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 esterific ation (39-74%) were incubated with the solubilized pure alpha and gamma iso forms of pectinmethylesterase, from mung bean hypocotyl (Vigna radiata), En zyme activity was determined at regular intervals along the deesterificatio n pathway at pH 5.6 and pH 7,6, It has been demonstrated that the distribut ion of the carboxyl units along the pectin backbone controls the activity o f the cell wall pectinmethylesterases to a much greater extent than the met hylation degree, with a random distribution leading to the strongest activi ty. Polygalacturonic acid was shown to be a competitive inhibitor of the al pha 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 activit y was shown to be correlated to the formation of deesterified blocks of 19 +/- 1 galacturonic acid residues through simulations of the enzymatic diges tion according to the mechanisms established previously (Catoire, L,, Pierr on, 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 sub strate had been totally deesterified, Comparison of alpha and gamma isoform cDNAs suggests that the N-terminal region of catalytic domains might expla in 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.