The Size of the Substrate‐Binding Site of an Aspergillus niger Extracellular Endopolygalacturonase

Abstract

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)

Kinetic studies of the polygalacturonase enzyme from Colletotrichum lindemuthianum

“…Consistent with this interpretation, previous studies have shown that PGA is broken down by PG to tri-, diand mono-galacturonic acids, while only trigalacturonic acid is inhibitory for the reaction [6,7]. Our figure of 55 % hydrolysis would be consistent with the equimolar production of these three products.…”

“…In all the experiments reported here, equal volumes (50 /,J) of enzyme and PGA solutions were mixed in the stopped-flow apparatus. As a routine check of the behaviour of different enzyme preparations, often from different fungal batches, the fluorescence profiles for turnover of 0.25 % PGA by 6 /uM enzyme were compared. The activity of different preparations was also checked for consistency by using the reducing-group assay described above.…”

“…Few enzymological studies have been performed upon these enzymes. However, there have been several reports that the hydrolysis rate of oligogalacturonic acids (OGAs) catalysed by PGs from different organisms decreases upon shortening of the substrate chain [6,7], a process that will ultimately lead to substrate/ product inhibition of the reaction. Several PGs, from different organisms, have been found to be inhibited by high-molecularweight inhibitors produced by plants [8,9].…”

Comparative biochemical and structural characterizations of fungal polygalacturonases

“…Fungal endoPGs also show considerable differences in action patterns on oligogalacturonates. These differences depend on the nature of active site of the enzyme but more specifically on the size of the substrate binding site and position of catalytic group (Rexova-Benkova 1973). Benen et al (1999) studied the kinetics of hydrolysis of oligogalacturonides by PG I, II and C produced by a recombinant strain of Aspergillus niger.…”

Identification of Disulfide Bonds in Protein Proteolytic Degradation Products Using de novo-Protein Unique Sequence Tags Approach

“…A different situation is shown in Figure 5C for plasminogen preactivation peptide. This preactivation peptide has sequence loops that are formed by two disulfide bonds (22). Degradation cleavage occurs not to the loop sequence, but to the C terminal truncation of the preactivation peptide.…”