12 Discussion 



Potter: Under carefully defined conditions. 



Chance : Yes. The product of that velocity constant multiplied by the 

 enzyme concentration is the velocity, which will vary. 



Krebs : The velocity constant and the concentration of an enzyme alone 

 do not determine reaction rates. These often depend on the amount of 

 substrate available. For example, in normal animal tissues the activity 

 of most intermediary enzymes is determined by the amount of substrate 

 which arises, because the enzymes have a much greater capacity than 

 is normally required. So you are quite right in drawing attention to the 

 terminology and the need to clarify what is meant when we speak of 

 reaction velocities. The ones that I had in mind are not those depending 

 on certain constants of the enzyme ; they refer to the overall reaction 

 which occurs under given conditions. 



Chance: It could be called velocity, and it is the consequence of a 

 number of factors. You mentioned the substrate-limited case where the 

 substrate determines the concentration of this intermediate. In other 

 words, the enzyme is not completely saturated with substrate, so that 

 the variable there is the amount of intermediate. 



Greville: In most cases where no substrate has been added to tissue 

 slices, DNP has no effect. Even when the endogenous respiration is as 

 high as the respiration in the presence of added substrate, DNP may 

 have no effect. If you add some, but not all, substrates and DNP, then 

 the respiration goes up. I am speaking of tissue slice conditions. 



Krebs : You have more experience in this field than I. I have not used 

 slices but, as the examples show, in homogenates also there was no 

 effect unless substrate were added. 



Greville : Exceptions were found in the case of slices from liver of fed 

 rats and of a chicken tumour of rather obscure origin. 



Chance: Would you say that this applies to excised muscle, i.e. 

 muscle cut away at the tendon and taken from the animal? 



Greville : Definitely not. I am speaking entirely of slices. 



Chance: It is important to make that distinction. Excised muscle is 

 completely different from tissue slices, and it will show a very large 

 response to DNP. 



Greville: Not only that; it also has the peculiarity that anaerobic 

 lactic acid production is vastly increased by addition of DNP. 



Krebs: We know that DNP acts in the intact body and causes a 

 raised heat production and oxygen consumption. What you mentioned 

 about slices is a matter of widespread, but perhaps not universal, 

 occurrence. 



Siekevitz : There is one example in which the supply of substrates is 

 not limiting, and that is the case of citrate oxidation by mitochondria. 

 You can pile up citrate in the mitochondria (Schneider, W. C, Striebich, 

 M. J., and Hogeboom, G. A. (1956). J. biol. Chem., 222, 969). There is a 

 very low rate of oxidation which is probably due to the DPNH-linked 

 isocitric dehydrogenase (Ernster, L., and Lindberg, O. (1958). Ann. 

 Rev. Physiol., 20, 13). 



Krebs : Citrate is a very special case for two reasons : firstly, because 

 the enzyme limiting its removal acts under physiological conditions 



