The Nature and Diversity of Catalytic Proteins 81 



soon becomes stable; the amounts of pyruvate kinase and lactate dehy- 

 drogenase present are sufficient to catalyze rapid attainment of 

 equilibrium. At this stage a comparatively large amount of pyruvate 

 kinase is added. If any appreciable pbosphoryl enzyme is formed from 

 reaction of the enzyme with ATP, the resultant ADP formation would 

 give an immediate drop in absorb ancy at 340 m/x; none such is de- 

 tected. The continued slow decline represents a side reaction catalyzed 

 by the very large excess of pyruvate kinase (26 ) . The sensitivity of the 

 system to ADP is indicated by the subsequent addition of an amount 

 of ADP equivalent to about one-half of the concentration of active 

 sites of the large amount of pyruvate kinase added. The results 

 demonstrate clearly the lack of formation of phosphoryl enzyme even 

 in the presence of all reactants and under conditions favorable to 

 rapid net reaction. 



(1) ATP 



R0 18 H + HX 



(2) ATP -^^__ ^^^r ADP + P/-0 18 



R0 18 H + HX -^ ^^^ R-X 



FIG. 4. The two general patterns for syntheses coupled to ATP cleavage. 



The lack of phosphoryl enzyme formation as shown by Fig. 3 could 

 result from the existence of the pyruvate kinase as a stable phosphoryl 

 enzyme. If any such phosphoryl group participated in the catalysis, 

 it would be rapidly replaced by the phosphoryl group from ATP. 

 This possibility is readily checked by the use of radioactive P 32 -labeled 

 ATP. Such experiments rule out the formation of a stable phosphoryl 

 enzyme as a catalytic intermediate (28 ) . 



Other phosphorylation enzymes which have been of interest are 

 those which catalyze syntheses coupled to cleavage of ATP. The two 

 patterns of ATP cleavage which have been found with this group of 

 enzymes are indicated schematically in Fig. 4. Examples of this im- 

 portant type of reaction are the synthesis of acyl coenzyme A deriva- 

 tives from fatty acids and coenzyme A, of glutamine from glutamate 

 and ammonia, and of glutathione from its component amino acids. 

 Studies with the stable isotope 1S have shown that in such syntheses 

 substrates and not water furnish the oxygen necessarj for ATP 



