Catalysis of Thiol/Disulfide Exchange

Abstract

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30 -180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.Members of the thioredoxin superfamily of redox proteins transfer reducing and oxidizing equivalents between proteins through thiol-disulfide exchange. With overall structural similarity, the classical thioredoxin and glutaredoxin members of the family all have an active site with the sequence CXXC (1). The two active site cysteines are able to cycle between dithiol and disulfide redox states. Despite the similarities in the active sites and a general mechanism involving thiol-disulfide exchange, thioredoxin family proteins serve a diverse set of biological functions. The family patriarch is thioredoxin, the principal function of which is to provide reducing equivalents for deoxyribonucleotide synthesis by reducing the active site disulfide of ribonucleotide reductase (2). Glutaredoxins (Grx), 1 which have a GSH binding site (3), serve as reductants of protein-SG mixed disulfides and also provide reducing equivalents to ribonucleotide reductase apart from their many other functions (4). Protein-disulfide isomerase (PDI) and its prokaryotic counterpart, DsbA, function primarily to introduce disulfides into protein substrates during oxidative protein folding (5, 6).In thioredoxin and PDI, the more N-terminal cysteine, CXXC, of the active site is exposed to solution (7) and acts as a nucleophile to attack substrate disulfides (8). The second ...

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…The k 1 values for GSH were 10-and 3-fold lower than the observed first order rate constants for E. coli Grx1 obtained by stopped flow kinetics (56). This might be due in part to our less accurate photometric assay, but it could also reflect specific properties of the individual Grxs.…”

“…Reduction of Grxs by GSH results in a mixed disulfide that is attacked by a second GSH molecule, leading to the reduced protein and GSSG (Scheme 1, left) (56). For E. coli Grx1, it has been discussed that possibly the Grx1-SSG intermediate is much more reactive toward GSH than the oxidized protein with intramolecular disulfide (56), which would render the latter reaction rate-limiting.…”

“…For E. coli Grx1, it has been discussed that possibly the Grx1-SSG intermediate is much more reactive toward GSH than the oxidized protein with intramolecular disulfide (56), which would render the latter reaction rate-limiting. The reaction with T(SH) 2 should also first generate a mixed disulfide intermediate.…”

Regulation of NAD(P)H Oxidase by Associated Protein Disulfide Isomerase in Vascular Smooth Muscle Cells

“…However, exposure to oxidants, which can occur in specific microenvironments (37), could promote PDI thiol oxidation, which is preferentially intra-rather than intermolecular, converting PDI into a thiol oxidase/isomerase (12). Of note, although the PDI oxidase activity requires only cysteines from the a or aЈ domains, its reductase and in most cases isomerase activity require the entire multidomain PDI structure (38). With respect to NAD(P)H oxidase, typical thioredoxin motifs are absent among known VSMC oxidase subunits, because a detailed search of the NCBI data base yielded no hits.…”

Insights into Deglutathionylation Reactions

“…These endoplasmic reticulum-resident enzymes are involved in native disulfide bond formation in vivo, an activity that may require deglutathionylation. Direct in vitro studies on PDI family member deglutathionylation are limited (29,43), but additional evidence for such an activity comes from in vitro refolding studies since PDI is able to form native disulfide bonds in fully glutathionylated protein substrates (for example, see Ref. 44), and the catalyzed folding pathway for reduced protein substrates in a glutathione buffer never results in the significant accumulation of glutathionylated protein (for example, see Ref.…”