A new affinity labeling reagent for the active site of glycogen synthase. Uridine diphosphopyridoxal.
A new affinity labeling reagent for glycogen synthase a from rabbit muscle, uridine diphosphopyridoxal, has been prepared. Incubation of the enzyme with this reagent resulted in a time-dependent, almost complete loss of activity. The inactivation was pseudo-first order, and the results of the kinetic analysis suggested the formation of a noncovalent enzyme-reagent complex prior to the covalent reaction, with a Kinact of 25 microM and a maximal rate constant of 0.22 min-1. The inactivation was pronouncedly protected by UDP-Glc and UDP, but not by the allosteric activator glucose 6-phosphate. The increase in a spectral peak at 425 nm and the decrease in enzymatic activity were well correlated, suggesting that the reagent causes the inactivation of the enzyme by the formation of a Schiff base. The rate of inactivation increased as the pH was raised, giving a pK of 8.85. Almost all the original activity was recovered by the treatment of the inactivated enzyme with cysteamine or any other aminothiol compound. No recovery of the activity, however, was observed with inactivated enzyme which had been treated with NaBH4. A peptide containing the labeled amino acid was isolated for inactivated enzyme after reduction with NaBH4, carboxymethylation, and chymotryptic digestion by fractionation on a Bio-Gel P-6 column and high performance liquid chromatographies. Manual Edman degradation established the sequence as Glu-Val-Ala-Asn-labeled Lys-Val-Gly-Gly-Ile-(Tyr). The introduction of an active site-directing moiety to pyridoxal 5'-phosphate makes the resultant reagent an effective probe for the active site of glycogen synthase.
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006006:Glycogen Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000032:analysis; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry; D014530:Uridine Diphosphate / Q000031:analogs & derivatives*
D000345:Affinity Labels; D010446:Peptide Fragments; D014530:Uridine Diphosphate; D011732:Pyridoxal Phosphate; C045612:uridine diphosphopyridoxal; D006006:Glycogen Synthase