Prenyltransferase; determination of the binding mechanism and individual kinetic constants for farnesylpyrophosphate synthetase by rapid quench and isotope partitioning experiments.
The 1'-4 condensation reaction catalyzed by farnesylpyrophosphate synthetase was examined by using rapid quench and isotope partitioning experiments. E.Mg2+-isopentyl-PP (PP = pyrophosphate) is not catalytically competent, as evidenced by failure to trap the complex with Mg2+-geranyl-PP at concentrations as high as 3.2 mM. In contrast, the concentration of Mg2+-isopentenyl-PP required for one-half maximal trapping (K 1/2) of E.Mg2+-geranyl-PP is 1.4 muM. The results strongly support an ordered mechanism for the 1'-4 condensation reaction, with addition of the allylic substrate before Mg2+-isopentenyl-PP. At short reaction times, a burst phase corresponding to accumulation of Mg2+-farnesyl-PP on the surface of the enzyme, followed by a slower, steady-state release of products, is observed. Evaluation of individual kinetic constants for the reaction indicates that the rates for addition of Mg2+-geranyl-PP to the enzyme (k1 = 4.9 X 10(6) M-1 s-1) and addition of Mg2+-isopentenyl-PP to E.Mg2+-geranyl-PP (k3 = 2 X 10(6) m-1 s-1) are below the diffusion-controlled limit. The rate-limiting step at steady state is isomerization of E.Mg2+-farnesyl-PP-Mg2+-PPi or release of products (k6 = 0.1 s-1). During the course of isotope partitioning experiments with E.Mg2+-geranyl-PP, a new dual isotope procedure was developed which minimizes difficulties encountered during workup. In addition, for enzymes such as farnesyl-PP synthetase that catalyze irreversible reactions, the dual isotope approach is both sensitive extremely easy to execute.
D000475:Alkenes / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001717:Birds; D002250:Carbon Radioisotopes; D004122:Dimethylallyltranstransferase / Q000378:metabolism*; D045782:Hemiterpenes; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011485:Protein Binding; D011865:Radioisotope Dilution Technique; D013997:Time Factors; D014166:Transferases / Q000378:metabolism*
D000475:Alkenes; D002250:Carbon Radioisotopes; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C004809:isopentenyl pyrophosphate; D014166:Transferases; D004122:Dimethylallyltranstransferase; D008274:Magnesium