Co-operative binding of nicotinamide-adenine dinucleotide to yeast glyceraldehyde-3-phosphate dehydrogenase

摘要: Abstract Binding studies using equilibrium dialysis are shown to confirm earlier evidence, obtained by spectrophotometric titration, that yeast glyceraldehyde-3-phosphate dehydrogenase binds four molecules of nicotinamide-adenine dinucleotide in a co-operative manner at pH 8.5 and 40 °C. Kinetic measurements with the aid more sensitive temperature-jump stopped-flow equipment have led detection five distinct relaxation processes under these conditions. Temperature difference spectra fully saturated complex dependence individual amplitudes on observation wavelength reveal very rapid initial absorbance change can be identified intrinsic temperature chromophore spectrum. The slowest process unequivocally assigned subform enzyme present crystalline preparation. remaining show same concentration as found earlier. failure find additional spite greatly improved instrumental sensitivity confirms basic degeneracy underlying mechanism. data rule out simplest sequential mechanism involving consecutive binding steps (Koshland, Nemethy & Filmer, 1966) being too simple. They fitted quantitatively other simple alternative, concerted (Monod, Wyman Changeux, 1965) leading evaluation all rate constants. kinetic discussed reference independent evidence consistent