l62 



c. F. coRi et al. 



VOL. 4 (1950) 



in rate between DPN concentrations of 4.4-10"^ and 4.4-10"^ M/ml that is consistent 

 with a dissociation constant of the order of 4-10"^ M/ml. 



The fact that depending upon whether or not one measures bound DPN or added 

 DPN, one gets apparent dissociation constants differing by a factor of at least 100 argues 

 for the existence of two types of catalytic sites. We will designate the still hypothetical 

 site with the higher DPN affinity as site I and the site with lower DPN affinity as site 

 II and proceed to examine the conditions that would hold during the course of a reaction. 



THE REACTION AT SITE I 



In Table II is shown an experiment in which the reduction of bound DPN is studied 

 as a function of glyceraldehyde concentration. The glyceraldehyde concentration in all 



TABLE II 



EFFECT OF CONCENTRATION OF GLYCERALDEHYDE 



Reaction mixture consisted (in moles per ml) of 2.4-10-® enzyme - DPN, e-io-® arsenate, 3-10-^ 

 cysteine, 5-10-* pyrophosphate (pn 8.3) and varying amounts of DL-glyceraldehyde. 



* K = 2.3/t log A (A — x), A = initial concentration of DPN. 



cases was sufficiently higher than that of DPN so that it was virtually constant during 

 the course of the reaction. Under these conditions the rate is described by a first order 

 velocity constant. The fact that the first order constants increase linearly with initial 

 glyceraldehyde concentration means that saturation of the enzyme with glyceraldehyde 

 has not been approached. The dissociation constant of enzyme-glyceraldehyde is there- 

 fore very large. 



At the concentrations of enzyme employed the amount of free DPN in equilibrium 

 with the protein would be negligible if the dissociation constant at site I is less than 

 I • io~^". The above reaction is therefore first order with respect to enzyme-DPN com- 

 plex. This means that each enzyme molecule behaves as though it reacted only once. 



When DPNH (in amounts equivalent to the bound DPN present) was added at the 

 beginning of the reaction, it exerted an inhibitory effect. This is indirect evidence that 

 DPNH as well as DPN is bound at site I. It is also possible to demonstrate in a direct 

 manner that DPNH is bound. This was done by reducing the bound DPN in a solution 

 containing 10 to 20 mg of enzyme per ml with excess glyceraldehyde phosphate and 

 References p. i6g. 



