Kinetic Analysis of the Mechanism and Specificity of Protein-disulfide Isomerase Using Fluorescence-quenched Peptides
1998; Elsevier BV; Volume: 273; Issue: 39 Linguagem: Inglês
10.1074/jbc.273.39.24992
ISSN1083-351X
AutoresVibeke Westphal, Jane C. Spetzler, Morten Meldal, Ulla Christensen, Jakob R. Winther,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoProtein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides revealed substantial similarities. Several of the peptides were synthesized in solution, and a quantitative characterization of pre-steady state kinetics was carried out. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 μg of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity. Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides revealed substantial similarities. Several of the peptides were synthesized in solution, and a quantitative characterization of pre-steady state kinetics was carried out. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 μg of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity. protein-disulfide isomerase human PDI reduced PDI oxidized PDI endoplasmic reticulum o-aminobenzoyl 3-nitrotyrosine scrambled ribonuclease dithiothreitol polyethylene glycol-poly-N,N-dimethyl acrylamide copolymer the s after a substrate name indicates that the substrate is in a soluble form, i.e. not bound to a bead. One of the reactions associated with folding of secretory proteins is the formation and isomerization of disulfide bonds. This is a slow process when uncatalyzed, but takes place rapidly in vivo, where the enzyme protein-disulfide isomerase (PDI)1 facilitates the formation and rearrangement of disulfide bonds. PDI is a 57-kDa protein present in the endoplasmic reticulum (ER) of eukaryotes. The protein is organized in an abb′a′c domain structure where thea and b domains are structurally related to thioredoxin and the c domain is rich in acidic amino acid residues (1Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 2Darby N.J. Kemmink J. Creighton T.E. Biochemistry. 1996; 35: 10517-10528Crossref PubMed Scopus (89) Google Scholar, 3Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Biochemistry. 1996; 35: 7684-7691Crossref PubMed Scopus (184) Google Scholar, 4Kemmink J. Darby N.J. Dijkstra K. Scheek R.M. Creighton T.E. Protein Sci. 1995; 4: 2587-2593Crossref PubMed Scopus (49) Google Scholar). The catalytic activity of PDI resides in thea and a′ domains, where the catalytically active cysteine residues are found in a WCGHCK motif in each domain. In each active site the two cysteines cycle between a reduced and an oxidized state, enabling the enzyme to exchange reducing equivalents with substrate sulfhydryls. PDI is capable of catalyzing several kinds of disulfide reactions: 1) oxidation reactions, in which the intramolecular disulfide bond of the CGHC motif is transferred to a pair of sulfhydryls in a substrate, 2) isomerization reactions in which disulfides are rearranged via the formation of a transient, mixed disulfide between the first cysteine residue of the CGHC motif and the substrate (5Darby N.J. Creighton T.E. Biochemistry. 1995; 34: 16770-16780Crossref PubMed Scopus (119) Google Scholar), and 3) reduction of mixed disulfides, in which PDI catalyzes the reductive cleavage of a disulfide bond (6Nakamura S. Matsushima M. Song H. Kikuchi M. J. Biochem. (Tokyo). 1996; 120: 525-530Crossref PubMed Scopus (8) Google Scholar, 7Hayano T. Inaka K. Otsu M. Taniyama Y. Miki K. Matsushima M. Kikuchi M. FEBS Lett. 1993; 328: 203-208Crossref PubMed Scopus (25) Google Scholar, 8Darby N.J. Freedman R.B. Creighton T.E. Biochemistry. 1994; 33: 7937-7947Crossref PubMed Scopus (82) Google Scholar). PDI has been studied in detail in vitro with a variety of polypeptide substrates such as bovine pancreatic trypsin inhibitor, insulin, lysozyme, and ribonuclease A (RNase) (recently reviewed by Ruddon and Bedows (45Ruddon R.W. Bedows E. J. Biol. Chem. 1997; 272: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and Creighton (46Creighton T.E. Biol. Chem. 1997; 378: 731-744PubMed Google Scholar)). The reactivation of “scrambled” RNase (sRNase) is the classic assay for measuring PDI activity (9Givol D. De Lorenzo F. Goldberger R.F. Anfinsen C.B. Biochemistry. 1965; 53: 676-685Google Scholar). Oxidation of reduced RNase in urea results in non-native pairing of the sulfhydryl groups, giving the inactive RNase derivative, sRNase. The conversion to the native protein requires the presence of a thiol reagent, and the process is catalyzed by PDI (9Givol D. De Lorenzo F. Goldberger R.F. Anfinsen C.B. Biochemistry. 