Effects of High Concentrations of KC1 and NaCl on Responses of Malate Dehydrogenase (Decarboxylating) to Malate and Various Inhibitors

Abstract

The effects of KC1 on catalytic and allosteric properties of malate dehydrogenase (decarboxylating) were studied. Chloride salts (50 mM and higher) strongly inhibited enzyme activity at low malate concentrations. At high malate (4 mM) , on the other hand, the degree of inhibition induced by chloride was very small. The s_0.5 value (i.e. the malate concentration required for half maximum velocity) was about 0.4 mM in the absence of KC1 and increased to 0.75 and 1.5 mM at 50 and 100 mM KC1 respectively. High chloride concentrations also removed a small degree of substrate inhibition, which in the absence of KC1 occurred at 8 mM malate. At low malate concentrations (< 0 . 5 mM) 50 mM KC1 increased the Hill number from 1.3 to 1.9. Thus chloride treatment revealed a strong degree of cooperativity for the enzyme. This potential for homotropic effects was much less realized in MES buffer (potassium salt), presumably because affinity of malate for individual active sites was already very high. These effects of KC1 were readily reversible and the enzyme showed no appreciable change in molecular weight in the presence of 200 mM KCl. At malate concentrations between 1 and 12 mM, inhibitions of malate dehydrogenase (decarboxylating) activity induced by oxaloacetate, D-malate, and phosphoglycerate were reduced by KCl. At low malate concentrations, on the other hand, the inhibitory effect induced by oxaloacetate changed to a strong stimulatory effect in the presence of KCl. The inhibitory effect due to oxaloacetate was greatly diminished in malate dehydrogenase (decarboxylating) which was eluted from a DE52 cellulose column, but the inhibitory effect due to KC1 was retained. Elution from DE52 cellulose also reduced the Hill number to 1 and increased the s_0.5 value. The above results suggest that KC1 reduced the affinity of both substrate and some allosteric inhibitors of malate dehydrogenase (decarboxylating).