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Malonate is the dianion of malonic acid, with two carboxylate groups separated by one methylene group. Its three-carbon skeleton resembles part of the four-carbon skeleton of succinate.

Malonate is a classical inhibitor of succinate dehydrogenase: structural similarity allows binding without the normal substrate transformation. The example illustrates how an enzyme inhibitor can probe the contribution of a particular reaction to a metabolic pathway.

Malonic acid is propanedioic acid, a dicarboxylic acid in which two carboxyl groups are separated by one methylene group:Its neutral molecular formula is ${\mathrm{C}}_3{\mathrm{H}}_4{\mathrm{O}}_4$ and its average molar mass is approximately $104.061\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$.^[ref]

Two of the four hydrogen atoms belong to carboxyl OH groups; the other two belong to the central carbon. Complete neutralization of both carboxyl groups therefore requires two equivalents of a suitable base, not four merely because the formula contains four hydrogens. An original stoichiometric model isFor $1.00\mathrm{mmol}$ acid, the two-equivalent amount of $0.100\mathrm{mol}{\mathrm{L}}^{-1}$ hydroxide solution is $20.0\mathrm{mL}$. The pH at an experimental endpoint also depends on acid-base equilibria and the measurement method; the equivalent count alone does not determine it. Malonate names the corresponding deprotonated system, whose protonation state should be specified.

Heating can promote decarboxylation, producing acetic acid and carbon dioxide. Malonic-acid derivatives underlie the carbon-chain construction described in the malonic ester synthesis.^[ref] For the unsubstituted acid, the stoichiometric balance isAn original ideal mass balance for $1.00\mathrm{mmol}$ converted by only this reaction gives about $60.052\mathrm{mg}$ acetic acid and $44.009\mathrm{mg}$ carbon dioxide from $104.061\mathrm{mg}$ starting material. Following evolved gas and residual material would test conversion, whereas a change in sample mass alone would not distinguish this reaction from evaporation or another decomposition pathway.

hydroxide attacks an ester carbonyl to form a tetrahedral intermediate. Collapse expels ethoxide, and acid–base transfer gives the carboxylate and ethanol. Repeating this nucleophilic acyl substitution at the other ester produces the malonate dianion; acid workup gives $\mathrm{H}{\mathrm{O}}_2\mathrm{C}-\mathrm{C}(\mathrm{L}{)}_2-\mathrm{C}{\mathrm{O}}_2\mathrm{H}$, with $L=\mathrm{C}{\mathrm{H}}_2\mathrm{CH}=\mathrm{C}{\mathrm{H}}_2$ as defined above.

On heating, one carboxylic acid group is lost through a six-membered cyclic proton-transfer/bond-cleavage arrangement. A carbonyl oxygen of the retained group accepts the proton from the departing carboxyl OH as its C–C bond breaks and carbon dioxide forms; electron redistribution gives the enol-like form of the retained acid. proton transfer/tautomerization then producesThe net decarboxylation is ${\mathrm{C}}_9{\mathrm{H}}_{12}{\mathrm{O}}_4\to {\mathrm{C}}_8{\mathrm{H}}_{12}{\mathrm{O}}_2+\mathrm{C}{\mathrm{O}}_2$. The central carbon gains a hydrogen, both allyl substituents survive, and only one carboxyl group is removed. It would be incorrect to draw hydrolysis directly giving the final acid without accounting for this carbon loss. The favourable cyclic pathway is characteristic of malonic acid decarboxylation.

The malonate carbon receives two allyl groups. With $L=\mathrm{C}{\mathrm{H}}_2\mathrm{CH}=\mathrm{C}{\mathrm{H}}_2$, attached through its first carbon, the product issodium hydride removes a methylene proton from diethyl malonate; molecular hydrogen formation accompanies production of an enolate stabilized by resonance in chemistry. Its carbon attacks the bromomethylene carbon of allyl bromide in an SN2 reaction, expelling bromide. The monoalkylated malonate still has one hydrogen between the carbonyls. Excess base removes that hydrogen, and a second equivalent of allyl bromide produces a second carbon–carbon bond. The final malonate carbon has no remaining hydrogen and cannot undergo a third analogous deprotonation there.

The two alkenes are preserved because the substitution occurs at the primary carbon bearing bromine. Counting the introduced groups gives ${\mathrm{C}}_{13}{\mathrm{H}}_{20}{\mathrm{O}}_4$ for the diallylated diethyl ester. Its two identical allyl groups also mean that the central carbon is not a stereocenter. This stage is the alkylation portion of the malonic ester synthesis.

Hans Adolf Krebs described the experimental reasoning in his 1953 Nobel lecture. Pigeon breast skeletal muscle retained strong oxidative activity after preparation in a Latapie mincing mill and suspension in aqueous medium. Small fumarate additions increased oxygen uptake by more than complete oxidation of the added fumarate could explain; added four-carbon acids could still be detected afterward. His Table 1 compares the increase in uptake with that calculated from complete consumption of the addition. These comparisons challenge the interpretation that the addition merely supplied extra fuel.^[ref]

The lecture identifies the preparation apparatus but does not describe the gas-measuring instrument behind Table 1. The result supports regeneration, not a unique complete reaction mechanism. Its later discussion combines citrate formation from oxaloacetate with breakdown back toward the four-carbon intermediates to close the cycle. Competitive inhibition supplies another comparison: malonate inhibits succinate dehydrogenase, and succinate accumulates. Such evidence links a blocked step to the surrounding metabolic pathway; damaged tissue and restricted inhibitor access can complicate interpretation.^[ref]

An increase above the added fuel's own oxygen demand is a particularly revealing control on the explanation: it compares the proposed cause with the largest effect that simple consumption could produce. The modern mitochondrial oxygen electrode assay asks a different question about a prepared mitochondrion, testing respiratory control through ADP additions. Neither an oxygen uptake signal nor an accumulating intermediate is by itself a direct measurement of ATP production.