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

Abstract

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 activemore » 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.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
980496
Report Number(s):
BNL-93414-2010-JA
TRN: US201015%%1881
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 48; Journal Issue: 1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; 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

Citation Formats

Crichlow, G., Lubetsky, J, Leng, L, Bucala, R, and Lolis, E. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions. United States: N. p., 2009. Web. doi:10.1021/bi8014423.
Crichlow, G., Lubetsky, J, Leng, L, Bucala, R, & Lolis, E. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions. United States. doi:10.1021/bi8014423.
Crichlow, G., Lubetsky, J, Leng, L, Bucala, R, and Lolis, E. Thu . "Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions". United States. doi:10.1021/bi8014423.
@article{osti_980496,
title = {Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions},
author = {Crichlow, G. and Lubetsky, J and Leng, L and Bucala, R and Lolis, E},
abstractNote = {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.},
doi = {10.1021/bi8014423},
journal = {Biochemistry},
number = 1,
volume = 48,
place = {United States},
year = {2009},
month = {1}
}