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...

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Bibliographic Details
Published in:Biochemistry (Easton) 1989-03, Vol.28 (5), p.2058-2065
Main Authors: Ehrlich, Robert S, Colman, Roberta F
Format: Article
Language:English
Subjects:
550601 - Medicine- Unsealed Radionuclides in Diagnostics
ALKALINE EARTH ISOTOPES
ALKALINE EARTH METAL COMPOUNDS
AMP
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
Bicarbonates - metabolism
Binding Sites
BIOCHEMISTRY
BIOLOGICAL PATHWAYS
BODY
Cadmium - metabolism
CADMIUM 113
CADMIUM ISOTOPES
CARBOXYLIC ACID SALTS
CARDIOVASCULAR SYSTEM
CATIONS
CHARGED PARTICLES
Chemical Phenomena
CHEMICAL SHIFT
CHEMISTRY
Chemistry, Physical
CITRATES
COENZYMES
Coenzymes - metabolism
DOMESTIC ANIMALS
ELECTRON SPIN RESONANCE
ENZYMES
EVEN-ODD NUCLEI
HEART
HEAVY WATER
HEMIACETAL DEHYDROGENASES
HYDROGEN COMPOUNDS
INTERMEDIATE MASS NUCLEI
IONS
Isocitrate Dehydrogenase - metabolism
ISOMERIC TRANSITION ISOTOPES
ISOTOPES
Ketoglutaric Acids
LIGHT NUCLEI
Magnesium - metabolism
MAGNESIUM 25
MAGNESIUM COMPOUNDS
MAGNESIUM ISOTOPES
MAGNESIUM SULFATES
MAGNETIC RESONANCE
Magnetic Resonance Spectroscopy
MAMMALS
Manganese - metabolism
MANGANESE COMPOUNDS
Metals - metabolism
Myocardium - enzymology
NADP
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
RADIOISOTOPES
RADIOLOGY AND NUCLEAR MEDICINE
RESONANCE
STABLE ISOTOPES
SULFATES
SULFUR COMPOUNDS
SWINE
TRANSITION ELEMENT COMPOUNDS
VERTEBRATES
VISCOSITY
WATER
YEARS LIVING RADIOISOTOPES
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00431a014