CHAPTER IV 



Chemical Pathways of Protein Biosynthesis 



A. ENERGY REQUIREMENT 



For many years, it was assumed that the synthesis of protein is brought 

 about by proteolytic enzymes. An enzyme, acting as a true catalyst, should 

 be able to promote a reaction in both directions. Since a mixture of pro- 

 teolytic enzymes split protein into smaller peptides and finally into amino 

 acids in vitro, it seemed reasonable to assume that the same agents are 

 responsible for amino acid condensation into proteins in vivo. The con- 

 ditions within the living cell were supposed to be such that the hydrolytic 

 process was reversed. Plastein formation in concentrated protein hydro- 

 lysate and the formation of relatively insoluble peptides under the action of 

 proteolytic enzymes, provided some support for this theory. However, it 

 was untenable on thermodynamical grounds. The standard free energy of 

 hydrolysis of a dipeptide is about —3 kcal. The figure becomes less 

 negative when the peptide chain becomes longer but it is still about —1 

 kcal for a soluble polypeptide of 'infinite length' (Borsook and Huffman, 

 1945; Linderstrom-Lang, 1949, 1952). The concentration of the amino 

 acids in the cell is low; the amount of a dipeptide in equilibrium with the 

 corresponding pair of amino acids must be very small indeed (Linder- 

 strom-Lang, 1949; Borsook, 1954), and for each further condensation the 

 equilibrium concentration will rapidly decrease and become vanishingly 

 small. These considerations led to the prediction that the condensation of 

 amino acids into elementary peptides and into polypeptides will not pro- 

 ceed to any appreciable extent unless it is driven by some process making 

 energy available for the reaction (Lipmann, 1941 ; Linderstrom-Lang, 

 1949, 1952). 



Borsook and Dunoff (1940) were the first to check this prediction ex- 

 perimentally in the case of hippuric acid formation ; they determined the 

 equilibrium constant of hippuric acid hydrolysis, and were able to show 

 that the amount of hippuric acid actually synthesized in vitro by kidney or 

 liver slices from various animals is 60-75 times greater than the amount 

 corresponding to the thermodynamic equilibrium. Coupling with an 

 energy-yielding process was also indicated by the fact that inhibition of 

 respiration either by cyanide or by oxygen deprivation completely in- 

 hibited the synthesis. Lipmann (1945) later showed that the acetylation of 



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