II ENERGY-RICH PHOSPHATE BONDS 29 



It should be noted that succinyl-CoA may also be generated from acetoacetyl-CoA by 

 a thiotransferase reaction. Thus, a substrate phosphorylation may be effected as a result 

 of the following reactions: 



a) 2 Acetyl-CoA -^ acetoacetyl-CoA + CoA, or 



DPN^ 



b) |3-Hydroxybutyryl-CoA > acetoacetyl-CoA 



c) Acetoacetyl-CoA + succinate -^ acetoacetate + succinyl-CoA 



d) Succinyl-CoA + GDP + Pi -> GTP + CoA + succinate 



Acetyl-CoA is generated as a result of the oxidative decarboxylation of pyruvate or 

 during the oxidation of fatty acids. The following additional mechanism for substrate 

 phosphorylation occurs in those microorganisms which contain the enzymes, coenzyme 

 A-transferase and phosphotransacetylase (Lynen, 1955): 



a) Acetyl-CoA + phosphate -^ acetyl phosphate + CoA 



b) Acetyl phosphate + ADP -^ ATP + acetate 



2. Electron transport phosphorylation 



Table 3 shows that the average potential between oxygen and a pair of average 

 svibstrate hydrogens is 1.2 V; 0.25 V corresponds to approximately 12 kcal, the 

 energy content of the pyrophosphate bond of ATP. Therefore a total of four high- 

 energy phosphate bonds theoretically could be generated as a result of the oxida- 

 tion of substrates and the transport of a pair of hydrogens to oxygen through the 

 mitochondrial electron transport system. The generation of 2-3 high-energy phos- 

 phate bonds per atom of oxygen utilized (P: O ^ 3) has in fact been demonstrated 

 during the oxidation of [3-hydroxybutyrate by intact mitochondria (Lehninger, 

 1 953-1 954; Chance, 1953- 1954) and by mitochondrial fragments obtained by 

 exposing freshly prepared mitochondria to digitonin. In order to demonstrate 

 phosphorylation reactions with exogenous DPNH as substrate (Cooper and 

 Lehninger, 1956a, 1956b; Devlin and Lehninger, 1956) instead of [3-hydroxy- 

 butyrate, it is necessary to increase mitochondrial permeability to DPNH by 

 pretreatment with ice cold hypotonic KCl or sucrose solutions for a short period 

 of time. Following the restoration of isotonicity, P:0 ratios of as high as 2.6 have 

 been observed accompanying the oxidation of exogenous DPNH2 by oxygen. 

 These experiments provided the first direct proof that phosphorylations occur 

 beyond the substrate level during the transport of electrons from DPNH-, to oxygen 

 via the respiratory chain of mitochondria (Lehninger, 1953- 1954). 



It is to be recalled that four dehydrogenation reactions of the tricarboxylic acid 

 cycle and two reactions of fatty acid oxidation are localized in the mitochondria. 

 However, many of the dehydrogenation reactions which result in the generation 

 of DPNH2 occur externally to the mitochondrion. Moreover, as in the case of 

 glyceraldehyde-phosphate dehydrogenase, the dehydrogenases of the soluble 

 cytoplasm are of particular importance in the energy economy of the cell. In 

 view of the selective permeability of the mitochondrial membrane, there is some 

 question as yet as to the manner of interaction of externally generated DPNHj 

 and mitochondria DPNHj-oxidase. 



Literature p. 124 



