Crystal Structure of Macrophage Migration Inhibitory Factor Complexed with (E)-2-Fluoro-p-hydroxycinnamate at 1.8 Å Resolution:  Implications for Enzymatic Catalysis and Inhibition^,

Abstract

Macrophage migration inhibitory factor (MIF) exhibits dual activities. It acts 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 structure 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 A resolution. The structure is nearly superimposable on that of the free protein indicating that the presence of the inhibitor does not result in any major structural changes. The inhibitor also confirms the location of the active site in a hydrophobic cavity containing the amino-terminal proline. Within this cavity, the inhibitor interacts with residues from adjacent subunits. At the back of the cavity, the side-chain carbonyl oxygen of 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 carboxylate 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 residues (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 insight into the enzymatic activity of MIF was obtained by carrying out kinetic studies using the enol isomers of phenylpyruvate and (p-hydroxyphenyl)pyruvate. The results demonstrate that MIF processes the enol isomers more efficiently than the keto isomers primarily because of a decrease in Km. On the basis of these results, a mechanism is proposed for the MIF-catalyzed tautomerization reaction.

Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites

“…Similar interactions between murine MIF and (E)-2-fluoro-p-hydroxycinnamate have been reported (31), and solution NMR studies support a direct interaction between model substrates and the Pro-1 located within the hydrophobic cavity (30). Of note, the environment of this cavity reduces the pKa of Pro-1 by almost 4 pH units relative to a proline amide (32), suggesting that this residue functions as a catalytic base to effect substrate tautomerization (33,34).…”

The Tautomerase Active Site of Macrophage Migration Inhibitory Factor Is a Potential Target for Discovery of Novel Anti-inflammatory Agents

“…MIF utilizes this active site in several non-physiological catalytic activities (27,28,50). This catalytic active site has been clearly identified through crystallographic studies of MIF complexed to a non-physiological substrate, HPP (31), and to the inhibitor, (E)-2-fluoro-p-hydroxycinnamate (32). A multiple sequence alignment of MIF orthologues and homologues indicates that the active site residues are among the most highly conserved residues (51).…”

“…More recently, phenylpyruvic acid, phydroxyphenylpyruvic acid (HPP), 3,4-dihydroxyphenylaminechrome, and norepinephrinechrome also have been found to be MIF substrates, but high Michaelis constant (K m ) values suggest that these also are unlikely natural substrates for MIF (28 -30). Human MIF⅐HPP and murine MIF⅐inhibitor crystal structures have identified a substrate-binding hydrophobic cavity that lies between two adjacent subunits of the homotrimer (31,32). Pro-1 appears to be a critical residue for enzymatic activity, as site-directed mutagenesis that substitutes a serine for this proline (P1S) is devoid of D-dopachrome tautomerase activity (33).…”

Molecular Interaction and Enzymatic Activity of Macrophage Migration Inhibitory Factor with Immunorelevant Peptides

“…Binding Analysis-Macrophage migration inhibitory factor functions as a tautomerase/isomerase and thiol reductase on small molecule substrates such as L-DOPA and insulin (11,12). Pro-1 and Cys-57-Cys-60 form the enzymatic catalytic sites.…”

“…MIF also possesses enzymatic activities as a tautomerase/isomerase (10) and thiol oxidoreductase (11). However, the natural substrates for MIF catalytic activity are unknown even though its enzymatic activity has been characterized in detail using insulin and L-DOPA as models (11,12).…”