Abstract
Aspartate beta-decarboxylase catalyzes abortive decarboxylation/transamination of [2-3H]aspartate with at least 17% internal transfer of tritium to the pro-S position at C-4' of the resulting pyridoxamine phosphate. In the normal beta-decarboxylation reaction, at least 1.06% of the tritium from the alpha-position of aspartate appears in the product alanine. The enzyme catalyzes slow hydrogen exchange from the beta-position of alanine but not aspartate. The replacement of the beta-carboxyl group of aspartate by hydrogen occurs in an inversion mode. These results are interpreted in terms of a two-base mechanism.
Highlights
From the +Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907 and the §Departments of Chemistry and Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
By are hydrogen occursainninversion interpreted in terms of a twoaminase was a gift from Dr LeVerne Schirch, Medical College of Virginia, L-aspartase was purchased from Sigma, and DzO from J.T
The chirality of the methyl group of acetate was determined by the method of Cornforth et al [8] and Arigoni et al [9] using a previously described procedure [10].The configuration at C-4' of [4"3H]pyridoxamine was determined by stereospecific equilibration of the pro-4's hydrogen with solvent protons catalyzed by pyridoxamine pyruvate transaminase as described by Voet et al [11]
Summary
Radioactivity was measured in Aquasol with ["C]- and r3H]toluene as an internal standard in a Beckman LS-7OOO liquid scintillation counter. Protein concentrations were measured at 280 nm. The chirality of the methyl group of acetate was determined by the method of Cornforth et al [8] and Arigoni et al [9] using a previously described procedure [10].The configuration at C-4' of [4"3H]pyridoxamine was determined by stereospecific equilibration of the pro-4's hydrogen with solvent protons catalyzed by pyridoxamine pyruvate transaminase as described by Voet et al [11]. Assay of Aspartate P-Decarboxylase-Enzyme activity could be measured by followingthe oxidation of NADH at 340 nm in a coupled assay: aspartate+
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