Investigations of the catalytic active site of macrophage migration inhibitory factor.

摘要

Macrophage migration inhibitory factor (MIF) is an immunoregulatory molecule with numerous biological activities that is a potential therapeutic target for a number of inflammatory diseases. Although considered a cytokine, MIF possesses a three-dimensional structure and active site similar to several bacterial enzymes. Moreover, a number of catalytic activities have been defined for MIF, although none of these are believed to be physiological. The role of the catalytic site of MIF in its biological activities remains controversial. To gain insight into its role, the catalytic active site was first characterized in more detail. The crystal structure of MIF complexed to p-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF, reveals that the binding sites for HPP in the MIF trimer lie at the interface between two subunits. Several conserved residues interact with the substrate through a series of aromatic-aromatic interactions, hydrogen bonds, and van der Waals contacts. This structure in combination with kinetic and structural studies of a Pro-1 to Gly mutant (P1G) which has reduced catalytic activity, provide insight into the mechanism of the catalytic reaction. Next, a catalytically inactive mutant in which an alanine is inserted between Pro-1 and Met-2 (PAM) was designed and characterized. Structural studies of this mutant define the basis for the reduced activity, whereas both in vitro and in vivo activity studies address any differences in behavior from wild-type protein. Finally, studies of four different classes of catalytic inhibitors are reported. Various small alkyl chains were screened for the inhibition of catalytic activity. These compounds were designed based on the known substrates of MIF, and the inhibition of catalytic activity by such MIF biological inhibitors was tested. Kinetic, biological, and structural studies of these inhibitors further establish a link between the catalytic active site and the biological role of MIF.