Molecular basis of negative co-operativity in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

The interactions of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with NAD⁺ and with its fluorescent derivative 1, N⁶-etheno-adenine dinucleotide were investigated using a variety of spectroscopic methods. These techniques included: difference spectroscopy, circular dichroism, fluorescence and circular polarized luminescence. It was found that the greatest structural change in the protein tetramer occurs upon binding of the first mole of coenzyme. We have also demonstrated that progressive structural changes occur at the adenine subsite in the NAD⁺ binding site as a function of coenzyme saturation. These conformational changes are probably responsible for the progressive decrease in the affinity towards the coenzyme. It was also found that every NAD⁺ molecule induces the same conformational change of the nicotinamide subsite. These results offer a molecular explanation for the negative co-operativity in the binding of the coenzyme, without a change in the catalytic power of the NAD⁺ site as a function of coenzyme saturation. These results also offer a new explanation for the fact that enzyme exhibits half-of-the-sites reactivity towards certain ligands and full-site reactivity towards others. It is suggested that those ligands interacting at the adenine subsite of the NAD⁺ binding site induce the half-of-the-sites reactivity.Our results support the view that both the negative co-operativity in coenzyme binding and half-of-the-sites reactivity are due to ligand-induced conformational changes on an a priori symmetric glyceraldehyde-3-phosphate dehydrogenase molecule.