CHEMICAL MODIFICATION OF GLYCOGEN-SYNTHASE FROM ESCHERICHIA-COLI-B USING THE SUBSTRATE-ANALOG 8-AZIDO ADP-GLUCOSE

Glycogen synthase (E.C.2.4.1.21) in bacteria utilizes ADPglucose as th e glucosyl donor to synthesize the a-l,l-glucan chains present in the glycogen. Results presented here show the specific labeling of the cat alytic site/s for ADPglucose of the glycogen synthase from Escherichia coli using the photoaffinity labeling agent 8-azido ADPglucose. It is shown that 1) 8-Azido ADPglucose can act as a glucosyl donor for the enzyme with V-max equivalent to a 0.05% of the V-max for the natural s ubstrate, ADPglucose and a Km of 0.39 mM (the Km for ADPglucose is 35 mu M). 2) Upon illumination with ultraviolet light (254 nm), the analo g binds to the enzyme in a covalent fashion, and labeling is correlate d with the irreversible loss of enzymatic activity. 3) Addition of ADP glucose (the natural substrate) or ADP (a competitive inhibitor) durin g irradiation of the enzyme-analog mix, protected the enzyme, to a lar ge extent, from loss of activity and from labeling. Conversely, AMP, G lucose-1-P and UDPglucose provided a lower degree of protection, indic ating that photoinactivation of the enzyme by 8-Azido ADPglucose is sp ecific, i.e., the analog binds to the catalytic site.