The mammalian glycogen debranching enzyme amylo-1,6-glucosidase/4-α-glucanotransferase is a eucaryotic enzyme which possesses two distinct activities on a single polypeptide chain. The transferase (1,4-α-D-glucan:1,4-α-D-glucan 4-α-glycosyltransferase, EC 18.104.22.168) and glucosidase (dextrin 6-α-glucosidase, EC 22.214.171.124) comprise the glycogen phosphorylase limit dextrin debranching system in muscle. The two activities appear to be located at separate catalytic sites on the enzyme molecule. The relationships between the catalytic and binding sites on this bifunctional enzyme have been investigated with the use of reversible substrate model inhibitors. Polyhydroxyamines were found to inhibit glucosidase activity. The best inhibitor was Nojirimycin (5-amino-D-glucose), with a Ki = 3.9 × 10-6 M, compared with a Km = 4.3 × 10-2 M for glucose. The amine inhibitors are noncompetitive with phosphorylase limit dextrin, but competitive with glucose. The strength of binding indicates that the amines are transition state analogues and apparently mimic the structure of an activated glucosyl ion complex formed by the glucosyl-enzyme intermediate during hydrolysis. The effectiveness of inhibition increases with increasing acidity of the conjugate acid of the amine, with a Brønsted slope of 1.3. Binding of inhibitor to enzyme occurs with proton transfer from the conjugate acid of the amine to an enzyme amino acid residue whose pKa is about 8.5. Transferase activity was not measurably inhibited by any of the compounds tested. Inhibition of glucosidase, without concomitant inhibition of transferase, provides further evidence for two distinct active sites on the debranching enzyme molecule. α-Schardingerdextrin (cyclohexaamylose) and glycogen were both competitive inhibitors of debrancher action on phosphorylase limit dextrin. Apparently, polymer binding for transferase and glucosidase action does not occur at two independent binding sites, but rather at a single site or at sites which overlap or interact very strongly. Based on these results we propose a mechanism of action for the debranching enzyme in which polysaccharide binding serves to coordinate the action of the two catalytic sites on the enzyme, so as to allow a concerted degradation of the branched chain of the limit dextrin structure.
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