Biosynthesis of the polygalacturonic acid chain of pectin by a particulate enzyme preparation from Phaseolus aureus seedlings.

Pectin is an important structural component of the cell walls of all higher plants and is formed primarily during the early stages of cellular growth.1 The basic building unit of this polymer is known to be D-galactopyranosyluronic acid in which the linear skeleton of these units is connected by a-1,4-D-glycosidic linkages.2 The carboxyl groups of this compound are methylated to various degrees, and polygalacturonate is associated with other carbohydrates, mainly with D-galactan and L-araban. Although a considerable amount of information is available pertaining to the hydrolytic action of a number of enzymes on pectin, virtually nothing is known concerning the mechanism of biosynthesis of this polymer. UDP-a-D-galacturonic acid was shown to exist in mung beans (Phaseolus aureus).3 A dehydrogenase capable of oxidizing UDP-D-glucose to UDP-D-glucuronic acid4 and a UDP-D-glucuronic acid 4-epimerase which interconverts UDP-D-glucuronic acid and UDP-D-galacturonic acid have been found in plants.5 Furthermore, enzymes that catalyze the formation of D-galacturonic acid 1-phosphate6 and UDP-D-galacturonic acid,7 leading to the formation of the same uronic acid nucleotide in plants by an alternate mechanism, have also been found in mung beans. The sequence of these enzymatic reactions has led to the hypothesis that UDP-Dgalacturonic acid is a precursor of pectin. In addition, preliminary experiments using nucleoside diphosphogalacturonic acid with different bases as precursors for pectin formation by particulate fractions of various plants indicated that UDP-Dgalacturonic acid was the most likely precursor.8 However, verification of this indication has been hindered thus far by the lack of a satisfactory cell-free system which would catalyze pectin formation. In this communication we wish to report the synthesis of the D-galacturonic acid chain of pectin with a cell-free enzyme preparation from mung beans. Methods.-Preparation of radioactive substrate: Uniformly labeled C14-D-galactose was obtained through the courtesy of D. McBrien and S. Abraham. It had a specific activity of 127 jsc/,umole and was prepared by a modification of the method described by Bean et al.9 C14-labeled a-Dgalactose 1-phosphate was prepared by incubation of a-D-galactose-CH with D-galactokinase from Saccharomycesfragilis.10 Conversion of C14-a-D-galactose 1-phosphate to labeled a-D-galacturonic acid 1-phosphate was accomplished with about 60% yield using a modification of the catalytic oxidation described by Marsh."1 The radioactive a-D-galacturonic acid 1-phosphate was quantitatively converted to UDP-D-galacturonic acid, labeled in the D-galacturonic acid moiety, by a mung bean pyrophosphorylase-catalyzed reaction with UTP.7 The final product was purified by paper electrophoresis in 0.05M phosphate buffer, pH 7.5, and by paper chromatography with a developing solvent consisting of 95% ethanol-1 M ammonium acetate (7:3), pH 3.8. Radioactive substrate purity: Hydrolysis of the UDP-D-galacturonic acid-C14 with snake venom phosphodiesterase produced galacturonic acid 1-phosphate as the sole radioactive product. A combination of phosphodiesterase and seminal phosphatase degraded this uronic acid nucleotide to galacturonic acid as the only radioactive compound. These results, in conjunction with the single radioactive bands obtained by subjecting the UDP-D-galacturonic acid-C14 to paper electrophoresis at pH 3.7 and 7.5, indicated that the radioactive substrate used in these experiments was pure with respect to radioactivity.