Kinetic Mechanism and Structural Requirements of the Amine-Catalyzed Decarboxylation of Oxaloacetic Acid

Abstract

The kinetic and chemical mechanism of amine-catalyzed decarboxylation of oxaloacetic acid at pH 8.0 has been reevaluated using a new and versatile assay. Amine-catalyzed decarboxylation of oxaloacetic acid proceeds via the formation of an imine intermediate, followed by decarboxylation of the intermediate and hydrolysis to yield pyruvate. The decrease in oxaloacetic acid was coupled to NADH formation by malate dehydrogenase, which allowed the rates of both initial carbinolamine formation (as part of the imination step) and decarboxylation to be determined. By comparing the rates observed for a variety of amines and, in particular, diamines, the structural and electronic requirements for diamine-catalyzed decarboxylation at pH 8.0 were identified. At pH 8.0, monoamines were found to be very poor catalysts, whereas some diamines, most notably ethylenediamine, were excellent catalysts. The results indicate that the second amino group of diamines enhances the rate of imine formation by acting as a proton shuttle during the carbinolamine formation step, which enables diamines to overcome high levels of solvation that would otherwise inhibit carbinolamine, and thus imine, formation. The presence of the second amino group may also enhance the rate of the carbinolamine dehydration step. In contrast to the findings of previous reports, the second amino group participates in the reaction by enhancing the rate of decarboxylation via hydrogen-bonding to the imine nitrogen to either stabilize the negative charge that develops on the imine during decarboxylation or preferentially stabilize the reactive imine over the unreactive enamine tautomer. These results provide insight into the precise catalytic mechanism of several enzymes whose reactions are known to proceed via an imine intermediate.

A theoretical study on ethylenediamine catalyzed decarboxylation of oxaloacetic acid

“…3). Such seven-membered ring transition structures were also estimated in Thalji's study [24]. The product of this step is the protonated imine IM5 (Fig.…”

“…Values without and with parentheses are the relative free energies (in kcal/mol) in the water solution and in the gas phase respectively. [24].…”

“…Thus, the role of the second amino group in efficient diamine-catalyzed decarboxylation of OAA may be 3-fold [24]: Firstly, it enables ethylenediamine to form carbinolamine easily (and thus imine); Secondly, the second amino group serves to catalyze the carbinolamine dehydration step by protonating the hydroxyl group to enhance its leaving group ability; finally, it assists in decarboxylation by preferentially stabilizing the imine tautomer over the enamine, thereby decreasing the active Gibbs free energy barrier of the decarboxylation step.…”

“…Catalysis by amines having more than one primary amino group have also been investigated, and ethylenediamine was reported to be a good catalyst for the decarboxylation of OAA by Leussing and Raghavan [23]. Just recently, a detailed mechanistic study for the diamines-catalyzed decarboxylation of OAA was carried out by Thalji et al [24], which showed that diamine catalyzed decarboxylation required their two amino groups to achieve high rates of decarboxylation, and ethylenediamine was remarkably active catalyst for the decarboxylation of OAA.…”

Mechanismus und Stammzellaktivität der Decarboxylierung von 5‐Carboxycytosin, bestimmt mittels Isotopenverfolgung

“…Für das Isotopenverfolgungsexperiment (Abbildung 1) wurde der DNA-Duplex D1 (ODN1 + ODN2) mit den zwei [ 15 [10,24] Um diese Idee näher zu untersuchen, reduzierten wir den 5-Carboxy-dC-methylester 11 mit NaBH 4 . Die reduzierte Verbindung 12 ist stabil.…”