Pro-1 of macrophage migration inhibitory factor functions as a catalytic base in the phenylpyruvate tautomerase activity

Macrophage migration inhibitory factor (MIF) is an important immunoregulato ry 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 3-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase. Moreover, a nu mber of catalytic activities have been defined for MIF. To gain insight int o 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 c rystal structure of MIF complexed with p-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF. The three binding site s for p-hydroxyphenylpyruvate in the MIF trimer lie at the interface betwee n 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 posit ioned to function as a catalytic base. There is no functional group that po larizes 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 reduc ed activity and provide insight into the mechanism of the catalytic reactio n.