Characterization of the conformational changes of acetohydroxy acid isomeroreductase induced by the binding of Mg2+ ions, NADPH, and a competitive inhibitor

Acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the parallel branched chain amino acid pathway, is a homodimer with an M-r of a pproximate to 114000 which in the presence of Mg2+ ions catalyzes an unusua l alkyl migration followed by an NADPH-dependent reduction. Prior binding o f NADPH and Mg2+ to the enzyme was shown to be required for substrate or co mpetitive inhibitor [N-hydroxy-N-isopropyloxamate (IpOHA)] binding [Dumas, R., et al. (1994) Biochem. J. 301, 813-820]. Moreover, crystallographic dat a 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 activ e site. These observations raised the question of how the reaction intermed iate analogue inhibitor can reach the active site and implied that conforma tional changes occurred during the binding process. With a view of characte rizing 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 t he two domains of each monomer. Binding of the two cofactors to isomeroredu ctase alters the structure of the active site to promote inhibitor (substra te) binding, in agreement with the ordered mechanism of the enzyme. Structu ral changes remote from the active site were also found. They were interpre ted as long-range structural effects on the two domains and on the two mono mers in the time course of the ligand binding process.