title: Pro-1 of macrophage migration inhibitory factor functions as a catalytic base in the phenylpyruvate tautomerase activity.
abstract: Macrophage migration inhibitory factor (MIF) is an important immunoregulatory molecule with a unique ability to suppress the anti-inflammatory effects of glucocorticoids. Although considered a cytokine, MIF possesses a three-dimensional structure and active site similar to those of 4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase. Moreover, a number of catalytic activities have been defined for MIF. To gain insight into the role of catalysis in the biological function of MIF, we have begun to characterize the catalytic activities in more detail. Here we report the crystal structure of MIF complexed with p-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF. The three binding sites for p-hydroxyphenylpyruvate in the MIF trimer lie at the interface between two subunits. The substrate interacts with Pro-1, Lys-32, and Ile-64 from one subunit and Tyr-95 and Asn-97 from an adjacent subunit. Pro-1 is positioned to function as a catalytic base. There is no functional group that polarizes the alpha-carbonyl of the substrate to weaken the adjacent C-H bond. Mutation of Pro-1 to glycine substantially reduces the catalytic activity. The insertion of an alanine between Pro-1 and Met-2 essentially abolishes activity. Structural studies of these mutants define a source of the reduced activity and provide insight into the mechanism of the catalytic reaction.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Lubetsky
authors.forename: M
authors.initials: M
authors.lastname: Swope
authors.forename: C
authors.initials: C
authors.lastname: Dealwis
authors.forename: P
authors.initials: P
authors.lastname: Blake
authors.forename: E
authors.initials: E
authors.lastname: Lolis
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000235:genetics; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008263:Macrophage Migration-Inhibitory Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010667:Phenylpyruvic Acids / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D046911:Macromolecular Substances; D008263:Macrophage Migration-Inhibitory Factors; D010667:Phenylpyruvic Acids; D011994:Recombinant Proteins; C010590:4-hydroxyphenylpyruvic acid; D011392:Proline; D008715:Methionine; D019746:Intramolecular Oxidoreductases; C022346:phenylpyruvate tautomerase; D005998:Glycine