Characterization of recombinant yeast dolichyl mannosyl phosphate synthase and site-directed mutagenesis of its cysteine residues.
Dolichyl mannosyl phosphate synthase is associated with membranes of the rough endoplasmic reticulum and catalyzes mannosyl transfer from GDP-mannose to the hydrophobic long-chain acceptor dolichyl-phosphate. The gene for the yeast enzyme encodes a protein with a molecular mass of 30.36 kDa containing three cysteine residues, at positions 93, 172 and 259 [Orlean, P., Albright, C. & Robbins, P. W. (1988) J. Biol. Chem. 263, 17499-17507]. Inhibition of the synthase by thiol-specific reagents, including N-ethylmaleimide, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2), and lucifer yellow iodoacetamide (LYI), suggests that sulfhydryl groups might play a role in the catalytic mechanism of the enzyme. Titration of the synthase with Nbs2 or LYI indicated that 1 mol sulfhydryl/mol protein was accessible to these reagents, and that saturation of this site completely inhibited enzyme activity. To ascertain the reactive group and its possible function in enzyme catalysis, each of the cysteine residues was replaced individually by site-directed mutagenesis. The mutant enzymes had specific activities comparable to that of the wild-type enzyme, demonstrating that none of the cysteine residues were essential for catalytic activity. All of the mutant proteins except those containing a substitution at Cys93 were inhibited by thiol-blocking reagents, indicating that Cys93 might be physically located near the catalytic site of the enzyme. GDP-mannose, dolichyl phosphate and substrate analogs were found to protect against Nbs2 inactivation, further suggesting that Cys93 was physically near, or within, the substrate-binding site of the enzyme.
Forsee
W T
WT
Department of Microbiology, University of Alabama at Birmingham, 35294-0019, USA.
McPherson
D
D
Schutzbach
J S
JS
D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004288:Dolichol Phosphates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005800:Genes, Fungal; D006155:Guanosine Diphosphate Mannose / Q000494:pharmacology; D008364:Mannosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010755:Organophosphates / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D004288:Dolichol Phosphates; D004791:Enzyme Inhibitors; D010755:Organophosphates; D010743:Phospholipids; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C028306:dolichol monophosphate; D006155:Guanosine Diphosphate Mannose; C074782:phenylphosphate; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D003545:Cysteine