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MIF is a pro\u2010inflammatory, pro\u2010tumorigenic protein that may be an attractive therapeutic target. MIF catalyzes the interconversion of the keto and enol forms of HPP by a tautomerase reaction. Although HPP is evidently not a physiological substrate of MIF, many compounds that inhibit this activity in enzymatic assays have been found also to inhibit physiological activities of MIF. Therefore, the MIF\u2010catalyzed HPP tautomerization reaction is used in initial screening of compounds in the search for inhibitors of MIF physiological activity. The neutron diffraction\u2010derived crystal structure reveals the position of a water molecule involved in the tautomerization reaction, and also confirms the charged state of lysine\u201032 in the active site. The structure confirms the previously proposed catalytic mechanism of MIF, with the N\u2010terminal Pro\u20101 abstracting a proton to generate an HPP enolate intermediate which is subsequently protonated. The structure reported herein reveals that this proton is supplied by a neighboring water molecule. Along with the neutron structure, a room\u2010temperature synchrotron x\u2010ray crystal structure reveals a covalent adduct between HPP and MIF. While this adduct is a result of radiation\u2010induced chemistry, its formation confirms the catalytic role of the active site residue because a covalent complex could only form if the reactive carbon of the substrate is correctly positioned by the enzyme.<\/jats:p>","DOI":"10.1002\/pro.70666","type":"journal-article","created":{"date-parts":[[2026,8,8]],"date-time":"2026-08-08T08:31:22Z","timestamp":1786177882000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Identification of a key water molecule involved in the macrophage migration inhibitory factor\u2010catalyzed tautomerization of\n                    <i>para<\/i>\n                    \u2010hydroxyphenylpyruvate using neutron crystallography"],"prefix":"10.1002","volume":"35","author":[{"given":"Gabriela C.","family":"Schr\u00f6der","sequence":"first","affiliation":[{"name":"Department of Molecular and Structural Biochemistry North Carolina State University  Raleigh North Carolina USA"},{"name":"Neutron Scattering Division Oak Ridge National Laboratory  Oak Ridge Tennessee USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0512-1031","authenticated-orcid":false,"given":"Gregg V.","family":"Crichlow","sequence":"additional","affiliation":[{"name":"Department of Pharmacology Yale University School of Medicine  New Haven Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ewelina","family":"Jablonowski","sequence":"additional","affiliation":[{"name":"Department of Pharmacology Yale University School of Medicine  New Haven Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5547-1406","authenticated-orcid":false,"given":"Georgios","family":"Pantouris","sequence":"additional","affiliation":[{"name":"Department of Pharmacology Yale University School of Medicine  New Haven Connecticut 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