i66 c. F. coRi et al. vol. 4 (1950) 



of two catalytic sites, the following considerations are of interest. In case (a) the enzyme 

 cannot contribute to the net free energy change which is lixed by the initial and final 

 states of the free reactants. In case (b) two of the reactants have been altered by com- 

 plex formation and the initial and final energy states are not the same as in case (a). 

 However, since only the difference in initial and final states determines the net free 

 energy change, case (b) may or may not have the same equilibrium constant as case (a). 

 These considerations apply irrespective of the physical nature of the bonding forces 

 involved and the number and type of binding sites. 



It may be inferred from the kinetics that the protein has the same affinity for 

 DPN as for DPNH*. Conclusions concerning the relative dissociation constants of 

 enzyme-DPN and enzyme-DPNH may also be drawn from a comparison of the equi- 

 librium constants in (a) and (b). If the binding of the other reactants does not alter 

 their energy differences then, from the equality of equilibrium constants, it follows that 

 the dissociation constants of enzyme-DPN and enzyme-DPNH are equal. 



Ph optimum 



The rate of the reaction of glyceraldehyde with enzyme DPN was measured at pn 

 8.4, 7.5, and 6.4 in cysteine-pyrophosphate buffer. The relative rates calculated from 

 the first order velocity constants were as 100:30:9. This agrees with the pn activity 

 curve as determined previously with small amounts of enzyme (6 y/ml) and addition 

 of DPN and glyceraldehyde phosphate as substrate^. 



REACTION WITH LACTIC DEHYDROGENASE 



It has been shown in a previous report^ that enzyme DPN, after reduction by 

 glyceraldehyde phosphate, was reoxidized by addition of sodium pyruvate and a purified 

 preparation of lactic dehydrogenase from rabbit muscle. The simplest explanation of 

 this result is that the bound DPNH has a small but finite tendency to dissociate and 

 that it is the dissociated DPNH which reacts with the pyruvate-lactic dehydrogenase 

 system. In these experiments lactic dehydrogenase was present in considerable excess, 

 so that the rate of the reaction could not be measured. 



The dissociation constant for lactic dehydrogenase and DPNH has been determined 



by KuBOWiTZ AND Ott^ who report a value of 5 • io~^ M/ml. In experiment A, Table VI, 



2.3-0.146 

 the initial concentration of bound DPNH was = 2.3-10-8 M/ml. If the DPNH- 



1.45 -107 



enzyme dissociation constant were i • io~i" M/ml, there would not be enough free DPNH 

 in solution to give 25% saturation of lactic dehydrogenase and the rate of reaction 

 would be much slower than in experiment C, where the concentration of added DPNH 

 was 3.i-io~8M/ml or enough to saturate the enzyme. The fact that such a difference** 



* This inference arises from the fact that in the presence of a large excess of glyceraldehyde 

 and arsenate the reduction of bound and of added DPN may be described by a first order velocity 

 constant. If one assumes that DPNH has the same affinity for the catalytic site as does DPN, then 

 the first order kinetics may be shown to be due to the formation of DPNH which acts as a com- 

 petitive inhibitor^. 



** Actually the rate was faster in A than in C. One possible explanation was that lactic dehydro- 

 genase in C was acting in the absence of "protective" protein. In order to compensate for this differ- 

 ence, lactic dehydrogenase was added in other experiments to a solution containing the same amount 

 of triosephosphate dehydrogenase the DPN of which had not been reduced. The rate of reaction of 

 lactic dehydrogenase with "bound" and with added DPNH was then approximately the same. 



References p. 169. 



