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The action pattern and kinetics of an Aspergillus niger extracellular endopolygalacturonase were studied with oligogalacturonic acids (digalacturonic acid through hexagalacturonic acid) and their derivatives in which the terminal aldehyde group was reduced. The rate of hydrolysis catalyzed by the enzyme decreases with the shortening of oligogalacturonide chain length; digalacturonic acid is not hydrolyzed. With tetragalacturonic acid one productive complex is formed resulting in (1 + 3) cleavage. Two and three modes of cleavage occur with pentagalacturonic acid and hexagalacturonic acid, respectively. The enzyme is competitively inhibited by trigalacturonic acid whereas digalacturonic acid does not affect the activity. Reduced pentagalacturonic acid forms one productive complex. Reducing trigalacturonic acid and nonreducing digalacturonic acid are the products of the reaction. Lower reduced oligogalacturonic acids are not degraded by the enzyme.The action pattern and k, values obtained with the oligouronides suggest that the binding site of the endopolygalacturonase contains four subsites and that the catalytic groups of the enzyme are located in the proximity of the first bond, counting from the reducing end, of the substrate segment bound in the complex. Pate1 and Phaff [3] have found that the hydrolysis rate of oligogalacturonic acids catalyzed by yeast endopolygalacturonase decreases with the shortening of the substrate chain. The extremely slow degradation of trigalacturonic acid and the immunity of digalacturonic acid against hydrolysis were ascribed to a protective effect of the terminal galacturonic acid unit.The specifity and action pattern of enzymes acting on homopolymer substrates are determined by the nature of the active centre, i.e. the size and structural properties of the binding site and the location of catalytic groups. A study of the action pattern of an enzyme acting on a series of homopolymer oligomeric substrates can give information about the character of the active centre.In the present study the action pattern of an Aspergillus niger extracellular endopolygalacturonase was investigated with the use of oligogalacturonic acids (digalacturonic acid through hexagalacturonic acid) and of their reduced derivatives as substrates. Further the relationship between the rate parameters and chain length of oligomeric substrates was studied and the results were interpreted with respect Eur. J. Biochem. 39 (1973)

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Nine forms of Aspergillus sp. polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing. Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical. Four forms with free alpha-amino groups have identical start positions but internal amino acid replacements. Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations. Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from other Aspergillus species (72-75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66-71% residue differences toward the Erwinia, tomato, and peach enzymes). Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties.

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