IV. BIOCHEMICAL SYSTEMS 435 



l)hosphate disappears during the i)ro('ess, and an equivalent amount of 

 adenosine polyphosphate is formed. Thus the oxidation generates an energy- 

 rich phosphate bond. The primary oxidation product of the pyruvic acid 

 Ix'haves like acetyl phosphate. Synthetic acetyl phosphate is able to trans- 

 fer its high-energy phosphate bond to adenylic acid. From these facts 

 Lipmann formulated the oxidation process. 



('II.COCOOII + II:a>04 + 0-2^ CH3COOPO3H2 + CO2 -I- II,>02 



Afterwards Lipmann^*' calculated the bond energy of acetyl phosphate. As 

 it is some 3 kcal. higher than the average energy of 12 kcal. of the energy- 

 rich bond, it will amount to around 15 kcal. The need of inorganic phos- 

 phate for pyru\'ate oxidation in brain also was demonstrated (Banga 

 et al.^^- '^). However, Ochoa et al.^^ could find no indication that acetyl 

 phosphate is an intermediate in the oxidation of pyruvic acid by brain or 

 by muscle. Thus there are many carboxjiases that require inorganic phos- 

 phate for their action. However, there are exceptions to this rule: Stumpf--' 

 studied the pyruvic acid oxidation by Proteus vulgaris, which contains an 

 enzyme that, in the presence of thiamine pyrophosphate and magnesium, 

 specifically catalyzes the oxidation decarboxylation of pyruvic acid to 

 acetic acid and carbon dioxide with liberation of considerable free energy: 



CH3COCOOH + hO'i -^ CH3COOH + CO2 



No disappearance of pyruvic acid could be observed under anaerobic 

 conditions and no carbon dioxide was produced. This enzj-^me from Proteus 

 vulgaris is equally active in the absence, as in the presence, of inorganic 

 phosphate. In several ways Stumpf could demonstrate that the assumption 

 that trace amounts of phosphate should react with pyruvic acid to form 

 acetyl phosphate, which could then act as a catalyst, was not in accordance 

 with the facts. Similarly the pyruvic oxidase of Escherichia coli (StilF^) does 

 not require the presence of inorganic phosphate. Ochoa^^ thoroughly studied 

 the coupling of phosphorylation with oxidation of pyruvic acid in the brain 

 and the oxidation of the a-ketoglutaric acid^" by a preparation from the 

 heart of the cat. He confirms the observation that inorganic phosphate 

 and adenylic acid or adenine triphosphate are required for the oxidation. 

 The rate of oxygen uptake by the dehydrogenase from cat heart depended 

 upon the concentration of inorganic phosphate. However, it is not certain 

 that this preparation contained a thiamine j^yrophosphate enzyme. 



Kalnitzky and Werkman,^- working with extracts of Escherichia coli, 

 found that inorganic phosphate was essential for the activity of its pyruvic 



"sS. Ochoa. R. .\. Peters, and L. A. Stocken, Xaliirc 144, 750 (1939). 

 " S. Ochoa, /. Biol. Chem. 138, 751 (1941). 

 '« S. Ochoa, J. Biol. Chem. 155, 87 (1944). 



