ROLE OF THE CYSTEINE RESIDUES IN THE ALPHA-1,2-MANNOSIDASE INVOLVED IN N-GLYCAN BIOSYNTHESIS IN SACCHAROMYCES-CEREVISIAE - THE CONSERVED CYS(340) AND CYS(385) RESIDUES FORM AN ESSENTIAL DISULFIDE BOND

The Saccharomyces cerevisiae alpha 1,2-mannosidase, which removes one specific mannose residue from Man(9)GlcNAc(2) to form Man(8)GlcNAc(2), is a member of a family of alpha 1,2-mannosidases with similar amino acid sequences. The yeast alpha 1,2-mannosidase contains five cysteine residues, three of which are conserved, Recombinant yeast alpha 1,2-m annosidase, produced as the soluble catalytic domain, was shown to con tain two disulfide bonds and one free thiol group using 2-nitro-5-thio sulfobenzoate and 5,5'-dithiobis(2-nitrobenzoate), respectively, Cys48 5 contains the free thiol group, as demonstrated by sequencing of labe led peptides following modification with [H-3]ICH2COOH and by high per formance liquid chromatography/mass spectrometry tryptic peptide mappi ng, A Cys(340)-Cys(385) disulfide was demonstrated by sequencing a pur ified peptide containing this disulfide and by tryptic peptide mapping , Cys(468) and Cys(471) were not labeled with [H-3]ICH2COOH and a pept ide containing these two residues was identified in the tryptic peptid e map, showing that Cys468 and Cys471 form the second disulfide bond, The alpha 1,alpha-mannosidase loses its activity in the presence of di thiothreitol with first order kinetics, suggesting that at least one d isulfide bond is essential for activity, Mutagenesis of each cysteine residue to serine showed that Cys(340) and Cys(385) are essential for production of recombinant enzyme, whereas Cys468, Cys(471), and Cys485 are not re quired for production and enzyme activity, These results i ndicate that the sensitivity to dithiothreitol is due to reduction of the Cys(340)-Cys(385) disulfide, Since Cys(340) and Cys(385) are conse rved residues, it is likely that this disulfide bond is important to m aintain the correct structure in the other members of the alpha 1,2-ma nnosidase family.