Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis.
The prenyltransferase involved in the biosynthesis of dolichyl phosphate has been characterized in Saccharomyces cerevisiae. Although the enzyme is predominantly membrane-bound, a significant percentage was found in the soluble fraction. The prenyltransferase preferentially utilizes farnesyl pyrophosphate as the allylic substrate and isopentenyl pyrophosphate as cosubstrate with half-maximal velocities obtained at 25 and 6.7 microM, respectively. The enzymatic activity is sensitive to sulfhydryl reagents and is inhibited by all detergents tested, except 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at concentrations less than 5 mM. The product of the reaction has been characterized as an alpha-unsaturated polyprenyl pyrophosphate, containing 12-15 isoprene units, approximately two isoprene units shorter than the endogenous yeast dolichyl phosphate. The stereochemistry of addition of isoprene units by the prenyltransferase was shown to be cis by a comparison of the HPLC retention time for a pentadecaprenyl phosphate derived from the in vitro reaction product with that for an authentic mixture of alpha-cis- and alpha-trans-pentadecaprenyl phosphates.
Adair
W L
WL
University of South Florida, College of Medicine, Department of Biochemistry, Tampa 33612.
Cafmeyer
N
N
D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D004122:Dimethylallyltranstransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004288:Dolichol Phosphates / Q000096:biosynthesis*; D004286:Dolichols / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*
D003902:Detergents; D004288:Dolichol Phosphates; D004286:Dolichols; D011106:Polyisoprenyl Phosphates; C028306:dolichol monophosphate; D014166:Transferases; D004122:Dimethylallyltranstransferase