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Publisher Summary This chapter focuses on purification and characterization of undecaprenylpyrophosphate synthetase. Undecaprenylpyrophosphate (C 55 PP) synthetase catalyzes the biosynthesis of the long-chain polyprenyl pyrophosphate required as a carbohydrate carrier in the biosynthesis of a variety of bacterial cell envelope components. The undecaprenylpyrophosphate synthetases catalyze the addition of only cis-isoprene residues instead of trans residues during product formation and require detergent or phospholipid for in vitro activity. Principle and procedure of assay method is described in the chapter. Undecaprenylpyrophosphate synthetase activity is determined by measuring the amount of an acid-labile polyprenyl pyrophosphate produced from the reaction of isopentenyl pyrophosphate (IPP) with the allylic isoprenyl pyrophosphate, farnesyl pyrophosphate (FPP). The assay solutions contain in a final volume of 0.5 ml: enzyme, I00 mM Tris-HCl buffer (pH 7.5), 0.5% Triton X-100, 200/μM MgCI2, 5 lμM trans,trans-FPP, and 30 μM IPP (0.5 mCi/mmol, 15,000 dpm). The enzyme is added last to start the reaction. This also results in more reproducible assays, because the enzyme is stabilized by detergent in dilute protein solutions. The enzyme may be assayed satisfactorily with 8-60/μM IPP and 5-50 lμM trans,trans-FPP.

Publisher Summary This chapter designs, prepares and tests photolabile analogs of the allylic pyrophosphate substrate of prenyltransferases as substrates, inhibitors, or covalent modifying agents for the long-chain prenyltransfcrase, C 55 PP synthetase. The synthesis of photolabile analogs of isoprenoid compounds provides a tool for probing the structure of the substrate binding sites of a variety of prenyltransferases. Recently, ones have synthesized two photolabile analogs of the allylic pyrophosphate substrates, geranyl and trans,trans-farnesyl pyrophosphate, which bear the 2-diazo-3-trifluoropropionyloxy (DATFP) moiety. DATFP containing lipids have been used subsequently as effective probes of lipid-lipid and lipid-protein interactions. The availability of photolabile prenyltransferase substrates now offers a number of other opportunities for their application in studying prenyltransferases. The ability to prepare long chain photolabile polyprenyl phosphates such as photoactivatable undecaprenyl- and dolichylmonophosphates would be of significant value in providing a tool to probe enzymes or enzyme complexes, which use the parent prenyl phosphates as glycosyl carriers in bacterial cell wall biosynthesis or mammalian glycoprotein biosynthesis.