Catalysis of Protein Disulfide Bond Isomerization in a Homogeneous Substrate

Abstract

Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a beta hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N and C terminus contain a fluorescence donor (tryptophan) and acceptor (N(epsilon)-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E(o') = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys-Gly-His-Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/K(M) = 1.7 x 10(5) M(-1) s(-1), which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude that catalysis of disulfide bond isomerization by PDI does not necessarily involve a cycle of substrate reduction/oxidation.

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

“…Despite not being the most effective disulfide redox catalyst, the importance of PDI in the secretory protein pathway, where the formation of native disulfides is a rate-limiting step, has been widely recognized (1)(2)(3). In recent years, PDI deletion has been related to diseases involving unfolded protein response, such as Alzheimer's, Parkinson's, and type II diabetes (4)(5)(6).…”

“…Understanding the PDI reaction with the GSH/GSSG buffer is critically important to (i) investigate the role of PDI in the redox balance of the ER; (ii) determine the kinetics and thermochemistry of the reaction catalyzed by PDI, complementing the rather scarce kinetic studies that can be found in the literature (2,12,13); (iii) draw mechanistic insights on PDI, because there are no known studies for the reaction cycle with atomistic detail for this enzyme; (iv) provide structural information (transition states) to allow for the rational discovery of therapeutic inhibitors; and (v) provide general insight on the disulfide oxidoreductase family of enzymes, which are responsible for the reduction and isomerization of disulfide bonds, through thiol-disulfide exchange.…”

Electrostatic Stabilization and General Base Catalysis in the Active Site of the Human Protein Disulfide Isomerase a Domain Monitored by Hydrogen Exchange

“…[27] Measurements by Gilbert and co-workers [26] provide an estimate of the kinetic stability for this mixed disulfide. In the absence of oxidized glutathione, they determined that concentrations of reduced glutathione above 0.5 mm are required in order to appreciably shift the fraction of reduced PDI from the steadystate values seen at lower glutathione concentrations during the unscrambling of oxidized RNase A.…”

“…In the absence of a redox buffer, Gilbert and co-workers [26] showed that the unscrambling of an oxidized protein substrate by PDI is sensitive to the levels of both the oxidized and reduced forms of the enzyme; this is consistent with isomerization via a reductionreoxidation cycle. On the other hand, more recently Raines and colleagues [27] utilized kinetic data obtained from a novel homogenous substrate to argue for the alternative mechanism of intramolecular isomerization (Scheme 1). In marked contrast to the reduction-reoxidation mechanism in which every step of the reaction is catalyzed by the enzyme, the central step of the intramolecular isomerization pathway is the uncatalyzed reaction between a cysteine thiolate and a disulfide linkage within the substrate protein.…”

Medidas das atividades da Dissulfeto Isomerase Proteica: uma análise crítica

“…One example is a peptide based on tachyplesin I (TI), a 17-residue antimicrobial peptide that crosses membranes (Kersteen et al, 2005). Scrambled TI (4 cysteines) is isomerized by PDI to its 'native' conformation (more stable) in which tryptophan2…”