Activation of GlcNAc-P-P-dolichol synthesis by mannosylphosphoryldolichol is stereospecific and requires a saturated alpha-isoprene unit.
Exogenous mannosylphosphoryldolichol (Man-P-Dol) has previously been shown to stimulate UDP-GlcNAc:dolichyl phosphate N-acetylglucosamine 1-phosphate transferase (GPT1), the enzyme catalyzing the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol). To define the structural specificity of the mannolipid-mediated activation of GPT1, the ability of a variety of mannosylphosphorylisoprenols to stimulate GlcNAc-lipid biosynthesis in microsomal preparations from retinas of the embryonic chick has been tested. For these comparisons several Man-P-isoprenols were synthesized enzymatically and chemically. The catalytic efficiency of activation expressed as the Vmax/Ka ratio was substantially higher for Man-P-Dol95 than for mannosylphosphorylpolyprenol95 (Man-P-Poly95), demonstrating that the saturated alpha-isoprene unit of the dolichyl moiety influences the mannolipid-enzyme interaction. The degree of activation increased with chain length and hydrophobicity of the dolichyl moiety when Man-P-dolichols containing 2, 11, and 19 isoprene units were evaluated. A strict stereospecificity was exhibited as beta-Man-P-Dol95 provided a 100-fold greater stimulation than the corresponding alpha-stereoisomer. The recognition of the saturated alpha-isoprene unit, the influence of chain length, and the strict stereospecificity of the interaction between beta-Man-P-Dol and GPT1 extend the description of the mannolipid-enzyme interaction and provide strong new evidence that Man-P-Dol levels can influence the rate of GlcNAc-P-P-Dol synthesis.
Kean
E L
EL
Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio 44106.
Rush
J S
JS
Waechter
C J
CJ
D000818:Animals; D002070:Butadienes / Q000737:chemistry; D002642:Chick Embryo; D004287:Dolichol Monophosphate Mannose / Q000737:chemistry / Q000494:pharmacology*; D045782:Hemiterpenes; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D010420:Pentanes; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism*; D012160:Retina / Q000187:drug effects / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D002070:Butadienes; D045782:Hemiterpenes; D010420:Pentanes; D011103:Polyisoprenyl Phosphate Monosaccharides; C005059:isoprene; D004287:Dolichol Monophosphate Mannose; C018439:N-acetylglucosaminylpyrophosphoryldolichol