title: Characterization of the conformational changes of acetohydroxy acid isomeroreductase induced by the binding of Mg2+ ions, NADPH, and a competitive inhibitor.
abstract: Acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the parallel branched chain amino acid pathway, is a homodimer with an Mr of approximately 114000 which in the presence of Mg2+ ions catalyzes an unusual alkyl migration followed by an NADPH-dependent reduction. Prior binding of NADPH and Mg2+ to the enzyme was shown to be required for substrate or competitive inhibitor [N-hydroxy-N-isopropyloxamate (IpOHA)] binding [Dumas, R., et al. (1994) Biochem. J. 301, 813-820]. Moreover, crystallographic data for the enzyme-NADPH-Mg2+-IpOHA complex [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] have shown that IpOHA was completely buried inside the active site. These observations raised the question of how the reaction intermediate analogue inhibitor can reach the active site and implied that conformational changes occurred during the binding process. With a view of characterizing these conformational changes, H-D exchange experiments combined with mass spectrometry were performed. Results demonstrated that Mg2+ ions and NADPH binding led to an initial conformational change at the interface of the two domains of each monomer. Binding of the two cofactors to isomeroreductase alters the structure of the active site to promote inhibitor (substrate) binding, in agreement with the ordered mechanism of the enzyme. Structural changes remote from the active site were also found. They were interpreted as long-range structural effects on the two domains and on the two monomers in the time course of the ligand binding process.
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry*; D050540:Ketol-Acid Reductoisomerase; D008274:Magnesium / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D018724:Spinacia oleracea / Q000201:enzymology