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

B K Gillard, R C White, R A Zingaro, T E Nelson
1980 Journal of Biological Chemistry  
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
more » ... 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 . 7 mg/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-, Calif. 90024. branching enzyme but not at the polymer binding site The temperature effect on the DATG inactivation ratc is quite large, A H # = 35 kcal/mol, AS$ = 54 e a . Thesc values suggest that DATG inactivation of the de brancher occurs with a significant conformationa change. Rabbit muscle glycogen debranching enzyme (amylo-1,6 glucosidase/4-a-glucanotransferase) possesses two differen active sites on a single polypeptide chain of M, = 160,OOO t c 170,000 (1, 2). It is the only known eukaryotic multicatalykic site enzyme which is active as a monomer (1-4). The twc activities, a transferase (1,4-a-~-glucan:l,4-a-~-glucan-4-a-gly cosyltransferase, EC 2.4.1.25) and a glucosidase (dextrin 6-a glucosidase, EC 3.2.1.33) form the glycogen phosphorylasc limit dextrin debranching system of skeletal muscle. Thc transferase disproportionates the branched symmetric limi dextrin of glycogen formed by phosphorylase (+-dextrin)' t c form an asymmetric structure having a single glucosyl residuc branch. This occurs by transfer of a maltotriosyl group fron the side chain to the main chain. The glucosidase then re moves the residual branch with retention of configuration t c produce free glucose and a debranched +-dextrin with an oute chain susceptible to further degradation by phosphorylasc (1,4-a-~-gh1can:orthophosphate a-glucosyltransferase, E( The transferase and glucosidase activities of debranchinl enzyme can function independently of each other (cf Ref. 8) For example, the glucosidase can catalyze removal of tht single unit glucose branch from 63-a-glucosylmaltotetraos~ ("fast E$,") (5) and from a-(1 + 6)-glucosyl cyclohexaamylose a-glucosyl a-Schardinger dextrin (a-GSD) (9). It will a l s c reincorporate glucose into polysaccharides ( 10,ll). The trans ferase can catalyze elongation of the outer tier chains o amylopectin, glycogen, and +-dextrin (12). The substrate spec ificity of the two individual activities as well as the combinec activity has been reviewed (13). Various assay systems havt been developed, based on these substrate specificities, fo. measurement of the transferase, glucosidase, and combine( transferase-glucosidase activities of debranching enzyme (13) Although the transferase and glucosidase actions occur a two separate catalytic sites on the debranching enzyme poly peptide chaii (8, 12,(14)(15)(16), the enzyme does not bind twc polysaccharide substrate molecules simultaneously (8). Ap parently, polymer binding for the transferase and glucosidasc 'The abbreviations used are: @-dextrin, glycogen phosphorylas limit dextrin; a-SD, a-Schardinger dextrin, cyclohexaamylose; a-GSI: a-glucosyl a-Schardinger dextrin, a-(1 -+ 6)-glucosyl cyclohexa amylose; bis-Tris, 2,2-bis-(hydroxymethyl)-2,2',2"-nitrilotriethano
pmid:6447697 fatcat:cgf7nw4livfi3ndy4z6nyepnbu