Crystal structure of macrophage migration inhibitory factor-complexed with(E)-2-fluoro-p-bydroxycinnamate at 1.8 angstrom resolution: Implications for enzymatic catalysis and inhibition

Macrophage migration inhibitory facto; (MIF) exhibits dual activities. It a cts as an immunoregulatory protein as Well as a phenylpyruvate tautomerase. To understand better the relationship between these two activities and to elucidate the structural basis for the enzymatic activity, a crystal struct ure of a complex between murine MIF and (E)-2-fluoro-p-hydroxycinnamate, a competitive inhibitor of the tautomerase activity, has been determined to 1 .8 Angstrom resolution. The structure is nearly superimposable on that of t he free protein indicating that the presence of the inhibitor does not resu lt in any major structural changes. The inhibitor also confirms the locatio n of the,active site in a hydrophobic cavity containing the amino-terminal proline. Within this cavity, the inhibitor interacts with residues from adj acent subunits. At the back of the cavity, the side-chain carbonyl oxygen o f Asn-97' interacts with the phenolic hydroxyl group of the inhibitor while at the mouth of the cavity the ammonium group of Lys-32 interacts with a c arboxylate oxygen. The other carboxylate oxygen of the inhibitor interacts with Pro-1. The hydroxyl group of Tyr-95' interacts,weakly with the fluoro group on the inhibitor. The hydrophobic side chains of five active-site res idues (Met-2, Ile-64, Met-101, Val-106, and Phe-113) and the phenyl moiety of Tyr-95' are responsible for the binding of the phenyl group. Further ins ight into the enzymatic activity of MIF was obtained by carrying out kineti c studies using the enol isomers of phenylpyruvate and (p-hydroxyphenyl)pyr uvate. The results demonstrate that MIF processes the enol isomers more eff iciently than the keto isomers primarily because of a decrease in K-m. On t he basis of these results, a mechanism is proposed for the MIF-catalyzed ta utomerization reaction.