Mechanism of negative cooperativity in glyceraldehyde-3-phosphate dehydrogenase deduced from ligand competition experiments.

Abstract

It is shown that the modulation in the negative cooperativity of ligand binding by another, competing ligand that binds noncooperatively is accounted for exclusively by the ligand-induced sequential model. It is therefore suggested that whenever such a phenomenon is observed it argues strongly in favor of the sequential model. The advantages and limitations of this approach are evaluated. The binding of the coenzymes NAD+ and nicotinamide-1-N6-ethenoadenine dinucleotide to rabbit muscle apo-glyceraldehydephosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating; EC 1.2.1.12] exhibits strong negative cooperativity, whereas acetylpyridine adenine dinucleotide, ATP, and ADPribose bind noncooperatively to the NAD+ sites. The strong negative cooperativity in coenzyme binding was found to be abolished in the presence of acetylpyridine adenine dinucleotide and strongly weakened by ATP, ADP, and AMP, but was not affected by addition of ADP-ribose. These findings demonstrate that the negative cooperativity in coenzyme binding to this enzyme results from sequential conformational changes and exclude the pre-existent asymmetry model as a possible explanation. These results also support the view that the structure of the pyridine moiety of the coenzyme analogs plays a role in orienting the adenine moiety at the adenine subsite, therefore affecting the cooperativity in the binding of the coenzyme analog which is mediated through the adenine subsites.

Kinetic and X-Ray Structural Evidence for Negative Cooperativity in Substrate Binding to Nicotinate Mononucleotide Adenylyltransferase (NMAT) from Bacillus anthracis

“…From NMR studies it was concluded that the allosteric mechanism for negative cooperativity is based on an increase in the energy required for the second subunit to reach the bound state, which is a similar mechanism described for GAPDH 10; 33. Both GAPDH and GCT have been shown to retain their apo conformations in the subunits not containing a bound ligand, providing evidence that the negative cooperativity in these systems is not derived from changes in interactions between subunits that are transmitted to the unoccupied binding sites 10; 31; 33.…”

“…The Koshland-Nemethy-Filmer (KNF) model31, as opposed to the Monod-Wyman-Changeux (MWC) model32, can be used to describe an enzyme with negative cooperativity associated with ligand binding. The KNF model assumes that the apo-protein is symmetric and has binding sites with equal affinities, but as ligands sequentially bind, the protein loses its symmetry and the relative affinities decrease producing negative cooperativity 31; 33. The application of the general sequential model for a dimeric protein, proposed by Koshland and Neet34, to the interaction of substrates and products with NMAT is illustrated in Figure 9.…”

“…For example, four well-studied proteins displaying negative cooperativity as described by the KNF model include: thymidylate synthase, the aspartate chemoreceptor, GCT, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 10; 33; 35; 36. In the case of thymidylate synthase, binding of the cofactor 5,10-methylene tetrahydrofolate to one monomer causes a conformational change in the second monomer that prevents cofactor binding 35.…”

Synthesis and Characterization of Stacked and Quenched Uridine Nucleotide Fluorophores

“…Kinetic manipulations can then provide information regarding bindng affinity, nunber of binding sites, and nature of cooperativity, if any. This was, in fact, essentially done for glycerldehyde-3-phosphate dehydrogenase from rabbit muscle with etheno-NAD as the probe (10). Folding studies can also be facilitated by using these probes to monitor the generation of ligand binding site during the folding process.…”

“…This stacked and quenched fluorophore was brilliantly used to establish negative co-operativity for glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle for the binding of the tetrameric apoenzyme to the coenzyme. The conformational transition of etheno-NAD from folded to stretched conformation as reflected by its enhanced fluorescence on interaction with the target protein was the monitoring parameter for this purpose (10). Although stacked fluorophore with quenched fluorescence can be of immense use in protein-ligand binding studies, as is exemplified by etheno-NAD, it is surprising to note that no deliberate effort has so far been made to design such compounds taking advantage of the potential aromatic interaction between the attached fluorophore and a suitable moiety of the desired biomolecule.…”