Multinuclear NMR studies of the divalent metal binding site of NADP-dependent isocitrate dehydrogenase from pig heart.
The metal activator site of NADP-dependent isocitrate dehydrogenase from pig heart has been probed by using 113Cd and 25Mg NMR as well as manganese paramagnetic relaxation of nuclei in the fast-exchanging ligands alpha-ketoglutarate and adenosine 2'-monophosphate. Cadmium NMR shows that cadmium, bound to the enzyme in the presence of isocitrate, has a resonance at 9 ppm relative to cadmium perchlorate, while the free Cd-isocitrate complex has a resonance at -23 ppm. Comparison with model compounds and previously studied proteins indicates that cadmium is coordinated with six oxygen ligands. Measurements as a function of cadmium concentration give a dissociation constant of 66 microM and a dissociation rate constant of 1.5 X 10(4) s-1 at pH 7.0. 25Mg NMR demonstrates that the line width of the magnesium resonance is increased upon binding to isocitrate dehydrogenase. A further increase in line width is observed upon addition of isocitrate. Measurement of line widths as a function of temperature reveals that in the binary complex between magnesium and enzyme, exchange is the major contributor to broadening while in the ternary complex containing isocitrate, the intrinsic relaxation in the bound state is also important, suggesting an increase in the dissociation rate constant for magnesium from the ternary complex. Paramagnetic relaxation studies of nuclei of alpha-ketoglutarate, bicarbonate, and adenosine 2'-monophosphate locate the divalent metal within the active site. The results with adenosine 2'-monophosphate show that atoms in the adenosine moiety of the coenzyme are at least 8 A from the metal site.(ABSTRACT TRUNCATED AT 250 WORDS)
Ehrlich
R S
RS
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003067:Coenzymes / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007554:Isotopes; D007656:Ketoglutaric Acids; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D013552:Swine
D001639:Bicarbonates; D003067:Coenzymes; D007554:Isotopes; D007656:Ketoglutaric Acids; D008670:Metals; D002104:Cadmium; D008345:Manganese; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D008274:Magnesium