Nature of sites hydrolyzable by endopolygalacturonase in partially-esterified homogalacturonans

Endopolygalacturonases (EPGs) hydrolyze glycosidic linkages between galacturonic acid (GalA) residues in polygalacturonans, a major fraction of plant pectins. However, most polygalacturonans occur naturally with some of the GalA residues methylesterified. Methylesterification is known to inhibit the activity of EPG. A commercial apple pectin sample was partially saponified to give three pectin samples of degree of methylesterification (DM) 52, 32, and 16. Each was digested to completion by a cloned EPG of Erwinia carotovora subsp. carotovora and the sizes of the resulting fragments were determined by HPLC. A mathematical model was devised to predict the distribution of fragment lengths expected from the three different DM pectins depending on how many adjacent GalA residues are necessary for the enzyme to be able to bind and act on its substrate. For all three DM pectins, the enzyme digestion pattern fit fairly well to that predicted if the enzyme needs four adjacent non-esterified residues to act. As an independent test for the substrate requirements for the enzyme, we determined the maximum cluster size of adjacent non-esterified GalA residues, which remained after complete EPG digestion of the three pectin samples. In each case it was three residues. Thus both methods show that the EPG of E. carotovora needs four adjacent non-esterified GalA residues within a partially-esterified region to be able to act. Copyright (C) 1996 Published by Elsevier Science Ltd