Evidence for ligand-induced conformational changes in rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase.
The tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle binds NAD+ and some of its analogues in a negatively cooperative manner, whereas other NAD+ analogues bind non-cooperatively to this enzyme. Subsequent to alkylation of a fraction of the active sites of the enzyme with the fluorescent SH reagent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine, it was found that the alkylated sites bind NAD+ and NAD+ analogues with a markedly reduced affinity as compared with non-alkylated sites. It was therefore feasible to measure the fluorescence and the circular polarization of the luminescence of the enzyme-bound alkyl groups as a function of binding of NAD+ and of NAD+ analogues to the non-alkylated sites. The changes observed indicate that ligand binding to the non-alkylated sites induces changes in the fluorescence properties of the alkyl groups bound to neighbouring subunits, most likely through the protein moiety. The nature of these changes appears to depend on the structure of the coenzyme analogue. The binding of the non-cooperative binders acetyl-pyridine--adenine dinucleotide, ATP and ADP-ribose induce different conformational changes in the neighbouring vacant subunit, as monitored by the spectroscopic properties of the bound alkyl group. These results in conjunction with other data support the view that the negative cooperativity in NAD+ binding to glyceraldehyde-3-phosphate dehydrogenase results from ligand-induced conformational changes. Furthermore, these results further support the view that subtle structural changes in the coenzyme molecule determine the nature of the conformational changes induced within the enzyme tetramer.
D000818:Animals; D001665:Binding Sites; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007460:Iodoacetamide / Q000031:analogs & derivatives; D008024:Ligands; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents
D008024:Ligands; D046911:Macromolecular Substances; D009282:Naphthalenesulfonates; D013439:Sulfhydryl Reagents; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007460:Iodoacetamide