Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue.
The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [3H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cation. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [14C]isopentenyl pyrophosphate, and MgCl2, the labeled polypeptide gave a ratio of 14C/3H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cation are present.
D000345:Affinity Labels / Q000494:pharmacology*; D019883:Alkyl and Aryl Transferases; D003979:Diazonium Compounds / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D014166:Transferases / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000345:Affinity Labels; D003979:Diazonium Compounds; C046509:2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C023266:undecaprenyl pyrophosphate synthetase