Subsite mapping of Aspergillus niger endopolygalacturonase II by site-directed mutagenesis

To assess the subsites involved in substrate binding in Aspergillus niger e ndopolygalacturonase II, residues located in the potential substrate bindin g cleft stretching along the enzyme from the N to the C terminus were subje cted to site-directed mutagenesis. Mutant enzymes were characterized with r espect to their kinetic parameters using polygalacturonate as a substrate a nd with respect to their mode of action using oligogalacturonates of define d length (n = 3-6). In addition, the effect of the mutations on the hydroly sis of pectins with various degrees of esterification was studied. Based on the results obtained with enzymes N186E and D282K it was established that the substrate binds with the nonreducing end toward the N terminus of the e nzyme. Asn(186) is located at subsite -4, and Asp(282) is located at subsit e +2. The mutations D183N and M150Q, both located at subsite -2, affected c atalysis, probably mediated via the sugar residue bound at subsite -1. Tyr( 291), located at subsite +1 and strictly conserved among endopolygalacturon ases appeared indispensable for effective catalysis. The mutations E252A an d Q288E, both located at subsite +2, showed only slight effects on catalysi s and mode of action. Tyr(326) is probably located at the imaginary subsite +3. The mutation Y326L affected the stability of the enzyme. For mutant E2 52A, an increased affinity for partially methylesterified substrates was re corded. Enzyme N186E displayed the opposite behavior; the specificity for c ompletely demethylesterified regions of substrate, already high for the nat ive enzyme, was increased. The origin of the effects of the mutations is di scussed.