Evidence for Ligand‐Induced Conformational Changes in Rabbit‐Muscle Glyceraldehyde‐3‐Phosphate Dehydrogenase

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Abstract
The tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle binds NAD+ and some of its analogs in a negatively cooperative manner, whereas other NAD+ analogs 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, the alkylated sites bind NAD+ and NAD+ analogs with a markedly reduced affinity as compared with non-alkylated sites. The fluorescence and the circular polarization of the luminescence of the enzyme-bound alkyl groups were measured as a function of binding of NAD+ and of NAD+ analogs 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 neighboring subunits, most likely through the protein moiety. The nature of these changes appears to depend on the structure of the coenzyme analog. The binding of the non-cooperative binders acetyl-pyridine - adenine dinucleotide, ATP and ADP-ribose induce different conformational changes in the neighboring vacant subunit, as monitored by the spectroscopic properties of the bound alkyl group. The negative cooperativity in NAD+ binding to glyceraldehyde-3-phosphate dehydrogenase probably results from ligand-induced conformational changes. The view that subtle structural changes in the coenzyme molecule determine the nature of the conformational changes induced within the enzyme tetramer was supported.