TY - JOUR

T1 - Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity

AU - Nørgaard, Per

AU - Winther, Jakob R.

PY - 2001

Y1 - 2001

N2 - Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during the folding of secretory proteins. PDI is composed of domains with structural homology to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild-type and mutant forms we assayed activity in oxidative refolding of reduced and denatured procarboxypeptidase Y as well as refolding of bovine pancreatic trypsin inhibitor. The wild-type protein showed very little activity, not only in oxidative refolding but also in assays where only isomerase activity was required. This was surprising, in particular since mutant forms of Eug1p containing a CXXC motif displayed activity close to that of genuine PDI. These results lead us to propose that general disulphide isomerization is not the main function of Eug1p in vivo.

AB - Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during the folding of secretory proteins. PDI is composed of domains with structural homology to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild-type and mutant forms we assayed activity in oxidative refolding of reduced and denatured procarboxypeptidase Y as well as refolding of bovine pancreatic trypsin inhibitor. The wild-type protein showed very little activity, not only in oxidative refolding but also in assays where only isomerase activity was required. This was surprising, in particular since mutant forms of Eug1p containing a CXXC motif displayed activity close to that of genuine PDI. These results lead us to propose that general disulphide isomerization is not the main function of Eug1p in vivo.

KW - Amino Acid Motifs

KW - Animals

KW - Aprotinin

KW - Binding Sites

KW - Carboxypeptidases

KW - Catalytic Domain

KW - Cathepsin A

KW - Cattle

KW - Disulfides

KW - Escherichia coli

KW - Fungal Proteins

KW - Glutathione

KW - Insulin

KW - Mutation

KW - Oxygen

KW - Plasmids

KW - Protein Disulfide-Isomerases

KW - Protein Folding

KW - Protein Structure, Tertiary

KW - Saccharomyces cerevisiae

KW - Saccharomyces cerevisiae Proteins

KW - Thioredoxins

KW - Time Factors

U2 - 10.1042/bj3580269

DO - 10.1042/bj3580269

M3 - Journal article

C2 - 11485577

VL - 358

SP - 269

EP - 274

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -