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.

Figure 1

Putative mechanism of disulfide bond isomerization by PDI. The reaction begins with nucleophilic attack of an enzymic thiolate on a nonnative disulfide bond. Thiol–disulfide interchange reactions within the substrate ultimately produce native disulfide bonds and regenerate PDI. If a mixed-disulfide intermediate becomes trapped, the other enzymic thiolate initiates an “escape” mechanism. Subsequent oxidation of the substrate and reduction of the enzyme would then be necessary to produce native disulfide bonds and regenerate PDI.

Figure 2

Structure of dns-nTI. Native TI is a _-hairpin stabilized by six hydrogen bonds and two disulfide bonds (53, 54). Side chains indicated with circles lie above the plane of the _-sheet; side chains with squares lie below the plane of the _-sheet. dns-nTI is identical to TI except with the N-terminus acetylated, Lys1 replaced with a glycine residue, Arg17 replaced with a lysine residue, and the C-terminal carboxamide replaced with a glycine residue. The assay described herein relies on FRET from Trp2 to the dansyl group attached to Lys17.

Figure 3

Scheme for the synthesis of dns-sTI. The peptide was synthesized on solid-phase using standard Fmoc-protection methods. The lysine residue was protected with a dansyl (dns) group. The N-terminal cysteine residues were protected with acetamidomethyl (Acm) groups; the C-terminal cysteines were protected with trityl (Trt) groups. In the first step, the peptide was cleaved from the resin with the removal of all but the Acm protecting groups. In the second step, the first disulfide bond was formed. In the final step, the Acm protecting groups were removed and the second disulfide bond was formed.

Figure 4

Fluoresence properties of dns-nTI and dns-sTI. (A) Fluorescence emission intensity of dns-nTI (1.1 μM, dashed line) and dns-sTI (1.1 μM, solid line) as a function of wavelength upon excitation at 280 nm. Emission intensity was corrected for that of the buffer (100 mM Tris–HCl buffer at pH 7.6, containing 1 mM EDTA). (B) Fluorescence emission intensity (emission 465 nm, excitation 280 nm) of dns-sTI (1.1 μM) as a function of time after addition of PDI (20 nM) and glutathione (116 μM GSH and 4 μM GSSG). Reaction was performed in 100 mM Tris–HCl buffer at pH 7.6, containing EDTA (1 mM) and IGEPAL CA-630 (0.01% w/v). dns-sTI was added at time-point 1; PDI and glutathione were added at time-point 2.

Figure 5

Fluorescence emission intensity (emission 465 nm, excitation 280 nm) of dns-nTI as a function of solution reduction potential. Thiol–disulfide exchange equilibria were established between dns-nTI (465 nM) and varying ratios of reduced to oxidized glutathione for 20 min prior to the measurement of fluorescence. The reduction potential of dns-nTI (E° = -0.313 V) was obtained by nonlinear least-squares analysis of the data with eq 1 and n = 2. Linear least-squares analysis of the data in a Hill plot (inset) indicated no cooperativity (h = 1.018 and E° = -0.314 V).

Figure 6

Typical assay of the isomerization of dns-sTI. Fluorescence emission intensity (emission 465 nm, excitation 280 nm) of dns-sTI (1.1 μM) was monitored after addition of wild-type human PDI (20 nM, solid line), CGHA:CGHA PDI (20 nM, dashed line), or glutathione alone (dotted line). Reactions were performed in 100 mM Tris–HCl buffer at pH 7.6, containing EDTA (1 mM), IGEPAL CA-630 (0.01% w/v), and glutathione (116 μM GSH and 4 μM GSSG).