6oO PETER MITCHELL 



by proteins. The concept of an enzyme implies that the substance which penetrates 

 is chemically changed and I should like to ask if changes in penetrating substances 

 occur ? 



Mitchell: Well, Dr. Dische, you are asking rather a big question. Let me 

 answer first of all with an example that stems from my paper. In the utilization of 

 glucose 6-phosphate by Bocteriiim coli, what actually passes through the membrane 

 is, on the one hand "glucose", and on the other hand "phosphate". We know 

 this because the rate of utilization of glucose 6-phosphate is the same as the rate 

 at which the externally available glucose 6-phosphatase can break it down. This is 

 a clear example of a case in which the first process in the overall transport reaction 

 is an enzymic one. We have to be very careful in speaking, for example, of the 

 transport of glucose 6-phosphate into the cell, because we know that the glucose 

 6-phosphate does not go in, although later glucose 6-phosphate is found in the cell 

 as a result of the separate entry of the " glucose " and the "phosphate " by different 

 molecular pathways. Moreover, there is evidence that the membrane is im- 

 permeable to glucose and phosphate and that these molecules pass across the 

 membrane as derivatives or chemical groups (hence the inverted commas). This 

 illustrates a general principle that I am trying to make, but I do agree with you, 

 that we must not jump to silly conclusions; we must not say that we have direct 

 evidence for the process of group translocation, and as I pointed out in my paper, 

 this evidence may well have to await the successful reconstruction of in vitro 

 membrane systems. All the same, the specificity and kinetics, and especially the 

 susceptibility to inhibitors, of a number of transport processes do strongly suggest 

 that they represent enzyme-catalyzed chemical reactions. Let us consider, for 

 example, the entry of " succinate " into micrococci. In this case we can examine the 

 catalytic system in rather an elegant way. Some years ago. Dr. Moyle and I 

 thought that the entry might be directly through succinic oxidase, and we argued 

 that as succinic oxidase is part of the membrane it could have its active centre 

 exposed on the outside. As we knew that the membrane is impermeable to suc- 

 cinate and malonate in the normal sense, we were able to investigate this possibility 

 by seeing whether external malonate would inhibit the succinic oxidase of intact 

 cells. In fact, malonate was found to have no effect at all unless at first you 

 depressed the pH to 5 to let the malonate in and then brought it back to 7. After 

 that the external malonate could be washed away, and succinate oxidation con- 

 tinued to be inhibited. This shows that the succinic oxidase is inhibitable by 

 malonate, but the active centre is facing inwards. It also shows that the specificity 

 of the process giving rise to "succinate" entry is such as to discriminate between 

 malonate and succinate. This is one of the reasons why we think that the kino- 

 synthetase may catalyze the first reaction for succinate entry, for this enzyme, 

 unlike succinic oxidase, does distinguish between succinate and malonate and is 

 not inhibited by the latter. Further, the substance that passes into the cyto- 

 plasm of the micrococcus while succinate passes into the outer surface of the 

 plasma membrane is not succinate ! This illustrates the background of my 

 approach to the closely related problems of transport and metabolism in whole 

 cells. 



In kinetic studies, the evidence obtained is circumstantial. One creates 

 hypotheses in order to disprove them. Our aim has been in the past to try to 



