The Interactions of Acetoacetate Decarboxylase with Carbonyl Compounds, Hydrogen Cyanide, and an Organic Mercurial (1970)

Abstract 1. Hydrogen cyanide inhibited acetoacetate decarboxylase only when incubated with the enzyme in the presence of carbonyl compounds. This resulted in an inhibitory synergism between hydrogen cyanide and carbonyl compounds. This synergism was used to investigate the comparative abilities of various carbonyl compounds to form Schiff's bases at the active site of the enzyme. The order of effectiveness among the compounds tested was acetaldehyde g acetone g cyclohexanone g methyl ethyl ketone g 3-hexanone g diethyl ketone. 2. The ability of hydrogen cyanide to inhibit the enzyme was optimal at pH 6 and decreased abruptly on both sides of this optimum. Although primarily an uncompetitive inhibitor of the enzyme, hydrogen cyanide caused perceptible decreases in the slopes of the lines obtained by plotting kinetic data on reciprocal coordinates. Thus, raising the concentration of substrate increased the degree of inhibition caused by a given level of hydrogen cyanide more than could be explained on the basis of combination of the inhibitor only with enzyme-substrate or enzyme-product complexes. 3. Continuous infusion of borohydride into well stirred and well buffered solutions of the enzyme resulted in no loss of activity unless carbonyl compounds, capable of forming Schiff's base compounds at the active site, were present. The rate of inactivation of the enzyme, under these conditions, could be used as an index of the degree of saturation of the active sites by the carbonyl compound being tested and of the reactivity with borohydride of the Schiff's base compounds so generated at the active site. Quantitative data were obtained in this way for acetone sulfonate, acetone, methyl ethyl ketone, and cyclohexanone. Nitrate was observed to protect the enzyme against inactivation by borohydride in the presence of acetone. 4. A number of diketones were compared for their abilities to inhibit the enzyme and the structural features essential for this action were discussed. β-Diketones, which inhibited the enzyme, generally did so with perceptible slowness which was accentuated by electron-withdrawing substituents adjacent to one of the carbonyl functions. The ability of acetylacetone to protect the enzyme against inactivation by borohydride, like its ability to inhibit the enzyme, developed slowly. 5. The reaction of acetoacetate decarboxylase with p-chloromercuriphenylsulfonate (CMS) was investigated both in terms of mercaptide formation and in terms of activity. One sulfhydryl group per subunit could be derivatized without loss of activity whereas reaction of a second sulfhydryl was associated with some inactivation. A third equivalent of CMS per subunit reached with an unidentified group to cause additional inactivation. Amounts of CMS in excess of three per subunit had no further effects and the enzyme retained activity which was impervious to additional CMS. The native enzyme retained 70% of its activity after treatment with CMS whereas the fully activated enzyme retained only 50% of its activity after exposure to CMS. Inhibition by CMS was reversed by cysteine.