Further experiments on the inhibition of aconitase by enzymically prepared fluorocitric acid

Abstract

A new method has been worked out for testing the activity of fluorocitrate on soluble aconitase. In this a cruder enzyme preparation is used, taken to an intermediate stage of fractionation, which does not require reactivation with ferrous salts and cysteine. When this preparation was used in 2-amino-2-hydroxymethylpropane-l,3-diol (tris) buffer solution instead of phosphate solution, 50% inhibition was observed with fluorocitrate (enzymically synthesized) at a concentration of 6.5 [mu]mM. This large increase in the inhibitory activity observed means that there is no longer a discrepancy between the effects of fluorocitrate upon aconitase in particle preparations and in a soluble form. It makes unnecessary the hypothesis of specific concentration or orientation previously proposed. The inhibitory effects were much reduced by the presence of phosphate or by preincubation with 5 mM-isocitrate. In the presence of NaCl the activity of the aconitase increased. With KC1 there was an increase followed by a fall. An increase in the concentration of both salts much reduced the inhibition of fluorocitrate. The percentage inhibition with fluorocitrate was the same for Ls(+)-isocitrate (D), though the rate for a given amount of isocitrate was doubled. There was no indication that the inactive isomer was an inhibitor. Variation of temperature within [plus or minus] 4[degree] was about 4% per degree. Variations in pH of [plus or minus] 0.6 caused little change in fluorocitrate inhibition, though a fall in activity was found on making more alkaline.