The effect of substrate modification on porcine pancreatic alpha-amylase subsite binding: hydrolysis of substrates containing 2-deoxy-D-glucose and 2-amino-2-deoxy-D-glucose.
Modified alpha-D-(1----4)-glucans containing a small proportion of 14C-labeled 2-deoxy-D-glucose or 2-amino-2-deoxy-D-glucose were examined as substrates for porcine pancreatic alpha-amylase (PPA). Cyclomaltoheptaose containing single 2-deoxy-D-glucose residues, synthesized by incubation of 2-deoxyglucosylglycogen with cyclomaltodextrin glucanotransferase in the presence of Triton X-100, was hydrolyzed by PPA to produce 2-deoxy-D-glucose; two isomers of 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxy-D-glucose may be productively bound at all five subsites of the PPA active site. Reaction kinetics and the distribution of products formed suggest, however, that productive binding of the modified residue does not occur readily at the point of catalytic attack (subsite 3) and that the preferred position of hydrolysis of modified substrates may be different from that of unmodified substrates. Results of PPA hydrolysis of glycogen containing [14C]-2-amino-2-deoxy-D-glucose showed that a modified trisaccharide and a modified disaccharide were the smallest substituted products formed. Analysis of these products indicated that they did not contain modified residues at their reducing ends. Formation of the observed 2-amino-2-deoxy-maltooligosaccharides is consistent with a scheme where productive binding of 2-amino-2-deoxy-D-glucose is allowed at subsites 1, 2, 4, and 5, but not at subsite 3, the subsite at which hydrolysis occurs.
Braun
P J
PJ
French
D
D
Robyt
J F
JF
D000818:Animals; D001345:Autoradiography; D003837:Deoxy Sugars / Q000378:metabolism*; D003847:Deoxyglucose / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D013552:Swine; D000516:alpha-Amylases / Q000378:metabolism*
D003837:Deoxy Sugars; D006003:Glycogen; D003847:Deoxyglucose; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D000516:alpha-Amylases; D005944:Glucosamine