164 



c. F. CORI et al. 



VOL. 4 (1950) 



to the total amount in the supernatant of B (1.57- io~' M) minus the amount arising 

 from residual triosephosphate, [(2.13 — 1.49) -lO"' = 0.64-10"'], minus unprecipitated 

 protein-DPNH (0.36-10"'). The net displaced DPNH is (1.57 — 0.64 — 0.36) -lO"' = 

 0.57-10"' M. A similar value is arrived at by comparing DPNH in the precipitated pro- 

 tein in A and B, namely (1.49 — 0.86) • io~' = 0.63- lO"' M. 



THE REACTION AT SITE II 



When reactions are studied with added DPN, site I is saturated, even at low enzyme 

 concentrations and site II is saturated to an extent which depends upon its dissociation 

 constant and the concentration of free DPN. Reaction will be expected to occur at both 

 sites but the DPNH formed at site I will be displaced by DPN in solution and site I 

 as well as site II will now have a "turnover". The reactions at both sites will be first 

 order provided that at each site the affinity for DPN is the same as that for DPNH. 

 Experimentally it was found that the rate remained first order when DPN was added. 

 Table IV. 



TABLE IV 



EFFECT OF ADDED DPN ON RATE OF REACTION 



The enzyme concentration corresponded to 3.4- lo-* M of bound DPN per ml, the pn was 8.3 and 

 the temperature 26°. No DPN was added in A, while in B and C, 3.4 and 7- 10—^ M per ml respectively 

 was added, giving the total of concentrations of DPN shown in the table headings. The reaction was 

 started by the addition of glyceraldehyde (final concentration as the D-form i.i-io-* M per ml). 

 K = 2.3/t log A (A — x), A being the initial concentration of DPN. Vq (initial velocity) — K times 

 the initial concentration of DPN. 



After addition of glyceraldehyde phosphate. 



By multiplying first order velocity constants, K, by the initial concentrations of 

 DPN one gets the initial velocity of the reaction, Vq, in terms of M. min"^ ml"^. The 

 observed increase in initial rate on addition of DPN can be seen to be approaching a 

 maximum value which would correspond to the saturation of both sites with DPN. 

 Because of the high concentration of enzyme, one cannot calculate the enzyme-coenzyme 

 dissociation constants by the usual methods (which are based on the assumption that 

 the concentration of free DPN is not appreciably diminished by combination with the 

 enzyme). It is furthermore not possible from this experiment to reach unambiguous 

 conclusions with respect to the number and type of catalytic sites. 

 References p. i6g. 



