Abstract
GM2 synthase is a homodimer in which the subunits are joined by lumenal domain 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 bonded 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 free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.
Highlights
GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s)
All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys429 and Cys476 forming a disulfide-bonded pair while Cys80 and Cys82 are disulfide bonded in combination with Cys412 and Cys529
When the soluble form of GM2 synthase was analyzed in this way, no biotinylated peptides were found, suggesting that no Cys residues with free thiol groups were present
Summary
Double mutation of Cys and Cys to Ser produced monomers but not dimers In summary these results demonstrate that Cys429 and Cys476 form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys and Cys with Cys412 and Cys529 are responsible for formation of the homodimer. In this report we have utilized protein chemistry experiments, coupled with mass spectrometric analyses, to determine: 1) that all Cys of the soluble form of GM2 synthase are involved in disulfide bonds; and 2) which disulfides are responsible for dimer formation. These results demonstrate that in the dimer the NH2 terminus of one subunit is close to the COOH terminus of the other subunit in space
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