Spectroscopic evidence for ligand-induced conformational change in NADP+:isocitrate dehydrogenase.
Conformational changes induced by binding of ligands to cytosolic NADP(+)-specific isocitrate dehydrogenase from lactating bovine mammary gland were assessed using circular dichroism and fluorescence techniques. The secondary structure of isocitrate dehydrogenase, as monitored by CD spectra in the far-UV region, is unaltered by enzyme-ligand interactions; in contrast, dramatic changes occur in the near-UV region (270-290 nm) assigned to tyrosine and/or solvent-exposed tryptophan residues. Both the coenzyme analog, 2'-phosphoadenosine 5'-diphosphoribose, and NADPH have an effect on the CD spectrum which is opposite to that produced by metal complexes of either isocitrate or citrate. A CD band at 292 nm assigned to approximately 2 tryptophan residues in a hydrophobic environment is unchanged by binding of substrate or coenzyme. Approximately 30% of the intrinsic fluorescence of isocitrate dehydrogenase, corresponding to approximately 2 tryptophan residues, is not quenched by acrylamide in the absence of 6.3 M guanidine hydrochloride and remains unquenched in the enzyme-substrate complex. The constancy in the proportion of buried and exposed tryptophan residues implicates tyrosine in the observed near-UV CD spectral changes. Since binding of ligands does not influence quaternary structure (Seery, V.L., and Farrell, H. M., Jr. (1989) Arch. Biochem. Biophys. 274, 453-462), activation of isocitrate dehydrogenase may be related to a substrate-induced conformational transition.
United States Department of Agriculture, Eastern Regional Research Center, Philadelphia, Pennsylvania 19118.
V L
VL
Seery
H M
HM
Farrell
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008321:Mammary Glands, Animal / Q000201:enzymology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D008024:Ligands; D007521:Isocitrate Dehydrogenase