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 13 C NMR spectroscopy, and the degree of esterification was probed by both 13 C NMR and potentiometric measurements. A dichotomy was observed in the behavior of the ␣ and ␥ isoforms when compared with that of the ␤ isoenzyme. Ideal blockwise deesterification mechanisms reproduced the experimental average structures (methylester distribution) throughout the course of the reaction. In the case of the ␣ and ␥ 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 ␤ 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.Pectins that represent around 30% of the primary plant cell walls play a key role in plant physiology as well as in plant pathology. The general structure of pectic polymers consists of homogalacturonan linear chains (smooth regions) interspersed with highly branched galacturonic chains (hairy regions). Some of the galacturonic residues linked by ␣-1,4 glycosidic bonds are methyl-esterified at the carboxyl group. The degree of methylesterification (DE) 1 varies greatly depending on the plant organ and the degree of differentiation of the cells. Young, plastic cell walls are generally characterized by a high content of highly methylated pectin that decreases in parallel with the loss of extensibility of the walls, whereas the amount of acidic residues increases (1-3). Moreover, the balance between high and low methylated pectins varies inside the wall of a single cell (4) generating microdomains. Not only the number but also the distribution of free, unesterified galacturonate carboxyl groups within the galacturonan regions will control the gellforming capacity of the pectin and thereby the porosity and the extensibility of the apoplasm (5, 6). It is commonly accepted that the polygalacturonic backbone is polymerized in the cisGolgi cisternae, methylesterified in the medial-Golgi, substituted with side chains in the trans-Golgi, and exported to the cell walls as a highly methylated polygalacturonan (7). At a later stage, it is deesterified in muro by cell wall pectinmethylesterases (PMEs). Many proteins exhibiting PME activity have been purified, and their biochemical features such as molecular weight, optimal pH, pI, and substrate specificity have been established (8). In some cases, the corresponding genes have also been cloned and sequenced (9 -11...

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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.

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Well-characterized pectin samples with a wide range of degrees of esterification (39 -74%) were incubated with the solubilized pure ␣ and ␥ 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 ␣ isoform activity at pH 5.6 and to inhibit the ␥ 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 ␣ and ␥ 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. Pectinmethylesterases (PMEs)1 are cell wall-bound proteins present in almost all plants and phytopathogenic microorganisms. They modify pectic homogalacturonan chains, generating free carboxyl groups along the polygalacturonan backbone and releasing protons into the apoplasm. Their action can therefore lead to antinomical effects, especially in primary cell walls. On one hand, the pH decrease should enhance expansin activity and in turn increase the cell wall extensibility (1), but, on the other hand, the generation of carboxyl blocks would be expected to allow the formation of multichain structures via calcium bridges (2). Such structures would greatly affect the physical properties of pectin, due to the assembly of pectic chains into expanded, highly hydrated gel networks decreasing cell wall porosity and also cell wall extensibility (3). Such Ca 2ϩ bridging requires the occurrence of nearly 10 successive carboxyl groups (4) and implies that the PMEs work processively along the galacturonan chain. Unfortunately, although PME biochemical and molecular characteristics have been widely investigated (5-8), a limited number of studies of their action pattern have been reported (9 -13). Recently, using partially depolymerized pectin samples and different PME isoforms extracted from mung bean hypocotyl cell walls, we (14) determined both enzyme activity and average product structure at regular intervals along the deesterification pathway. Simulations of different mechanisms...