Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III |
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Authors: | Nina Jajčanin - Jozić Sigrid Deller Tea Pavkov Peter Macheroux Marija Abramić |
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Affiliation: | 1. Division of Organic Chemistry and Biochemistry, Ru?er Boškovi? Institute, Bijeni?ka, cesta 54, PO Box 180, HR-10002 Zagreb, Croatia;2. Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria;3. Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50-3, A-8010 Graz, Austria |
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Abstract: | Dipeptidyl peptidases III (DPPs III) form a distinct metallopeptidase family characterized by the unique HEXXGH motif. High susceptibility to inactivation by organomercurials suggests the presence of a reactive cysteine residue(s) in, or close to, their active site. Yeast DPP III contains five Cys, none of which is absolutely conserved within the family. In order to identify reactive residue(s), site-directed mutagenesis on yeast His6-tagged DPP III was employed to substitute specifically all five cysteine residues to serine. The variant enzymes thus obtained were enzymatically active and showed an overall structure not greatly affected by the mutations as judged by circular dichroism. Analysis by native and SDS-PAGE under non-reducing conditions revealed the existence of a monomeric and dimeric form in all DPP III proteins except in the C130S, implying that dimerization of yeast DPP III is mediated by the surface-exposed cysteine 130. |
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Keywords: | Dipeptidyl peptidase III Peptidase family M49 Saccharomyces cerevisiae Reactive cysteine residue Site-directed mutagenesis |
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