Oxidative folding and N-terminal cyclization of onconase.
Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally.
Welker
Ervin
E
Institute of Biochemistry, Biological Research Centre of the Hungarian Academy, H-6701 Szeged, TemesvAri krt. 62, Hungary.
Hathaway
Laura
L
Xu
Guoqiang
G
Narayan
Mahesh
M
Pradeep
Lovy
L
Shin
Hang-Cheol
HC
Scheraga
Harold A
HA
D000818:Animals; D004220:Disulfides / Q000737:chemistry; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry / Q000378:metabolism; D011894:Rana pipiens; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D012260:Ribonucleases; D011761:Pyrrolidonecarboxylic Acid; C058703:ranpirnase