1965; 53: 676-685Google Scholar, 10Ruoppolo M. Lundstrom-Ljung J. Talamo F. Pucci P. Marino G. Biochemistry. 1997; 36: 12259-12267Crossref PubMed Scopus (56) Google Scholar, 11Goldberger R.F. Epstein C.J. Anfinsen C.B. J. Biol. Chem. 1964; 239: 1406-1410Abstract Full Text PDF PubMed Google Scholar). In most in vitro studies of PDI, activity has been investigated using a redox-buffer containing reduced and oxidized glutathione; GSH and GSSG, respectively. In the ER, the active site cysteine residue of PDI presumably reacts with endogenous glutathione, which is present in mm amounts with a [GSH]/[GSSG] ratio in the range 1–3 (12Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1605) Google Scholar). Similarly, the redox state of PDI is also determined by the relative levels of GSH and GSSG in most in vitro assays. The likely mechanism of the catalysis of disulfide bond formation involves the transfer of an active site disulfide bond from PDI to the substrate via a PDI-substrate disulfide intermediate (2Darby N.J. Kemmink J. Creighton T.E. Biochemistry. 1996; 35: 10517-10528Crossref PubMed Scopus (89) Google Scholar). PDI cycles between reduced and oxidized states and the re-oxidation of the reduced PDI by GSSG limits the overall reaction rate in vitro (2Darby N.J. Kemmink J. Creighton T.E. Biochemistry. 1996; 35: 10517-10528Crossref PubMed Scopus (89) Google Scholar, 13Darby N.J. Creighton T.E. Biochemistry. 1995; 34: 3576-3587Crossref PubMed Scopus (92) Google Scholar). This suggests that GSSG is a poor substrate of PDI. New evidence suggests that the yeast ERO1gene product is required for oxidation of protein thiols in the ER. Loss of Ero1p function results in the accumulation of proteins whose folding is dependent upon disulfide bond formation. Together, these observations argue that Ero1p in vivo may be directly responsible of reoxidation of PDI (14Pollard M.G. Travers K.J. Weissman J.S. Mol. Cell. 1998; 1: 171-182Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 15Frand A.R. Kaiser C.A. Mol. Cell. 1998; 1: 161-170Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). Even though the nature of PDI and its substrate binding has been investigated in various ways (16Klappa P. Hawkins H.C. Freedman R.B. Eur. J. Biochem. 1997; 248: 37-42Crossref PubMed Scopus (84) Google Scholar, 17Morjana N.A. Gilbert H.F. Biochemistry. 1991; 30: 4985-4990Crossref PubMed Scopus (79) Google Scholar, 18Noiva R. Freedman R.B. Lennarz W.J. J. Biol. Chem. 1993; 268: 19210-19217Abstract Full Text PDF PubMed Google Scholar, 19Otsu M. Omura F. Yoshimori T. Kikuchi M. J. Biol. Chem. 1994; 269: 6874-6877Abstract Full Text PDF PubMed Google Scholar, 20Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (297) Google Scholar), little is known about the specificity of PDI in catalysis of disulfide rearrangement and redox reactions. This, and the lack of small, well defined substrates with high affinity for PDI prompted us to carry out the present study. In analogy with substrates of proteolytic enzymes (21Meldal M. Shmuel C. Combinatorial Peptide Libraries. Humana Press, Totowa, NJ1998: 51-82Google Scholar), a fluorescent and internally quenched disulfide-linked library of peptides (22Spetzler J.C. Westphal V. Winther J.R. Meldal M. J. Pept. Sci. 1998; 4: 128-137Crossref PubMed Google Scholar) was used to identify a series of heptameric peptides that are extremely sensitive substrates of PDI. A surprisingly high degree of similarity was found in the amino acid sequences identified by screening the library of peptides on resin beads. Human PDI (hPDI) favored substrates containing the following features: small/helix breaker-cysteine-X-hydrophobic/basic-hydrophobic. A quantitative kinetic characterization was carried out by measuring the time-dependent change in fluorescence due to reduction of soluble substrates by hPDI. This analysis suggests a higher degree of specificity of hPDI than previously anticipated. The main difference between the substrates was seen for the dissociation constant, which showed a 10-fold difference. This indicates that the specificity of hPDI is primarily determined by the association between enzyme and substrate rather than catalytic effectiveness. The gene encoding hPDI was a cDNA clone isolated by K. Kivirikko (University of Oulu, Oulu, Finland). hPDI was purified from the Escherichia coli strain BL21(DE3) carrying the plasmid pET12a-PDI2 (23Darby N.J. Creighton T.E. Biochemistry. 