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

Protein folding catalysed by protein disulphide isomerase (PDI) has been st udied 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 serin e residues. The activity of the mutant forms of yPDI was determined quantit atively by following the refolding of bovine pancreatic trypsin inhibitor i n vitro. Ln this assay the activity of the wild-type yPDI is quite similar to that of human PDI, both in rearrangement and oxidation reactions. Howeve r, while the a domain active site of the human enzyme is more active than t he 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 nativ e yeast substrate, procarboxypeptidase Y. In this assay, however, the a' do main active site also appeared to be much more potent than the a-site. Thes e results were unexpected, not only because of the difference with human PD I, but also because analysis of folding of procarboxypeptidase Y in vivo ha d shown the a-site to be most important. We furthermore show that the appar ent difference between in vivo and in vitro activities is not due to cataly tic contributions from the other PDI homologues found in yeast. (C) 1999 Ac ademic Press.