Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast.
Preparation and purification of substrate amounts of radioactive as well as non-radioactive dolichyl diphosphate N-acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I-III). The test conditions are described in detail. All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35-fold and 70-fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+. The Km values for UDP-N-acetylglucosamine for enzyme I and II were 15 and 10 muM, respectively, and for GDP-mannose (enzyme III) 7 muM. The apparent Km values for the lipophilic acceptor was 180 muM for enzyme I (dolichyl phosphate), 40 muM for enzyme II (dolichyl diphosphate N-acetylglucosamine) and 17 muM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol X h-1 X mg protein-1. All reactions were inhibited by nucleoside diphosphates.
D002462:Cell Membrane / Q000201:enzymology; D004224:Diterpenes / Q000378:metabolism*; D004286:Dolichols / Q000378:metabolism*; D005964:Glucosyltransferases / Q000302:isolation & purification*; D006602:Hexosyltransferases / Q000302:isolation & purification*; D008364:Mannosyltransferases / Q000302:isolation & purification*; D017351:N-Acetylglucosaminyltransferases; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012995:Solubility; D017855:Transferases (Other Substituted Phosphate Groups)
D004224:Diterpenes; D004286:Dolichols; D005964:Glucosyltransferases; D006602:Hexosyltransferases; D008364:Mannosyltransferases; D017351:N-Acetylglucosaminyltransferases; C035783:UDP-N-acetylglucosamine-dolichyldiphosphate N-acetylglucosamine, N-acetylglucosamine transferase; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase; D017855:Transferases (Other Substituted Phosphate Groups); C023222:UDPacetylglucosamine-dolichyl-phosphate acetylglucosamine-1-phosphate transferase