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Macrophage migration inhibitory factor (MIF), known as a cytokine, is a multifunctional protein that is ubiquitously expressed in a variety of cells and tissues; however, enzymatic function of MIF still remains elusive in cells. In this study, we assessed details of the tautomerase activity of MIF. We established rapid purification condition for MIF by using pGEX system and compared the L-dopachrome tautomerase activity of GST-MIF, tMIF, and MIF. The results show that GST (glutathione S-transferase)-epitope tag or N-terminal amino acids flanking the essential P²almost completely abrogated L-dopachrome tautomerase activity of MIF. Subsequently, to determine whether the N-terminal tags have effects on oligomerization of MIF, protein cross-linking products were analyzed on 15% SDS-PAGE. The result demonstrates that N-terminal tags are dispensable for the formation of MIF’s homooligomers. Thus, the results imply that exposure of P²containing hydrophobic pocket in the active site is critical for L-dopachrome tautomerase activity of MIF. In addition, our study suggest that the MIF’s tautomerase activity might be influenced by interacting with cellular partners.
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