Photoaffinity Labeling of Undecaprenyl Pyrophosphate Synthetase

The prenyl transferase undecaprenyl p rophosphate 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 Cas to Czs. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [‘H]2-diazo-3trifluoropropionyloxy 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 sulfatepolyacrylamide 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 MgClz 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 ‘H-photoprobe, [14C]isopentenyl pyrophosphate, and MgCl2, the labeled polypeptide gave a ratio of 14C/’H 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.