42 1. MALONATE 



arsenicals require at least 30 min to reach their maximal inhibition. Such 

 protection on a competitive basis was called interference inhibition by 

 Ackermann and Potter (1949). Once the sulfhydryl reagents have reacted 

 with the enzyme, malonate will not reverse the inhibition, but only slows 

 down the rate at which the substance acts on the enzyme. (I find it difficult 

 to understand how, in some cases, such low concentrations of malonate 

 afford protection against irreversible inhibitors. For example. Potter and 

 DuBois reported that 0.33 mM malonate protects quite well against 

 p-quinone, and yet this concentration of malonate inhibits only around 

 20%, showing most of the enzyme uncombined with malonate). 



Succinate dehydrogenase also contains nonheme iron and flavin dinucleo- 

 tide but the locations of these components relative to the succinate-binding 

 site are not known. Since the iron and the flavin both participate in the 

 electron transfer, it is reasonable to assume that at least one of them is 

 close, or even part of the active center. Most formulations have pictured 

 the initial step as a transfer of hydrogen atoms from succinate to the fla- 

 vin; if this is so, the topography of the active center must be rather 

 complex. 



We shall now turn to the energetics of the binding of malonate in order 

 to determine if the ionic interactions generally assumed are reasonable. 

 An immediate difficulty is the variability in the values of K^ reported, even 

 for the same tissue; for example, 0.0076 mM (Thorn, 1953) and 0.05 milf 

 (Hellerman et al., 1960) for the enzyme from pig heart, and 0.041 mM for 

 beef heart (Kearney, 1957). A K^ of 0.0076 mM would indicate an over all 

 binding energy of 7.24 kcal/mole, or 3.62 kcal/mole for each carboxylate 

 interaction assuming only ion-ion contribution. This is a reasonably high 

 value for the interaction of C00~ and NH3+ groups and corresponds to 

 an intercharge separation of around 4.30 A (Fig. 1-6-16), which is not far 

 from contact of the groups. It is unlikely that other types of interaction 

 are important for malonate. The corresponding K, for succinate is 0.0028 

 mM, giving an interaction energy of 3.28 kcal/mole per carboxylate group 

 and a separation of 4.45 A. A difference of fit of 0.15 A would thus account 

 for the relative bindings of malonate and succinate. However, in the case 

 of succinate it is more likely that other energy terms are involved. There 

 is undoubtedly some interaction between the — CH2CH2 — region and the 

 enzyme, and it is probable that distortion of the succinate molecule occurs 

 upon binding. In any event, these rough estimates point to a fairly close 

 fit of malonate to the enzyme cationic groups for the pig heart enzyme. 

 The ability of malonate to bind to the active center of succinate dehydro- 

 genases from bacteria is apparently much less (Table 1-8). 



The next question is: what is the most probable distance between the 

 two enzyme cationic binding sites? Information on this must be obtained 

 from the relative bindings of substances having negatively charged groups 



