VOL. 4 (1950) DPN AND GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE 



165 



EQUILIBRIUM CONSTANTS 



The equilibria of reactions with free DPN and substrate using catalytic amounts 

 of enzyme and of reactions between bound DPN and substrate with the protein present 

 in quantities equivalent to the DPN may be formulated respectively as follows: 



a) DPN + 3-glyceraldehyde phosphate + HPOr ^^^ DPNH + H+ + 1,3" 

 diphosphoglyceric acid 



b) DPN-enzyme + 3-glyceraldehyde phosphate + HP04~^=^DPNH-enzyme + 

 H+ + 1.3-diphosphoglyceric acid. 



In the former case which is a true catalytic reaction, the enzyme forms transient 

 intermediates with a minute fraction of the substrate at any given time. Case (b) is in 

 effect a different reaction in which not free DPN and DPNH but the corresponding 

 protein complexes are reactants. 



Meyerhof and Oesper* have carried out a detailed study of the reaction as re- 

 presented by (a). Since one hydrogen ion enters the equilibrium, the equilibrium con- 

 stant showed a dependence upon pn- Equilibrium measurements were made with added 

 DPN under conditions similar to those employed by Meyerhof and Oesper. About 

 30 y of enzyme per ml were used so that equilibrium was reached within one minute 

 after addition of glyceraldehyde phosphate, even at low p^ values. Concentrations of 

 DPN and glyceraldehyde phosphate in the stock solutions were determined optically 

 by enzyTnatic methods, pn was measured with a glass electrode in the reaction mixture 

 at the end of the experiment. The values found for the equiHbrium constants fall well 

 within the range reported by Meyerhof and Oesper, Table V. 



TABLE V 



EQUILIBRIUM OF REACTION AT DIFFERENT PH 



The equilibrium is compared for catalytic amounts of enzyme (C) plus added DPN, and large amounts 

 of enzyme (L) containing bound DPN. The initial and final concentrations are given in moles per 

 liter. GAP = glyceraldehyde phosphate. 



* Calculated from Meyerhof and Oesper's* data by means of their complete equilibrium 

 equation. 



For equilibrium measurements under the conditions of case (b) two parallel reaction 

 mixtures were prepared which differed only in that one contained phosphate and the 

 other arsenate. The former was used for equilibrium determination while the latter 

 served for determination of the amount of DPN present in the enzyme. The value of 

 the equilibrium constants that were obtained agree within experimental limits with those 

 found with small amounts of enzyme and added DPN. 



Although one cannot derive from these measurements evidence for the existence 



References p. i6g. 



