Abstract
A general model of co-operativity, which combines the concepts of preferential ligand binding, molecular constraints, and nearest-neighbor constraints, is used to analyze quantitatively the co-operative properties of yeast and rabbit muscle glyceraldehyde 3-phosphate dehydrogenase.
The model is found to fit equilibrium dialysis and spectrophotometric measurements of NAD binding, measurements of the NAD dependence of proteolytic inactivation, and measurements of the NAD dependence of enzyme activity with consistent sets of parameter values. It is concluded that this enzyme has two quaternary conformations and that the change from the quaternary conformation of the apoenzyme to the quaternary conformation of the holoenzyme occurs roughly when the first NAD molecule is bound; this change is responsible for the fact that in the yeast enzyme the second NAD molecule has a higher binding affinity than the first and that the resistance of the yeast and rabbit muscle enzymes to proteolysis increases sharply when the first one or two NAD molecules bind. In addition, within the quaternary conformation of the holoenzyme, NAD binding at different sites is coupled by a tetrahedral (rather than square or dimer) pattern of interactions; these interactions are responsible for negative co-operativity in glyceraldehyde 3-phosphate dehydrogenase. Pairwise asymmetry (induced or pre-existing) is excluded as the basis for the co-operativity observed in NAD binding.
Analysis of the temperature-dependence of model parameters shows that the change from the apo to the holo quaternary conformation is a strongly endothermic process, accompanied by an increase in entropy, whereas binding of NAD to the enzyme in the holo quaternary conformation is a strongly exothermic process, accompanied by a decrease in entropy. The enthalpy and entropy associated with nearest-neighbor constraints change with temperature, suggesting that an effector is present which influences the strength of these constraints.
When the enzyme is in the quaternary conformation characteristic of the apoenzyme, it is essentially inactive. Half-of-the-sites reactivity can be explained by supposing that modification of the cysteine-149 sulfhydryl groups stabilizes an inactive quaternary conformation of the enzyme. In contrast to other proposed explanations of half-of-the-sites reactivity, the explanation given here does not assume molecular asymmetry or direct interactions between sites, and is therefore also applicable to enzymes which show no negative co-operativity. Numerous phenomena related to half-of-the-sites reactivity are also shown to be consistent with the present model.
Whereas in the past different models have often been used to explain different properties of glyceraldehyde 3-phosphate dehydrogenase the present model consistently explains a very wide variety of data.