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 s hown to stimulate UDP-GlcNAc:dolichyl phosphate N-acetylglucosamine 1- phosphate transferase (GPT1), the enzyme catalyzing the biosynthesis o f N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol). To defi ne the structural specificity of the mannolipid-mediated activation of GPT1, the ability of a variety of mannosylphosphorylisoprenols to sti mulate GlcNAc-lipid biosynthesis in microsomal preparations from retin as of the embryonic chick has been tested. For these comparisons sever al Man-P-isoprenols were synthesized enzymatically and chemically. The catalytic efficiency of activation expressed as the V-max/K-a ratio w as substantially higher for Man-P-Dol(95) than for mannosyiphosphorylp olyprenol(95) (Man-P-Poly(95)), 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 stereospecifici ty was exhibited as P-Man-P-Dol(95) provided a 100-fold greater stimul ation than the corresponding alpha-stereoisomer. The recognition of th e saturated alpha-isoprene unit, the influence of chain length, and th e strict stereospecificity of the interaction between P-Man-P-Dol and GPT1 extend the description of the mannolipid-enzyme interaction and p rovide strong new evidence that Man-P-Dol levels can influence the rat e of GlcNAc-P-P-Dol synthesis.