Disulfide bonds of GM2 synthase homodimers - Antiparallel orientation of the catalytic domains

GM2 synthase is a homodimer in which the subunits are joined by lumenal dom ain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length:form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonde d with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to fre e thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cle avage at amino acid 330 produced a monomer-sized band under nonreducing con ditions which was converted upon reduction to a 40-kDa fragment and a 24-kD a myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser prod uced monomers but not: dimers, In summary these results demonstrate that Cy s(429) and Cys(476) form an intrasubunit disulfide while the intersubunit d isulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangemen t results in an antiparallel orientation of the catalytic domains of the GM 2 synthase homodimer.