Structural basis for a change in substrate specificity: Crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of i socitrate and has negligible activity toward other (R)-malate-type substrat es. The S113E mutant of IDH significantly improves its ability to utilize i sopropylmalate as a substrate and switches the substrate specificity (k(cat )/K-M) from isocitrate to isopropylmalate. To understand the structural bas is for this switch in substrate specificity, we have determined the crystal . structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg2to 2.0 Angstrom resolution. On the basis of a comparison with previously de termined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates. The S113E complex exhibits a lterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the acti ve site. In addition, the angle of the hinge that relates the two domains w as altered in this structure, which suggests that the S113E substitution an d the binding of substrates act together to promote catalysis of isopropylm alate. Ligand binding results in reorientation of the active site helix tha t contains residues 113 through 116. E113 exhibits new interactions, includ ing van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, which in turn forms a hydrogen bond with NADP. I n addition, the loop and helix regions that bind NADP are altered, as is th e loop that connects the NADP binding region to the active site helix, chan ging the relationship between substrates and enzyme. In combination, these interactions appear to provide the basis for the switch in substrate specif icity.