1995; 34: 11725-11735Crossref PubMed Scopus (143) Google Scholar). Five hours after induction with isopropyl–1-thio-β-d-galactopyranoside at 30 °C, the cells were harvested and resuspended in 50 mm Tris-HCl (pH 8), 1 mm EDTA, 0.1 mm phenylmethanesulfonyl fluoride, and 1 mm dithiothreitol (DTT). The cell suspension was sonicated 3 × 30 s/0.5 liter culture. After centrifugation, ammonium sulfate was added to the supernatant to 25% saturation and centrifuged at 10000 × g for 20 min. Additional ammonium sulfate was added to the supernatant to 100% saturation, and the precipitated protein was recovered by centrifugation. The pellet was dissolved in 50 mm Tris-HCl (pH 8), 1 mm EDTA and the cleared extract was dialyzed against 50 mm Tris-HCl (pH 8), 1 mm EDTA. After dialysis, NaCl was added to the sample to a final concentration of 1m and the sample was applied to an Octyl-Sepharose column equilibrated with 1 m NaCl in 50 mm Tris-HCl (pH 8), 1 mm EDTA. After a wash with 1 m NaCl in 50 mm Tris-HCl (pH 8), 1 mm EDTA, the protein was eluted in one step with 50 mm Tris-HCl (pH 8),1 mm EDTA. This resulted in more than 80% pure hPDI estimated by SDS-polyacrylamide gel electrophoresis. The hPDI was further purified by applying the dialyzed sample to a Mono Q ion exchange column equilibrated with 50 mm Tris-HCl (pH 8), 1 mm EDTA and was subsequently eluted with a linear gradient from 0.1 to 0.5 m NaCl in the same buffer. The hPDI eluted at ∼0.3 m NaCl and was at least 95% pure, estimated by SDS-polyacrylamide gel electrophoresis. The pooled fractions were dialyzed against 50 mm Tris-HCl (pH 8), 1 mmEDTA and stored frozen at −80 °C until use. The concentration of hPDI was determined using an absorbance coefficient of 56399 cm−1m−1 (23Darby N.J. Creighton T.E. Biochemistry. 1995; 34: 11725-11735Crossref PubMed Scopus (143) Google Scholar). The yield was approximately 5 mg/0.5 liter culture. The hPDI used in the reduction assays was reduced by adding 10-fold molar excess of DTT. The sample was incubated in an argon atmosphere for at least 20 min at room temperature before removal of the DTT on a NAP-5 column (Pharmacia Biotech Inc.) equilibrated with 50 mm Tris-HCl (pH 8.0) 1 mm EDTA. The total sulfhydryl content of hPDI was determined by Ellman's reaction (24Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21604) Google Scholar, 25Riddles P.W. Blakeley R.L. Zerner B. Methods Enzymol. 1983; 91: 49-60Crossref PubMed Scopus (1073) Google Scholar). Between 3.3 and 3.9 free cysteines were found per molecule of hPDI. Two peptides linked by an interchain disulfide bond, a fluorescent probe, and a quencher group were synthesized as described (22Spetzler J.C. Westphal V. Winther J.R. Meldal M. J. Pept. Sci. 1998; 4: 128-137Crossref PubMed Google Scholar, 26Meldal M. Breddam K. Anal. Biochem. 1991; 195: 141-147Crossref PubMed Scopus (140) Google Scholar). The library was synthesized on 100–300-μm polyethylene glycol-poly-N,N-dimethyl acrylamide copolymer (PEGA) beads (from Polymer Laboratories, UK) for libraries (27Renil, M., Ferreras, M., Delaisse, J. M., Foged, N. T., and Meldal, M. (1998) J. Pept. Sci., in pressGoogle Scholar) using the split synthesis method ensuring an unique peptide on each bead (28Lam K.S. Salmon S.E. Hersh E.M. Hruby V.J. Kazmierski W.M. Knapp R.J. Nature. 1991; 354: 82-84Crossref PubMed Scopus (1743) Google Scholar, 29Meldal M. Methods. 1994; 6: 417-424Crossref Scopus (30) Google Scholar). The library consists of a cysteine residue flanked by random amino acid residues and the fluorescento-aminobenzoyl (Abz) group at the amino-terminal of the peptide. All 20 natural amino acids except cysteine were employed in the library. Cysteine was omitted in the random amino acid (Xaa) pool in order to avoid nonspecific disulfide bridge formation. This peptide was coupled to a peptide containing a constant amino acid sequence and the quenching group, 3-nitrotyrosine (Tyr(NO2)). The peptide library has been described in detail (22Spetzler J.C. Westphal V. Winther J.R. Meldal M. J. Pept. Sci. 1998; 4: 128-137Crossref PubMed Google Scholar). The bead-linked peptide library was analyzed under an Optical Star fluorescence microscope with a 320-nm band pass filter for the excitation and a 410-nm filter for the detection of emitted light. Approximately 30,000 PEGA beads (∼1000 beads at a time) containing