The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase.
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.
Hawkins
H C
HC
Biological Laboratory, University of Kent, Canterbury, U.K.
Freedman
R B
RB
D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000378:metabolism* / Q000494:pharmacology; D019807:Iodoacetic Acid; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D019704:Protein Disulfide-Isomerases; D051381:Rats
D004220:Disulfides; D007461:Iodoacetates; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D004229:Dithiothreitol; D019807:Iodoacetic Acid