A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities

Alberto Puig, Todd P. Primm, Rajendran Surendran, James Lee, Kevin D. Ballard, Ralph S. Orkiszewski, Vladimir Makarov, Hiram F. Gilbert

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Abstract

A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The k(cat) for ribonuclease oxidative folding by weePDI is 0.26 ± 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full- length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.

Original languageEnglish (US)
Pages (from-to)32988-32994
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number52
DOIs
StatePublished - Dec 26 1997

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Protein Disulfide-Isomerases
Isomerases
Thioredoxins
Disulfides
Catalytic Domain
Cysteine
Isomerization
Serine
Protein folding
Biochemistry
Molecules
Protein Folding
Accessories
Mutant Proteins
Enzymes
Ribonucleases
Dimers
Monomers
Amino Acids
Catalysts

ASJC Scopus subject areas

  • Biochemistry

Cite this

Puig, A., Primm, T. P., Surendran, R., Lee, J., Ballard, K. D., Orkiszewski, R. S., ... Gilbert, H. F. (1997). A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities. Journal of Biological Chemistry, 272(52), 32988-32994. https://doi.org/10.1074/jbc.272.52.32988

A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities. / Puig, Alberto; Primm, Todd P.; Surendran, Rajendran; Lee, James; Ballard, Kevin D.; Orkiszewski, Ralph S.; Makarov, Vladimir; Gilbert, Hiram F.

In: Journal of Biological Chemistry, Vol. 272, No. 52, 26.12.1997, p. 32988-32994.

Research output: Contribution to journalArticle

Puig, A, Primm, TP, Surendran, R, Lee, J, Ballard, KD, Orkiszewski, RS, Makarov, V & Gilbert, HF 1997, 'A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities', Journal of Biological Chemistry, vol. 272, no. 52, pp. 32988-32994. https://doi.org/10.1074/jbc.272.52.32988
Puig, Alberto ; Primm, Todd P. ; Surendran, Rajendran ; Lee, James ; Ballard, Kevin D. ; Orkiszewski, Ralph S. ; Makarov, Vladimir ; Gilbert, Hiram F. / A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 52. pp. 32988-32994.
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abstract = "A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The k(cat) for ribonuclease oxidative folding by weePDI is 0.26 ± 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full- length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.",
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AU - Lee, James

AU - Ballard, Kevin D.

AU - Orkiszewski, Ralph S.

AU - Makarov, Vladimir

AU - Gilbert, Hiram F.

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