104 Discussion 



certain step which is rate-controUing for the pacemaking reaction. In a 

 heterogeneous S3^stem, as in the cell, there must be for each individual 

 enzymatic stage a number of steps which are physicochemically dis- 

 tinct, such as diffusion of substrate to the enzyme and diffusion of the 

 product away from the enzyme in addition to the actual chemical reac- 

 tion occurring on the enzyme. Any one of these steps could determine 

 the overall rate of the chemical reaction. Could you tell us whether the 

 rate-controlling step of the individual enzyme reactions in the cell is likely 

 to be the actual chemical reaction on the enzyme or a diffusion process ? 



Krehs: I don't think that one can make a general statement about it. 

 What one can tackle experimentally is the identification of pacemaker 

 steps, by determining the steady-state level of the intermediate meta- 

 bolites, especially of the substrate of the pacemaker. If a substance 

 reacts at a variable rate, its steady-state concentration must vary, 

 because it will be produced at the same rate but removed at a different 

 rate. A number of people have started to determine the steady-state 

 level of intermediate metabolites, such as DPN, or reduced DPN, 

 organic phosphates. This may lead to the identification of a pacemaker. 

 But what changes the rate of such a reaction is a different matter. 

 Before this can be answered the mechanism of the pacemaker reaction 

 must be known. We just don't know enough about such mechanisms to 

 say what role diffusion might play. 



Dale: Do you consider it useful to do model experiments which 

 may show effects on surfaces which bind enzymes and perhaps sub- 

 strates at the same time, e.g. to imitate perhaps internal cell boundaries 

 or cell surfaces with model experiments, such as burning of glucose on 

 charcoal ? 



Krehs: In general I would think that the scope of model experiments 

 is nowadays limited. The earlier models, like the oxidation of sugars on 

 charcoal, would be largely irrelevant. W^e should study the real thing 

 for preference. 



Dale: It must be extremely difficult to determine the steady state in 

 the cell for one of these recommended steps, because of their very small 

 amounts present at a given time. 



Krehs: It is indeed a great problem but it is being tackled and is 

 being successfully solved in some cases. With paper chromatography 

 and isotope techniques it is possible to determine quantitatively 

 metabolites in very small amounts. 



Dale: With regard to radiation it is difficult; you have to irradiate 

 practically at the same time, because the steady state may change as 

 soon as radiation stops. 



Krehs: One can stop reactions very quickly under most conditions, by 

 liquid air and other means. 



Iladdow: Did I understand j^ou to mention hexokinase in relation to 

 the cell surface? 



Krehs: Yes, there is a good deal of evidence showing that the entry 

 of sugar into the cell is not by passive diffusion but an active process. 

 We have recently carried out experiments on the true sugar content of a 

 number of tissues, using chromatographic separation, and they confirm 



