I. LIPOIC ACID 579 



mide pyrophosphate" (LTPP) were active in the oxidative decarboxylation 

 of pyruvate. These materials were obtained in a reaction mixture of a- 

 lipoic acid or its acid chloride with thiamine and thiamine pyrophosphate, 

 respectively. The biologically active products possessed Rf values identical 

 with those of two of the forms present in natural materials, lioth ga\e nega- 

 tive thiochrome tests and positive azo tests, and the authors suggested that 

 the carboxyl group of lipoic acid was conjugated with the pyrimidine amino 

 group of thiamine. The test system used was a mutant strain of Escher- 

 ichia coli that would not respond to either vitamin, alone or mixed together, 

 but which grew luxuriantly on LT or LTPP. Soluble enzyme preparations 

 from the mutant catalyzed the anaerobic dismutation of pyruvate or the 

 oxidation of a-ketoglutarate only when LTPP was supplied (LT was inac- 

 tive in the latter system). These reactions thus reveal a definite enzymatic 

 role for hpoic acid in oxidation of a-keto acids, as a molecular conjugate 

 with thiamine, and point to LTPP as the actual coenzyme. The authors-" 

 proposed, partly on the basis of previous knowledge of the mechanism of 

 pyruvate oxidation in E. coli,^^- ^^ that LTPP participates according to re- 

 action 1: 



Pyruvate (a-Ketoglutarate) + LTPP -f DPN+ -^ 



Acetyl LTPP (succinyl LTPP) -f CO-. + DPNH + H+ (1) 



In the presence of coenzyme A, the acyl group could then be transferred 

 as follows : 



Acetyl LTPP (succinyl LTPP) -1- CoA -^ 



Acetyl CoA (succinyl CoA) + LTPP (2) 



This scheme thus provided a plausible order of participation for LTPP 

 and CoA, although it did not per se clarify the question of Avhether LTPP 

 is needed for decarboxylation or dehydrogenation. Ex-idence bearing on 

 this is provided in other studies on E. coli and S. faecalis,^--^^ as follows: 



I. The complete system for the pyruvate -^ acetate + CO2 conversion 

 in E. coli requires DPT, diphosphopyridinenucleotide (DPN), CoA, Mg++, 

 and lipoic acid (presumably as LTPP). 



" L. J. Reed and B. G. De Busk, J. Am. Chem. Soc. 74, 3964 (1952). 



28 L. J. Reed and B. G. De Busk, /. Biol. Chem. 199, S73 (1952). 



2" L. J. Reed and B. G. De Busk, /. Biol. Chem. 199, 881 (1952). 



3" S. Korkes, J. R. Stern, I. C. Gunsalus, and S. Ochoa, Nature 166, 439 (1950). 



" S. Korkes, A. del Campillo, I. C. Gunsalus, and S. Ochoa, J. Biol. Chem-. 193. 721 



(1951). 

 '2 D. J. O'Kane, J. Bacteriol. 60, 449 (1950). 

 " M. I.Dolin and I. C. Gunsalus, ./. Bacteriol. 62, 199 (1951). 

 " M. I. Dolin and I. C. Gunsalu.s, Federation Proc. 11, 203 (1952). 

 " L C. Gunsalus, /. Cellular Comp. Physiol. Suppl. 1, 41, 113 (1953). 

 '« B. G. De Busk and L. J. Reed, Federation Proc. 12, 193 (1953). 



