Local conformational changes induced by successive nicotinamide adenine dinucleotide binding to dissociable tetrameric D-glyceraldehyde-3-phosphate dehydrogenase. Quantitative analysis of a two-step dissociation process.
Covalent binding of FITC up to 2 mol/mol of tetrameric enzyme does not affect the enzymatic activity and dissociation properties of pig muscle D-glyceraldehyde-3-phosphate dehydrogenase (GAPD). The binding of NAD to dehydrogenase-FITC complex partially reverts the quenching caused by the binding of dye to apo-GAPD. This phenomenon, as well as the formation of a characteristic absorption difference spectrum caused by the binding of NAD, makes it possible to follow the NAD-induced local conformational changes near the dye-binding region. The time course of NAD-induced spectral changes shows biphasic kinetics: a burst and a slow phase. The amplitude of burst phase as a function of NAD equivalents has sigmoidal shape due to the cooperative interaction between subunits. The same conclusion could be drawn from fluorescence anisotropy measurements. In the presence of excess NAD a slow conformational change can be detected, the amplitude of which is a function of NAD concentration. This phenomenon can be attributed to the binding of further NAD molecules to the holoenzyme. The slow phase follows first-order kinetics, and the rate constant depends on enzyme concentration. The specific fluorescence intensity and the fluorescence anisotropy of fluorescent dye labeled apo-GAPD and GAPD saturated with NAD are also dependent on enzyme concentration. We suggest that NAD binding induces major changes in the steric structure of tetrameric enzyme without influencing remarkably the interacting forces between the contact surfaces of subunits. Data are quantitatively interpreted in terms of a two-step dissociation model.
D000818:Animals; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D005454:Fluorescence Polarization; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013552:Swine; D013861:Thiocyanates / Q000378:metabolism
D005452:Fluoresceins; D046911:Macromolecular Substances; D013861:Thiocyanates; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D016650:Fluorescein-5-isothiocyanate