Cooperative and non-cooperative conformational effects of the coenzyme on yeast glyceraldehyde-3-phosphate dehydrogenase.

Abstract

Changes induced on addition of the cofactor, NAD, to glyceraldehyde-3-phosphate dehydrogenase from yeast have been investigated by hydrodynamic and spectroscopic techniques. As shown by light-scattering and sedimentation and diffusion studies, the tetrameric structure of the holoenzyme persists under all but strong dissociating conditions: there is thus no change in the quaternary structure on binding. On the other hand, binding is accompanied by an increase of some 4% in the sedimentation coefficient, which is too large to be explicable in terms of the increase in particle weight by bound ligand. A conformational change is also observed by optical rotatory dispersion and circular dichroism measurements. The magnitude of the Cotton effect associated with the aromatic chromophores of the protein, near 280 nm, increases, whereas the rotation in the neighbourhood of 233 nm undergoes a small diminution as the cofactor is added. It is shown that these changes are conformational in origin, rather than a consequence of NAD attachment per se. Increasing the temperature in the range 20–40° leads to a transition from non-cooperative to cooperative binding of NAD to the apoenzyme. At 40°, where NAD binding is weakly sigmoidal, the change in optical rotation is likewise sigmoidal and parallels the increase in the sedimentation coefficient. At 20° both the uptake of NAD and the structural changes are hyperbolic. In the cooperative situation the data are shown to be consistent with the two-state “concerted” model for ligand-binding and incompatible with a sequential mechanism.