Structure of isocitrate dehydrogenase with isocitrate, nicotinamide adenine dinucleotide phosphate, and calcium at 2.5-A resolution: a pseudo-Michaelis ternary complex.
The structure of isocitrate dehydrogenase (IDH) with a bound complex of isocitrate, NADP+, and Ca2+ was solved at 2.5-A resolution and compared by difference mapping against previously determined enzymatic complexes. Calcium replaces magnesium in the binding of metal-substrate chelate complex, resulting in a substantially reduced turnover rate. The structure shows the following: (i) A complete, structurally ordered ternary complex (enzyme, isocitrate, NADP+, and Ca2+) is observed in the active site, with the nicotinamide ring of NADP+ exhibiting a specific salt bridge with isocitrate. The binding of the cofactor nicotinamide ring is dependent on this interaction. (ii) Isocitrate is bound by the enzyme with the same interactions as those found for the magnesium/substrate binary complex, but the entire molecule is shifted in the active site by approximately 1 A in order to accommodate the larger metal species and to interact with the nicotinamide ring. The distances from isocitrate to the bound calcium are substantially longer than those previously found with magnesium. (iii) NADP in the Escherichia coli IDH has a novel binding site and conformation as compared to previously solved dehydrogenases. (iv) The orientation and interactions of the nicotinamide ring with the substrate are consistent with the stereospecificity of the enzyme-catalyzed reaction.
Stoddard
B L
BL
Department of Molecular and Cellular Biology, University of California, Berkeley 94720.
Dean
A
A
Koshland
D E
DE
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D007523:Isocitrates / Q000737:chemistry*; D046911:Macromolecular Substances; D009249:NADP / Q000737:chemistry*; D014961:X-Ray Diffraction
D007523:Isocitrates; D046911:Macromolecular Substances; D009249:NADP; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; D002118:Calcium