Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
The effect of substrate modification on the action of PPA has been determined for five (alpha)-(1(--->)4)-glucans in which a small proportion of the glucose residues (15% or less) contained modified functional groups. Chemical and enzymatic techniques were used to synthesize four substrates (6-deoxyamylose, mono-6-deoxy-6-fluoro-(beta)-cyclodextrin, mono-2-deoxy-(beta)-cyclodextrin, and amylose containing D-allose residues) in which hydrogen bond-forming capabilities were altered or removed. Hydrolysis of these substrates by PPA produced two isomers of modified disaccharides and modified monosaccharide. These results indicate that modifications of this type may be productively bound at subsites 1 through 4 of the five-subsite active site. Evidence for productive binding at subsite 5 was also obtained for substrates containing D-allose and 2-deoxy-D-glucose. For each of these modified substrates, formation of the modified disaccharide substituted in the nonreducing residue is favored over formation of the disaccharide substituted in the reducing residue, suggesting that modified residues are bound less readily than D-glucose at subsite 3, the subsite where catalytic attack occurs. Kinetic data for 2-deoxy-oligosaccharides indicated that PPA hydrolysis of glucosidic linkages of 2-deoxy-D-glucose is slower than enzymolysis of normal glucosidic bonds. A modified glycogen which contained 2-amino-2-deoxy-D-glucose was also hydrolyzed by PPA. This reaction produced no modified monosaccharide, a single disaccharide in the nonreducing residue, two trisaccharides modified in the nonreducing and middle residues, and larger products. Analysis of the substituted products indicated that 2-amino-2-deoxy-D-glucose may be productively bound at subsites 1, 2, 4, and 5, but not at subsite 3. These results are compared with data obtained from other modified PPA substrates and interpreted in terms of the enzyme-substrate binding requirements.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Paul J. Braun
Braun, Paul J., "Porcine pancreatic α-amylase hydrolysis of modified substrates and the specificity of subsite binding " (1984). Retrospective Theses and Dissertations. 8149.