ACTION PATTERNS AND MAPPING OF THE SUBSTRATE-BINDING REGIONS OF ENDO-(1-]5)-ALPHA-L-ARABINANASES FROM ASPERGILLUS-NIGER AND ASPERGILLUS-ACULEATUS

The substrate binding sites of endo-(1 --> 5)-alpha-L-arabinanases (EC 3.2.1.99) from Aspergillus niger and Aspergillus aculeatus were inves tigated using reduced and regular (1 --> 5)-alpha-L-arabino-oligosacch arides and high performance anion exchange chromatographic analysis. C alculation of bond cleavage frequencies and k(cat)/K-m parameters for these substrates enabled the determination of the number of arabinofur anosyl binding subsites and the estimation of the binding affinities o f each subsite. The A. aculeatus endo-arabinanase has six subsites arr anged symmetrically around the catalytic site, while the A. niger endo -arabinanase has five subsites; two from the catalytic site towards th e non-reducing end of the bound substrate and three toward the reducin g end. The two subsites directly adjacent to the catalytic sites in bo th the A. niger and A. aculeatus endo-arabinanase have near-zero net f ree energy of binding. These results are unlike most glycopyranosyl en do-hydrolases studied which have net negative (unfavourable) energies of interaction at these two subsites, and may be related to the greate r conformational flexibility of arabinofuranosyl residues than glycopy ranosyl residues. The complete Subsite maps are also rationalized with regard to the observed action patterns of these enzymes on linear (1 --> 5)-alpha-L-arabinan. (C) 1997 Elsevier Science Ltd.