66 1. MALONATE 



Inhibition Due to Chelation with Metal Ion Cofactors 



Many enzymes are dependent on dissociable metal ions for their activity 

 and the operation of most of the important metabolic systems thus requires 

 the presence of these cofactors. The list of enzymes requiring Mg++ is a 

 long one and includes the oxidases and decarboxylases for the keto acids, 

 most of the enzymes involved in phosphate metabolism (e.g., the kinases, 

 the transphosphorylases, the phosphatases, and the acyl — CoA synthetases), 

 some dehydrogenases e.g., phosphoglucose dehydrogenase, phosphogluco- 

 nate dehydrogenase, and isocitrate dehydrogenase), some peptidases, 

 phosphoglucomutase, and enolase. Several enzymes require Zn++, such as 

 lactate dehydrogenase, glutamate dehydrogenase, alcohol dehydrogenases, 

 carboxy peptidase, and carbonic anhydrase. Since malonate is able to chelate 

 effectively with these metal ions, inhibition may result from the reduction 

 of metal ion concentration in the medium or the removal of the metal 

 ions from the enzyme. The ability of malonate to inhibit by this mechanism 

 will depend on the affinity of the enzyme for the metal ion. The binding 

 of Zn++ to enzymes is usually rather strong and it is difficult for malonate 

 to deplete the enzyme of this metal, but Mg++ is more loosely bound in most 

 cases and the activity of enzymes dependent on it is generally related to 

 the Mg+'*" concentration in the medium. The effect of malonate on Mg++- 

 dependent enzymes will thus depend on the concentrations of Mg++ and 

 malonate, and on the relationship between enzyme activity and Mg+*^ 

 concentration. In the use of malonate, especially at higher concentrations, 

 it is imperative to consider the possibility of such effects. It is likely that 

 some of the inhibitions in Table 1-12 are due to metal ion depletion. 



Inhibition by reaction with an activator was discussed briefly in Chapter 

 1-3 and it was seen that in the general case no simple expression for the 

 inhibition is possible. Nevertheless, it should be reasonably easy to deter- 

 mine if the inhibition is purely the result of activator depletion, since the 

 concentration of free activator can be calculated from the dissociation 

 constant of the activator-inhibitor complex by an equation similar to 

 Eq. 1-3-72: 



(Mg) = ^ V [(I,) - (Mg,) + K-\- + 4(Mg)if - -^ [(I,) - (Mg,) + K] (1-3) 



If the effect on the enzyme is only to reduce the Mg++ concentration, the 

 addition of malonate should bring the activity to that value corresponding 

 to the reduced free Mg++. Another possibility is that the Mg-malonate 

 complex is the active inhibitor, in which case the kinetics should be investi- 

 gated with the calculated concentrations of this complex at different con- 

 centrations of Mg++ and malonate. 



The most thorougly studied instance of the possible relationship of Mg++ 

 to inhibition by malonate is the work on the utilization of oxalacetate 



