Mechanism of activation of glucocerebrosidase by co-beta-glucosidase (glucosidase activator protein).
The nature of the stimulatory action of the protein 'coglucosidase' on glucocerebrosidase was investigated with the use of highly purified cofactor from bovine spleen, radioactive glucosyl ceramide and methylumbelliferyl-beta-glucoside. A complex between glucosidase and either substrate could not be detected under equilibrium and non-equilibrium binding conditions. Complex formation between stimulating protein and the enzyme could be shown by the binding of the enzyme to an affinity column containing coglucosidase. This binding could be blocked by adding phosphatidylserine to the enzyme. The lipid also stimulated the enzyme. Additional evidence for binding of the enzyme to the two kinds of stimulators was the finding that they protected the enzyme against inactivation by N-ethylmaleimide and chloromercuriphenylsulfonate. A role for lipids in the stimulatory action of coglucosidase was shown by extracting lipids from the enzyme; this resulted in a loss of basal enzyme activity and of sensitivity to activation by the protein. Adding back to the lipids or phosphatidylserine increased the sensitivity of the delipidated enzyme to coglucosidase. Using the crude, unextracted enzyme we could show that low concentrations of phosphatidylserine augmented the effectiveness of coglucosidase but high concentrations of the lipid blocked the effect of the protein. It is proposed that lipids, particularly acidic ones, act on solubilized glucocerebrosidase to produce an enzyme conformation which allows binding and stimulation by coglucosidase. At higher lipid concentrations, the acidic lipids bind, in competition with coglucosidase, to the latter's binding site on the enzyme.
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D005959:Glucosidases / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D005962:Glucosylceramidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005963:Glucosylceramides / Q000378:metabolism; D006023:Glycoproteins; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D010718:Phosphatidylserines / Q000494:pharmacology; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology*; D049231:Saposins; D013154:Spleen / Q000201:enzymology
D005960:Glucosides; D005963:Glucosylceramides; D006023:Glycoproteins; D010718:Phosphatidylserines; D011506:Proteins; D049231:Saposins; C014110:4-methylumbelliferyl glucoside; D006923:Hymecromone; D002731:4-Chloromercuribenzenesulfonate; D005959:Glucosidases; D005962:Glucosylceramidase; D005033:Ethylmaleimide