28 INTERMEDIARY METABOLISM AND GROWTH I 



of "reduced'' inuscle glyceraldehyde-phosphate dehydrogenase contains three 

 moles of DPN"^, and two moles of glutathione. The glutathione may be liberated 

 from the enzyme by tryptic digestion. If prior to tryptic digestion, the enzyme is 

 treated with the sulfhydryl binding agent, iodoacetate, no free glutathione is 

 formed. Iodoacetate is also an inhibitor of this enzyme. 



As shown in reaction b), the reaction of 1,3-diphosphoglycerate with ADP 

 results in the formation of ATP : 



b) 1,3-Diphosphoglycerate + ADP >• 3-phosphoglycerate + ATP 



The DPNH, generated by reaction a) may be reoxidized in dismutation reactions 

 or by DPNH2 oxidase. When one mole of DPNHj is reoxidized by mitochondrial 

 DPNH oxidase enzymes, three additional moles of ATP are generated by electron 

 transport phosphorylation (see below). 



(b) Phosphoenol pyruvate 



The second substrate phosphorylation of the glycolytic sequence takes place at 

 the level of phosphoenolpyruvate. The dehydration of 2-phosphoglycerate, 

 results in the formation of phosphoenol pyruvate in which part of the energy of 

 the molecule is distributed in the labile phosphate linkage (Fig. 12). 



In the event that phosphoenol pyruvate next reacts with ADP, ATP and pyru- 

 vate are formed. 



Fig. 12. Phosphoenolpyruvate and substrate phosphorylation. 



{c) Phosphorylation coupled to the oxidation of oi-ketoglutarate 



A substrate phosphorylation occurs during the oxidative decarboxylation of 

 a-ketoglutaric acid to succinic acid (Reactions a-c). 



a) a-Ketoglutarate + DPN" + CoASH -^ succinyl-CoA + DPNH ^ H* + CO2 



Mg- 



b) Succinyl-CoA + GDP + Pi -> succinate + CoASH + GTP 



Mg"" 



c) GTP + ADP > GDP + ATP 



The enzymes of this phosphorylation system have been purified from beef 

 heart mitochondria (Sanadi et al., 1956). GDP or IDP rather than ADP is re- 

 quired in the phosphorylation step b). ATP is then generated from the GTP by 

 a nucleotide transphosphorylation reaction (step c). 



