Amylo-1,6-glucosidase/4-alpha-glucanotransferase. Reaction of rabbit muscle debranching enzyme with an active site-directed irreversible inhibitor, 1-S-dimethylarsino-1-thio-beta-D-glucopyranoside.

Mammalian glycogen debranching enzyme, amylo-1,6-glucosidase/4-a-glucanotransferase, consists of a single polypeptide chain with two different active sites.Studies with reversible substrate model inhibitors have shown that the active sites share a common or overlapping polysaccharide binding site.To define further the reaction mechanism of this multicatalytic site enzyme, the effect of a new irreversible inhibitor has been studied.The inhibitor l-S-dimethylarsino-l-thio-8-D-glucanopyranoside (DATG) reacts with the debranching enzyme to inactivate both the glucosidase and the transferase activities.The two activities are lost at different rates.The rate of inactivation of each activity is first order in both enzyme and inhibitor concentration.Glucosidase and combined glucosidase-transferase activity are lost at the same rate, indicating that the glucosidase is rate-limiting for the combined action on glycogen phosphorylase limit dextrin.The relative rate of inactivation of the glucosidase and transferase activities by DATG is a function of the protein concentration.At high enzyme concentration (>6 mg/ml), the transferase is inactivated twice as rapidly as the glucosidase; at low enzyme concentration ( ~0 . 7mg/ml), the transferase is inactivated only one-half as fast.The rate of inactivation of both activities at high enzyme concentration is much slower, indicating that aggregation of the protein at these concentrations affects the accessibility of DATG to the active sites.The sites of DATG reaction with the debrancher were deduced from the protective effect of a series of substrates and substrate analogs.Glycogen phosphorylase limit dextrin protects both the glucosidase and transferase activities from inactivation by DATG.bis-"ris, (2,2-bis(hydroxy-methyI)-2,2',2"-nitrilotriethanol), a reversible inhibitor which binds specifically at the glucosidase active site, protects the glucosidase but not the transferase against inactivation.a-Schardinger dextrin, which binds to the polymer site, has no protective effect against the action of DATG.The results indicate that DATG acts as an active site-directed irreversible inhibitor at both the glucosidase and the transferase active sites on the de-