The reaction between NAD + and rabbit-muscle glyceraldehydephosphate dehydrogenase

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1. 1. The A 280 mμ : A 260 mμ ratio of NAD +-free rabbit-muscle glyceraldehydephosphate dehydrogenase was found to be 2.00–2.03. 2. 2. Ultracentrifugation showed that 4 moles of NAD + may be bound to the enzyme per mole (mol. wt. 145 000). The first two molecules are bound stoicheiometrically within the experimental error ( K D < 0.05 μM), while the third and fourth molecules are bound with dissociaton constants of 4 and 35 μM, respectively. 3. 3. The first three molecules of NAD + bound to the enzyme cause the formation of a band at 360 mμ of about equal intensity for each molecule. The fourth molecule causes little further increase of absorption at 360 mμ. 4. 4. A plot of the rate of reduction of NAD + by glyceraldehyde in the presence of arsenate against the NAD + concentration shows a sharp break in the curve at 4 moles NAd + per mole enzyme. 5. 5. Stopped-flow experiments showed that when up to 1 mole of NAD + is added to the enzyme, maximum absorbance increase at 360 mμ is reached within 3 msec. This corresponds to a second-order reaction constant of more than 10 8 M −1·sec −1. With 2 moles of NAD + per mole of enzyme, 81% of the reaction is over in 3 msec, and with 3 or more moles NAD + about 75%. The reaction requires about 1 sec for completion. 6. 6. Prior treatment with NAD + speeds the change of absorbance obtained with subsequent additions of NAD +. The final value obtained, however, is unaltered. 7. 7. For the muscle enzyme under our experimental conditions, a model in which the binding of 1 NAD + molecule to one protomer affects the conformation of a second protomer, either before or after binding with NAD +, appear more appropriate than the allosteric model, which requires that the binding of any one ligand molecule is intrinsically independent of the binding of any other.