Multifunctional behavior as an aid in deducing metalloenzyme and model reaction mechanisms

Abstract The ability of a substance to participate in competing reactions presents difficulties but if the problems can be resolved a clearer picture of the behavior in all reaction modes may be forthcoming. Oxalacetate is involved in a number of different biological reactions which are catalyzed by metalloenzymes. Many of these reactions are amenable to independent investigation in model systems employing metal ions. Decarboxylation is one reaction mode of oxac2− which has attracted interest for many years owing to strong resemblances between the rate dependencies of the enzymic and model systems on metal ion concentration. The Steinberger-Westheimer mechanism had early been accepted as the means by which metal ions catalyzed oxac2− decarboxylation: Prevailing evidence indicates that the activation barrier is lowered by the complexation of the high energy enolate of pyruvate. Enolization and hydration reactions of oxac2− procede within the same time frame and are closely entwined. Resolved rate data show that the reactions are subject to acid and base catalysis. Proton catalysis appears to be equally effective for both, but large differences are evident in base catalysis. Hydration rates are sensitive to possessing an oxygen donor atom. OH− is a very efficient catalyst and even H2O catalyzed rates are appreciable. Tertiary amines are found to be weak catalysts. Enolization appears to be more susceptible to softer bases. The rate constant for the OH− catalyzed path is 1 6 as large as that determined for hydration, and H2O catalysis is negligible; however, tertiary amines are potent catalysts and the more basic ones exceed OH− in activity. different sites are involved in catalysis. Enolization involves the removal of a CH2 proton from oxac2−, while in hydration the base attacks the keto carbon atom through an H2O molecule, C(O)CH2 + HOH··B ⇌ C(O−)(OH)CH2 + BH+. In either case, an oxyanion is formed at the 2-carbon atom. Metal ion complex formation has a relatively small effect on the rate constant for acid catalyzed enolization, suggesting that there is little interaction between the metal ion and the site for proton attack, the keto group oxygen atom. In contrast, the rates along the base catalyzed pathways are strongly influenced by a metal ion and exhibit increases that appear more dependent on the nature of the catalyst than on the reaction mode. The presence of Mg(II) induces increases of three orders of magnitude in the rates of OH− catalyzed enolization and hydration, while the H2O catalyzed pathway for hydration and the tertiary amine catalyzed pathways for both reactions undergo rate increases of 20–30 fold. A charge is apparent in these figures, but, especially in the case of OH−, the metal ion must interact with, and stabilize the intermediate oxyanions in ways similar to that by which decarboxylation is promoted. Rates of ketonization of enolpyruvate are also being investigated. Consistent with the charge differences acid catalysis is slower, and base catalysis is faster with enolpyruvate than with oxac2−. Complexing metal ions show very strong cooperativity with base catalysts. Here again, the metal ion seems to function as stabilizer of the intermediate oxyanion. The coordinating abilities of metal ions are paramount in their causing activation barriers to be lowered in these model reactions. However, recent evidence obtained in a detailed study of enzymic decarboxylation suggest that an enzyme may not utilize this function of a metal ion, but may achieve greater catalytic efficacy through other mechanisms, such as proton transfer.