Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III.
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 His(6)-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. The investigation of the effect of thiol reagent 4,4'-dithiodipyridine (DTDP) on all five Cys to Ser single protein variants showed that Cys639 and Cys518 are more reactive than the remainder. Only the C639S mutant protein displayed the remarkable resistance against p-hydroxy-mercuribenzoate (pHMB) indicating that modification of Cys639 is responsible for the fast inactivation of yeast DPP III by this sulfhydryl reagent.
Jajcanin-Jozić
Nina
N
Ruder Bosković Institute, Zagreb, Croatia.
Deller
Sigrid
S
Pavkov
Tea
T
Macheroux
Peter
P
Abramić
Marija
M
D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004220:Disulfides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011725:Pyridines / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D004220:Disulfides; D011725:Pyridines; D013438:Sulfhydryl Compounds; C007542:4,4'-dipyridyl disulfide; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D003545:Cysteine