Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

Journal Article · Thu Jan 01 04:00:00 EST 2009 · Biochemistry

DOI:https://doi.org/10.1021/bi8014423· OSTI ID:980496

Crichlow, G; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

• Research Organization: Brookhaven National Laboratory (BNL) National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 980496
• Report Number(s): BNL--93414-2010-JA
• Journal Information: Biochemistry, Journal Name: Biochemistry Journal Issue: 1 Vol. 48
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
BIOLOGICAL FUNCTIONS
COMPLEXES
DISEASES
ELECTRON DENSITY
ENZYMES
FLUORIDES
GLUCOCORTICOIDS
IMINES
INHIBITION
INTERACTIONS
KINETICS
MACROPHAGES
MIGRATION
NEOPLASMS
NITROGEN
ORAL CAVITY
PROLINE
PROTEINS
SUBSTRATES
SULFUR
national synchrotron light source