Yeast mannosyl transferases requiring dolichyl phosphate and dolichyl phosphate mannose as substrate. Partial purification and characterization of the solubilized enzyme.
The first mannosyl unit of manno-oligosaccharides of fungal mannoproteins is transferred in a dolichyl-phosphate-dependent reaction sequence to serine/threonine residues of the protein. The two membrane-bound enzymes catalyzing this transfer in the yeast Saccharomyces cerevisiae have been solubilized by detergents. The enzyme transferring mannose from guanosine diphosphate mannose to dolichyl phosphate has been purified 18-fold when based on membrane protein and 140-fold when based on total cell protein. The enzyme transferring mannose from dolichyl phosphate mannose to protein has been purified 48-fold and 380-fold, respectively. A HCl-treated cell-wall mannoprotein from yeast served as acceptor protein for the second enzyme. The solubilized enzyme catalyzing the formation of dolichyl diphosphate mannose has a Km for guanosine diphosphate mannose of 7 x 10(-6) M and is saturated with about 0.15 mM yeast dolichyl phosphate. The metal requirement, pH-optima, and the detergent concentration necessary for optimal activity have been determined for both solubilized enzymes.
D002845:Chromatography; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism*; D004288:Dolichol Phosphates / Q000378:metabolism*; D006602:Hexosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008364:Mannosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012995:Solubility
D004288:Dolichol Phosphates; D011105:Polyisoprenyl Phosphate Sugars; D011106:Polyisoprenyl Phosphates; D011092:Polyethylene Glycols; D008345:Manganese; D004287:Dolichol Monophosphate Mannose; D017830:Octoxynol; D006602:Hexosyltransferases; D008364:Mannosyltransferases; D008274:Magnesium