Affinity labeling of phosphoglycerate kinase by 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine.
Yeast phosphoglycerate kinase is irreversibly inactivated upon incubation with 5'-[p-(fluorosulfonyl)-benzoyl]-1-N6-ethenoadenosine (5'-FSB epsilon A), an analogue to the nucleotide substrate. Marked protection against inactivation occurs with MgATP, ATP, MgADP, ADP, and 3-phosphoglycerate, suggesting that a part of the catalytic center is modified. The time dependence of the inactivation is characterized by a nonlinear kinetic profile. Curve fitting of various models for ligand binding to the enzyme suggested a two-site model. Modification of one of the sites appears to protect the catalytically essential site from modification. Stoichiometric studies show that the relationship between moles of 5'-FSB epsilon A incorporated per mole of enzyme and the residual enzymatic activity also shows nonlinear behavior. An extrapolated value of 1.5 mol of bound label/mol of enzyme corresponds to complete inactivation. The apparent overall pseudo first-order rate constant for the reaction between phosphoglycerate kinase and 5'-FSB epsilon A, as well as the separate rate constants for the modification, exhibit saturation behavior with respect to the concentration of 5'-FSB epsilon A, indicative of a rapid reversible binding of the reagent to the enzyme prior to modification.
Wiksell
E
E
Department of Chemistry and Molecular Biology, Swedish University of Agricultural Sciences, Uppsala.
Larsson-Raźnikiewicz
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M
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D001665:Binding Sites; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008962:Models, Theoretical; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000345:Affinity Labels; C029342:5'-(4-fluorosulfonylbenzoyl)-1,N(6)-ethenoadenosine; D010735:Phosphoglycerate Kinase; D008274:Magnesium; D000241:Adenosine