IV. mOCllEMlCAL SYSTEMS 429 



cordance with the observation of Lipmann,-'' confirmed by Lipton and 

 Elvohjein^" and by Westenbrink, that thiamine is witliout any eCt'ect when 

 it is added to the yeast suspension some minutes after the addition of co- 

 carboxykise, for then the cocarboxylase is destroyed l)y the phosphatase. 

 Westenbrink ct al.^^ demonstrated the high enzymic activity of this phos- 

 phatase: in 10 seconds it dephosphorylatetl a large part of the cocarboxy- 

 lase. The objection of Lipton and Elvehjem'" that phosphate, the other 

 product of the splitting of thiamine pyrophosphate, did not intiibit the 

 phosphatase action on cocarboxylase could be answered by the assumption 

 that in this case there is no shift of the equilibrium according to the law 

 of mass action, but a competitive inhibition. It appeared later that ani- 

 mal phosphatases are not inhibited by thiamine but by phosphate ions 

 (Westenbrink et aU^). 



The synthesis of carboxylase by bringing together alkali-washed yeast, 

 magnesium, and thiamine pyrophosphate was thoroughly studied by 

 Parv^.^^ Even when a large amount of thiamine is added, a great deal of 

 the thiamine pyrophosphate is hydrolyzed before it is bound to the protein 

 and becomes resistant to the phosphatase. Therefore, only part of the thia- 

 mine pyrophosphate is resynthesized to carboxylase ; at most about 25 %. 



In all respects, the effect of washing at pH 6.2 or 5.6, the resynthesis at 

 pH 6.8, the dissociation at pH 7.8, and the activity per microgram of thia- 

 mine pyrophosphate left the resynthesized carboxylase equally as stable 

 as the original holoferment. Only one difference was found. The maximum 

 acti\dty, measured by the production of carbon dioxide from pyruvate, is 

 about pH 5.7 for the native carboxylase and pH 6.2 for a mixture of alkali- 

 washed 3^east + magnesium ions + thiamine pyrophosphate. This differ- 

 ence is easily ex-plained, for the optimum pH for the resynthesis is 6.8. In 

 the mixture of alkali-washed yeast + coferment, two reactions are going 

 on simultaneously; the synthesis of the holoenzyme and the action of this 

 enzyme on pyruvate. So pH 6.2 is a compromise between pH 5.7 and 6.8. 



Lipton and Elvehjem gave another explanation for the stimulation of the 

 formation of carboxylase by thiamine; they postulated that a substance in 

 yeast, probably a protein other than the enzyme protein, adsorbs the co- 

 carboxylase and in this way inhibits the formation of the holoenzyme car- 

 bo.xylase. The addition of excess thiamine saturates this material and thus 

 permits the adsorption of the thiamine pyrophosphate on the apoenzyme. 



Most of their experiments could be interpreted just as well by the supposi- 



" F. Lipmann, Enzymologia 7, 142 (1939). 



'» M. A. Lipton and C. A. Elvehjem, /. Biol. Chem. 136, 637 (1940). 



'' H. G. K. Westenbrink, D. A. van Dorp, and M. Gruber, Rcc. trav. chim. 60, 185 



(1941). 

 " H. G.K. Westenbrink, E. P. S. Parvd, and J. Goudsmit, Enzijinologia 11, 26 (1943). 

 " E. P. S. Parvd, Thesis, Scheltema en Holkema, .\mstcrdam, 1945. 



