Kinetic Analysis of the Mechanism and Specificity of Protein-disulfide Isomerase Using Fluorescence-quenched Peptides

Abstract

Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides revealed substantial similarities. Several of the peptides were synthesized in solution, and a quantitative characterization of pre-steady state kinetics was carried out. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 g of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity.One of the reactions associated with folding of secretory proteins is the formation and isomerization of disulfide bonds. This is a slow process when uncatalyzed, but takes place rapidly in vivo, where the enzyme protein-disulfide isomerase (PDI) 1 facilitates the formation and rearrangement of disulfide bonds. PDI is a 57-kDa protein present in the endoplasmic reticulum (ER) of eukaryotes. The protein is organized in an abbac domain structure where the a and b domains are structurally related to thioredoxin and the c domain is rich in acidic amino acid residues (1-4). The catalytic activity of PDI resides in the a and a domains, where the catalytically active cysteine residues are found in a WCGHCK motif in each domain. In each active site the two cysteines cycle between a reduced and an oxidized state, enabling the enzyme to exchange reducing equivalents with substrate sulfhydryls. PDI is capable of catalyzing several kinds of disulfide reactions: 1) oxidation reactions, in which the intramolecular disulfide bond of the CGHC motif is transferred to a pair of sulfhydryls in a substrate, 2) isomerization reactions in which disulfides are rearranged via the formation of a transient, mixed disulfide between the first cysteine residue of the CGHC motif and the substrate (5), and 3) reduction of mixed disulfides, in which PDI catalyzes the reductive cleavage of a disulfide bond (6 -8). PDI has been studied in detail in vitro with a variety of polypeptide substrates such as bovine pancreatic trypsin inhibitor, insulin, lysozyme, and ribonuclease A (RNase) (recently reviewed by Ruddon and Bedows (45) and Creighton (46)). The reactivation of "scrambled" RNase (sRNase) is the classic assay for measuring PDI activity (9). Oxidation of reduced RNase in urea results in non-native pairing of the sulfhydryl groups, giving the inactive RNase deriva...

Hormone Binding by Protein Disulfide Isomerase, a High Capacity Hormone Reservoir of the Endoplasmic Reticulum

“…PDI, like some other folding assistants (47), must interact with a large number of different substrates with few recognizable sequence cues except for a small preference for peptides that contain cysteine (10,11). Recently, Winther and colleagues (13) found that peptide sequence can affect the rate at which PDI reacts with peptide disulfides with some preference for a sequence that has the features, small/helix breaker-Cys-X-hydrophobic/basic-hydrophobic. Peptide binding sites have been difficult to localize on PDI.…”

“…PDI interacts covalently with substrate cysteines (9), but it also binds a variety of peptide/protein substrates (10). There are few strict sequence/structure requirements for binding (11,12), although there is some effect of sequence on reactivity with PDI (13). PDI activity can be inhibited by a number of peptides (11), including the cyclic peptide antibiotic, bacitracin (14,15).…”

TROSY-NMR reveals interaction between ERp57 and the tip of the calreticulin P-domain

“…15 N, 2 H labeling of the protein was obtained by expressing it in 0.5 liters of M9 minimal medium supplemented with 1 g Celtone dN powder (Martek Biosciences, Columbia, MD) and 2 g͞liter glucose. Recombinant human PDI was expressed and purified as described (10), except for an additional, final purification step on a Superdex 200 gelfiltration column (Amersham Pharmacia). For the recombinant expression of human ERp57 we used Escherichia coli BL21(DE3) cells freshly transformed with the pHisERp57 expression vector, which encodes a N-terminal 22-aa affinity tag containing a deca-histidine sequence and a factor X a cleavage site.…”

Mechanistic insights on the reduction of glutathione disulfide by protein disulfide isomerase

“…2). Although GSSG is not a substrate for which PDI exhibits a high affinity, it is one of the smallest and more important substrates of PDI (59). The role of this enzyme in controlling the GSH/GSSG buffer has been a subject of research in the near past (13,26,60).…”