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 d etermined at intervals along the deesterification pathway at pH 5.6 and 7,6 , The latter analyses were performed by C-13 MMR spectroscopy, and the degr ee of esterification was probed by both C-13 MMR and potentiometric measure ments. A dichotomy was observed in the behavior of the alpha and gamma isof orms when compared with that of the beta isoenzyme. Ideal blockwise deester ification mechanisms reproduced the experimental average structures (methyl ester distribution) throughout the course of the reaction. In the case of t he alpha and gamma isoforms, a single chain mechanism associated with a fre e carboxyl group at the second nearest neighbor position could be postulate d at pH 5.6, whereas some multiple attack character was required to reprodu ce 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 value s. Both the nature of the polysaccharides produced in these reactions and t he role of pectinmethylesterase in the cell wall-stiffening process along t he growth gradient are discussed.