the peptides (∼50 pmol of peptide/bead corresponding to 1 mm concentration) were incubated in a nitrogen atmosphere at room temperature in a 50 mm Tris-HCl (pH 8), 1 mm EDTA buffer with ∼1.0 mm GSH for 3 h. After treatment with GSH, some beads particularly susceptible to uncatalyzed reduction became fluorescent and were removed and defined as false positive. The rest of the beads were incubated under the same conditions with catalytic amounts of hPDI (0.2 μm), and the beads that turned fluorescent within 1 h were collected. These beads were washed with DTT in order to completely reduce the substrate so only the random peptide chain remained attached to the bead. This peptide was re-coupled with the Tyr(NO2)-containing peptide via a disulfide bond (22Spetzler J.C. Westphal V. Winther J.R. Meldal M. J. Pept. Sci. 1998; 4: 128-137Crossref PubMed Google Scholar), and the procedure was repeated. Beads in the original screen that were not fluorescent after 3 days were also collected in order to identify poor substrates for hPDI. The amino acid sequence of peptides on individual beads was determined directly using a protein sequencer (model 470A) from Applied Biosystems. As a control, the peptides from two pools of 2000 beads were sequenced collectively. This showed, as expected, that all amino acids except cysteine were present at about equal amounts at each position in the library (data not shown), confirming that the library was random. Soluble peptide substrates were synthesized as described previously (22Spetzler J.C. Westphal V. Winther J.R. Meldal M. J. Pept. Sci. 1998; 4: 128-137Crossref PubMed Google Scholar). The reduction of internally quenched disulfide-linked substrates was measured as a function of time using a Perkin-Elmer Luminescence Spectrometer LS50B with excitation at 320 nm and emission at 420 nm. The excitation and emission slit widths were 15 nm each. The assay was performed at 25 °C in a 3-ml 1 × 1-cm quartz cuvette from Hellma using a thermostated, stirred single-cell holder. Complete mixing of the sample was achieved in approximately 1 s. The buffer (50 mm Tris-HCl (pH 8), 1 mm EDTA) was filtered and purged with argon, and the assays were performed in an argon atmosphere in order to avoid uncontrolled re-oxidation. Soluble peptide substrates were synthesized as described (22Spetzler J.C. Westphal V. Winther J.R. Meldal M. J. Pept. Sci. 1998; 4: 128-137Crossref PubMed Google Scholar) and purified by semi-preparative reverse phase high performanc liquid chromatography to more than 98% purity as determined by amino acid analysis. The substrate concentrations were determined by amino acid analysis, and confirmed by titration with reduced hPDI as follows: Reduced hPDI with known active site concentration,c PDI (approximately 6 nm), was added to a surplus of substrate (100–300 nm) and the increase in fluorescence was measured. When all the reducing equivalents from hPDI were transferred to the substrate, the change in intensity of fluorescence corresponding to the amount of reduced substrate was determined (ΔF PDI). Total reduction of the substrate was obtained by addition of DTT (10 mm final concentration) (ΔF total). The substrate concentration [S]0 is obtained from: [S]0 =c PDIΔF total/ΔF PDI Solutions of the substrates (1 mm) were made in N, N-dimethylformamide and further diluted to 10 μm in water. The amount of N, N-dimethylformamide in the assay (<0.5%) did not affect the activity of hPDI (data not shown). The substrate concentrations were in the range 10–500 nm, and the concentration of reduced hPDI was 3 nm. The buffer and substrate were mixed in the cuvette before the addition of reduced hPDI. The rate of reduction was determined by the increase in fluorescence as a function of time. In each experiment the reduction of the substrate was followed for at least 600 s. The background fluorescence signal caused by the buffer and the non-reduced substrate was small and constant during the time course of the assay. In all cases, Equation 1, given under “Results,” described a satisfying fit to the measured data. The concentration dependence of the kinetic parameters,k obs and F ∞, obtained from fits of Equation 1, using the program Kaleidagraph (Synergy Software Ltd.), were further analyzed. As expected, allF ∞ values were similar (data not shown), whereas k obs showed a non-linear dependence on the concentrations. To develop a simple quantitative assay for investigating hPDI activity and