Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo

TY - JOUR

T1 - Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo

AU - Westphal, V

AU - Darby, N J

AU - Winther, Jakob R.

N1 - Copyright 1999 Academic Press.

PY - 1999

Y1 - 1999

N2 - Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (yPDI) has been analysed by exchanging the active-site cysteine residues for serine residues. The activity of the mutant forms of yPDI was determined quantitatively by following the refolding of bovine pancreatic trypsin inhibitor in vitro. In this assay the activity of the wild-type yPDI is quite similar to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast substrate, procarboxypeptidase Y. In this assay, however, the a' domain active site also appeared to be much more potent than the a-site. These results were unexpected, not only because of the difference with human PDI, but also because analysis of folding of procarboxypeptidase Y in vivo had shown the a-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast.

AB - Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (yPDI) has been analysed by exchanging the active-site cysteine residues for serine residues. The activity of the mutant forms of yPDI was determined quantitatively by following the refolding of bovine pancreatic trypsin inhibitor in vitro. In this assay the activity of the wild-type yPDI is quite similar to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast substrate, procarboxypeptidase Y. In this assay, however, the a' domain active site also appeared to be much more potent than the a-site. These results were unexpected, not only because of the difference with human PDI, but also because analysis of folding of procarboxypeptidase Y in vivo had shown the a-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast.

KW - Aprotinin

KW - Binding Sites

KW - Carboxypeptidases

KW - Catalysis

KW - Cathepsin A

KW - Disulfides

KW - Mutation

KW - Oxidation-Reduction

KW - Protein Denaturation

KW - Protein Disulfide-Isomerases

KW - Protein Folding

KW - Saccharomyces cerevisiae

U2 - 10.1006/jmbi.1999.2560

DO - 10.1006/jmbi.1999.2560

M3 - Journal article

C2 - 10047493

SN - 0022-2836

VL - 286

SP - 1229

EP - 1239

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 4

ER -