Control of Mung bean pectinmethylesterase isoform activities. Influence of pH and carboxyl group distribution along the pectic chains.

Well-characterized pectin samples with a wide range of degrees of esterification (39-74%) were incubated with the solubilized pure alpha and gamma isoforms of pectinmethylesterase, from mung bean hypocotyl (Vigna radiata). Enzyme activity was determined at regular intervals along the deesterification pathway at pH 5.6 and pH 7.6. It has been demonstrated that the distribution of the carboxyl units along the pectin backbone controls the activity of the cell wall pectinmethylesterases to a much greater extent than the methylation degree, with a random distribution leading to the strongest activity. Polygalacturonic acid was shown to be a competitive inhibitor of the alpha isoform activity at pH 5.6 and to inhibit the gamma isoform activity at both pH 5.6 and pH 7.6. Under these conditions, the drop in enzyme activity was shown to be correlated to the formation of deesterified blocks of 19 +/- 1 galacturonic acid residues through simulations of the enzymatic digestion according to the mechanisms established previously (Catoire, L., Pierron, M., Morvan, C., Herve du Penhoat, C., and Goldberg, R. (1998) J. Biol. Chem. 273, 33150-33156). However, even in the absence of inhibition by the reaction product, activity dropped to negligible levels long before the substrate had been totally deesterified. Comparison of alpha and gamma isoform cDNAs suggests that the N-terminal region of catalytic domains might explain their subtle differences in activity revealed in this study. The role of pectinmethylesterase in the cell wall stiffening process along the growth gradient is discussed.

Investigation of the action patterns of pectinmethylesterase isoforms through kinetic analyses and NMR spectroscopy. Implications In cell wall expansion.

Well characterized pectin samples were incubated with cell wall-bound and -solubilized pure isoforms of pectinmethylesterase from mung bean hypocotyls (Vigna radiata). Both enzyme activity and average product structure were determined at intervals along the deesterification pathway at pH 5.6 and 7.6. The latter analyses were performed by 13C NMR spectroscopy, and the degree of esterification was probed by both 13C NMR and potentiometric measurements. A dichotomy was observed in the behavior of the alpha and gamma isoforms when compared with that of the beta isoenzyme. Ideal blockwise deesterification mechanisms reproduced the experimental average structures (methylester distribution) throughout the course of the reaction. In the case of the alpha and gamma isoforms, a single chain mechanism associated with a free carboxyl group at the second nearest neighbor position could be postulated at pH 5.6, whereas some multiple attack character was required to reproduce the data at pH 7.6. Several mechanisms that differed from the preceding ones were compatible with the data for the beta isoform at the two pH values. Both the nature of the polysaccharides produced in these reactions and the role of pectinmethylesterase in the cell wall-stiffening process along the growth gradient are discussed.

An efficient procedure for studying pectin structure which combines limited depolymerization and 13C NMR.

A protocol for partial thermally-induced depolymerization of differently methoxylated pectin samples is described. The resulting macromolecules have been fully characterized with various complementary techniques, such as size exclusion chromatography (SEC), potentiometry, viscometry and 13C NMR. Optimum conditions afford samples at 50-80% yield with weight-average molecular weights in the 4 to 20 kDa range. The major fraction of these polysaccharides adopts the random-coil conformation and such samples are suitable for 13C NMR structural studies at room temperature. The methoxyl distributions of two apple pectin samples with a degree of esterification (DE) between 54 and 74% and a citrus pectin (DE, 72%) were shown to be random in nature, whereas that of a lightly methoxylated apple pectin (DE 39%) was partially blockwise. The carbon relaxation parameters of the depolymerized pectins attain asymptotic values for Mw > 4 kDa. The Mw values estimated from intrinsic viscosity data with the Mark-Houwink relationship reported for native pectins are in good agreement with those obtained by either end-group analysis (NMR) or SEC. Thus, all the physicochemical data indicate that the secondary structure of the isolated chains of depolymerized pectin is closely related to that of the parent polymers. Finally, pectinmethylesterase activity towards the depolymerized pectins was similar to that of the untreated samples.