Secondary 18O and Primary 13C Isotope Effects as a Probe of Transition-State Structure for Enzymic Decarboxylation of Oxalacetate

Abstract

A new method for directly measuring 18O isotope effects on decarboxylation reactions has been developed. By running the reaction under high vacuum (10(-5) torr), CO2 leaves the solution before exchange with the oxygens of water to an extent greater than 2%. Thus, the method permits determination of 18O isotope effects with the precision of the isotope ratio mass spectrometer, and without the necessity of resorting to the remote label method and its attendant required syntheses. The method is used to determine 18O isotope effects for decarboxylation of oxalacetate (OAA) by Mg2+, and enzymatically by OAA decarboxylase from Pseudomonas putida; 13C isotope effects are also reported for this enzyme, as well as for decarboxylation of OAA by pyruvate kinase. Initial velocity patterns and pH profiles are reported for the P. putida enzyme, and all available data are used to discuss the kinetic and chemical mechanism of decarboxylation.

Structural Analysis of the Active Site Geometry of N5-Carboxyaminoimidazole Ribonucleotide Synthetase from Escherichia coli,

“…The pseudo-chair conformation of carboxyphosphate allows for the phosphate group to act as an intramolecular base to remove the proton from the acid resulting in the generation of CO 2 and HPO 4 −2 . Such a reaction has precedent in the decomposition of carbamoyl phosphate (25) and in product inhibition studies on biotin carboxylase, which have shown that HPO 4 −2 is the product released from the enzyme (26). …”

Mutants of 4-Oxalocrotonate Tautomerase Catalyze the Decarboxylation of Oxaloacetate through an Imine Mechanism

“…These enzymes include the naturally occurring enzyme, [30] and representative helical-bundle-forming, synthetic, catalytic peptides, YLK-18 (YKLLKELLAKLKWLLRKL-CONH 2 ) [26] and oxaldie 1 (LAKLLKALAKLLKK-CONH 2 ) [25] (Table 1). As can be seen, both 4-OT mutants are significantly better catalysts for this reaction than the de novo designed peptides that employ the imine mechanism.…”

Detailed Reaction Mechanism of Macrophomate Synthase

“…The dissociation constant for Mg(II) showed that macrophomate synthase contains a precisely stoichiometric amount of tightly bound Mg(II). The tight binding of Mg(II) and the high specificity for Mg(II) are relatively unusual in metal-dependent ␤-ketoacid decarboxylases such as malic enzyme (18) and oxalacetate decarboxylase from Pseudomonas putida (19) and Acetobacter xylinum (20), in which both Mg(II) and Mn(II) are equally effective in their catalysis. The K d is at least 20 times lower than those for the enolase superfamily (21), mandelate racemase (8 M) (22) and enolase (1.8 M) (23), which possess relatively higher affinity for divalent metals.…”