The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase

Abstract

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum

“…The two thioredoxin-like (CGHC) sequences of PDI have been characterized as the active sites for its isomerase activity [28]. In vitro experiments have shown that PDI can accelerate oxidation and can unscramble inappropriate disulfide bonds [27].…”

Enzymes as chaperones and chaperones as enzymes

“…The two thioredoxin-like -CGHC-sequences and the peptide segment 451^476 in the C-terminal region of PDI have been characterized to be the active sites for its isomerase activity [15] and the peptide binding site [16] respectively. PDI alkylated at the active site thiols shows little isomerase activity but retains nearly full ability for increasing reactivation and decreasing aggregation during refolding of denatured GAPDH as that of native PDI [17], suggesting that the chaperone activity of PDI is independent of its -CGHC-active sites.…”

“…In addition, PDI suppresses aggregation of rhodanese during thermal denaturation. The above properties are entirely consistent with the de¢nition by Ellis of chaperones [1] and fully meet the four criteria proposed by Jakob and Buchner [14] for characterization of a protein as a molecular chaperone.The two thioredoxin-like -CGHC-sequences and the peptide segment 451^476 in the C-terminal region of PDI have been characterized to be the active sites for its isomerase activity [15] and the peptide binding site [16] respectively. PDI alkylated at the active site thiols shows little isomerase activity but retains nearly full ability for increasing reactivation and decreasing aggregation during refolding of denatured GAPDH as that of native PDI [17], suggesting that the chaperone activity of PDI is independent of its -CGHC-active sites.…”

Synthesis of Aromatic Monothiols and Aromatic Dithiols to Increase the Folding Rate and Yield of Disulfide Containing Proteins

“…The pK a value of the aromatic thiols were expected to be similar to that of PDI. 135 As most protein folding assays are performed in aqueous media close to neutral pH, the aromatic thiols selected were expected to be soluble at pH 7 in both the thiol and disulfide form.…”