20 1. MALONATE 



of these two substances with the enzyme surface, undoubtedly at different 

 sites. At low concentrations of the acceptor, or with weak acceptors, the 

 over-all rate may not be determined by the rate at which the hydrogen atoms 

 are removed from the succinate, but may depend also on the rate of transfer 

 to the acceptor. The malonate inhibition will thus vary with the degree of 

 saturation of the enzyme with acceptor. On this basis it would seem reason- 

 able to use those acceptors which are the most active and react with 

 sites closest to the succinate site. A further consideration is the inhibition 

 produced by the acceptors themselves. Both ferricyanide and iV-methylphe- 

 nazine begin to inhibit succinate dehydrogenase as the concentration is 

 raised above certain levels. Such systems would then constitute examples 

 of multiple inhibition and the kinetics of the inhibition due to malonate 

 alone may be distorted. The choice of the acceptor and its concentration 

 is thus of some significance. 



Site of Inhibition by Malonate in the Succinate Oxidase Sequence 



The results discussed in the preceding section point clearly to the site 

 of inhibition as succinate dehydrogenase. Indeed, inhibition of soluble 

 succinate dehydrogenase by malonate has been demonstrated. The competi- 

 tive nature of the inhibition, to be treated in the following section, indicates 

 the inhibition to be at the active site at which succinate is bound. There is 

 thus no question but that the major site of inhibition is at the very begin- 

 ning of the electron transport sequence in succinate oxidase. The question 

 that now must be considered is whether malonate can inhibit at any other 

 step of the electron transport chain. 



There are two obvious ways to examine this. One is to test the action 

 of malonate on the succinate oxidase system, using substrates that donate 

 electrons at more distal sites than succinate. The other way is to determine 

 the response of other oxidases that utilize most of the electron carriers 

 in the succinate oxidase. Quastel and Wheatley (1931) observed that ma- 

 lonate at 67 mM does not inhibit the oxidation of p-phenylenediamine 

 and hence concluded that the cytochrome region of the sequence is immune 

 in their preparations. Actually, many oxidases, comprising varying segments 

 of the electron transport chain, have been found to be insensitive to malo- 

 nate at concentrations from 25 mlf to 50 mM. All this evidence points to 

 a rather specific action on the dehydrogenase. However, there are data in 

 the literature which indicate that, at least in some species and at high enough 

 malonate concentrations, inhibition at other sites may occur. Although 30 

 mM malonate does not inhibit NADH oxidation in beet mitochondria 

 (Wiskich et al., 1960), some inhibition has been observed in mosquito parti- 

 cles (10-15% at 1-10 mM) (Gonda et al, 1957) and in Tetrahymena NADH 

 oxidase (35% at 6.2 mM and 70% at 18 mM) (Eichel, 1959). Such inhi- 

 bition could, of course, be on the NADH dehydrogenase rather than on 



