,

Abstract

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 A 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 beta-sheet with two antiparallel alpha-helices on one side. Examination of the structure also 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 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.

trans -3-Chloroacrylic Acid Dehalogenase from Pseudomonas pavonaceae 170 Shares Structural and Mechanistic Similarities with 4-Oxalocrotonate Tautomerase

“…In contrast to the presence of potentially six active sites in the highly symmetrical 4-OT molecule (45), there are three potential active sites in CaaD. From the crystal structure of 4-OT inactivated by 2-oxo-3-pentynoate (45), the substrate should interact with Pro-1 of one subunit and Arg-11 from an adjacent subunit within the same homodimer.…”

Structural Basis of Perturbed pKa Values of Catalytic Groups in Enzyme Active Sites

“…Pro-1 can function as a general base under physiological conditions because of its unusually low pK a of 6.4, which is 3 units lower than the pK a of the model compound, proline amide (70). The X-ray structure of 4-OT (71) shows that within a sphere of 9Å radius, Pro-1 is surrounded by hydrophobic residues, creating a site with a low dielectric constant. In addition, Arg-11 and Arg-39 are positioned 11Å and 7Å, respectively, from Pro-1 and are located at opposite sides of the active site, providing binding specificity to the terminal carboxyl groups of the substrate and product.…”

Biodegradation of Aromatic Compounds by Escherichia coli

“…The catalytic mechanism involves a single catalytic base that is the N-terminal proline residue. Two arginine residues interact with the substrate in the active-site region (304,312). Although the HpcD isomerase catalyzes a reaction analogous to that catalyzed by the 4-oxalocrotonate tautomerase of the meta-cleavage pathway of phenol and toluene on a chemically similar substrate, the two enzymes do not have any apparent sequence similarity.…”

“…Nevertheless, the two isomerases are specific, and each shows a preference for its own substrate. This substrate specificity in the isomerases seems to be determined by steric constraints in the catalytic cleft (304,312).…”