Alpha-ketoglutaric dehydrogenase. V. Guanosine diphosphate in coupled phosphorylation.

SUMMARY The phosphorylation of ADP coupled to the breakdown of succinyl CoA in animal tissues is a two-step reaction involving two different enzymes and GDP. The product of the first reaction catalyzed by the GDP phos- phorylation enzyme is GTP. The second step, transphosphorylation from GTP to ADP, is catalyzed by NDP kinase. GTP has been isolated from the reaction and identified. For the synthesis of succinyl CoA from ATP both enzymes and GDP are necessary. However, only the phosphoryl- ation enzyme is necessary for succinyl CoA synthesis when GTP is used instead of ATP. IDP can substitute for GDP in the system but less ef- fectively. 4 The DPNH oxidizing activity (28) of such a preparation was only 10 to 20 per cent of that observed by Huennekens et al. Before this step the activity was 2- to 3-fold higher and in agreement with our earlier calculations that the “diaphorase” contamination amounted to 1 to 2 per cent of the total protein (10). It may be appropriate to emphasize that Huennekens et al. have compared the DPNH oxidiz- ing and KG dehydrogenase (DPN reduction) activities of the enzyme preparation at two different pH values, the former at pH 10 and the latter at pH 7.4. The observed activity, if assumed to be due to “diaphorase,” would amount to less than 1 per cent of the total protein in the preparation.