substrate preference, we tested the following simple notions: (a) Tyr(NO2) would quench the fluorescent probe Abz when these groups were placed in individual peptides linked by a disulfide bond, and (b) reduction of the disulfide bond would result in a significance increase in the fluorescence yield. A model substrate (VW0s), composed of two peptide chains linked by a disulfide bond, was synthesized as described in Ref. 22Spetzler J.C. Westphal V. Winther J.R. Meldal M. J. Pept. Sci. 1998; 4: 128-137Crossref PubMed Google Scholar (Fig. 1). We found that VW0s was readily reduced by DTT, giving a 20-fold increase in fluorescence. This indicated that the system could in principle be used as a sensitive tool to study the reduction of disulfide bonds. However, the model substrate was not easily reduced by hPDI and therefore not a sensitive substrate of hPDI. In order to identify a better substrate of hPDI, an immobilized fluorescence-quenched peptide library was generated by the split synthesis method to obtain one peptide compound per bead (28Lam K.S. Salmon S.E. Hersh E.M. Hruby V.J. Kazmierski W.M. Knapp R.J. Nature. 1991; 354: 82-84Crossref PubMed Scopus (1743) Google Scholar). Like the compound VW0s, each consisted of two peptides linked together by a disulfide bond: one, with the sequence Abz-Gly-Xaa-Cys-Xaa-Xaa-Xaa-Met, synthesized on a methionine residue linked to the resin bead. Xaa indicates an amino acid residue except cysteine, varying from bead to bead. The other peptide contained the sequence Ala-Tyr(NO2)-Cys-Ala-NH2 (22Spetzler J.C. Westphal V. Winther J.R. Meldal M. J. Pept. Sci. 1998; 4: 128-137Crossref PubMed Google Scholar) (Fig. 2 A). This relatively small peptide chain was chosen as the non-random quenching peptide chain because VW0s, containing a similar sequence, was an extremely poor substrate. This ensured that the hPDI specificity would be mainly dependent on the random amino acid chain. Beads containing suitable substrates quickly showed fluorescence when treated with reduced hPDI (Fig. 2 B). These beads were picked up under a stereo microscope, completely reduced, washed, and the amino acid sequence determined (Table I). Thirteen peptides were picked up as fluorescent beads within 1 h, 2 were collected the next day, and finally 2 peptides were picked up as being non-fluorescent after 3 days of incubation with reduced hPDI (Table I).Table IThe amino acid sequences obtained by sequence analysis of substrates attached to resin beads collected by screening of the fluorescent peptide libraryAbz C3Gly C2Xaa C1CysXaa C1′Xaa C2′Xaa C3′Met C4′VW1AbzGlySerCysThrPheIleMetVW3AbzGlyAlaCysMetLysValMetVW4AbzGlyIleCys?AlaTyrMetVW6AbzGlyAlaCysThrMet?MetVW7AbzGlySerCys?Lys?MetVW10AbzGlyGlyCysMetAlaLeuMetVW11AbzGlyProCysAsn?AspMetVW18AbzGlyGlyCysLeuLysLeuMetVW19AbzGlyProCysIleLysLeuMetVW20AbzGlyArgCysValMetGluMetVW21AbzGlyProCysAlaArgSerMetVW23AbzGlyGlyCysPheLysProMetVW24AbzGlyLeuCysProHisLeuMetVW17AbzGlyTyrCysSerGlyHisMetVW22AbzGlyIleCysAsnIleThrMetVW30AbzGlyIleCysTrpTrpTrpMetVW31AbzGlyTyrCysPheTrpMetMetThe first 13 sequences were coupled to beads that were picked up within the first hour of incubation with reduced hPDI.VW17 and VW22 were picked up the following day as fluorescent beads.Beads containing VW30 and VW31 did not react upon treatment with reduced hPDI.A question mark in the sequence indicates that the residue could not be identified during the sequencing. Open table in a new tab The first 13 sequences were coupled to beads that were picked up within the first hour of incubation with reduced hPDI. VW17 and VW22 were picked up the following day as fluorescent beads. Beads containing VW30 and VW31 did not react upon treatment with reduced hPDI. A question mark in the sequence indicates that the residue could not be identified during the sequencing. When we inspected the first 13 sequences in Table I, certain amino acid residues seemed to be over-represented at certain positions. The first substrates to fluoresce had an over-representation of helix-breakers (such as proline and glycine) and small amino acid residues at the C1 position. 2To facilitate the discussion of substrate preference we wish to introduce a nomenclature similar to that of protease substrates; C1 and C1′ indicate the residues directly NH2- and COOH-proximal, respectively, to the cysteine residue engaged in disulfide bond formation. The subscripts indicate the distances of the residues from the cysteine residue.There is an even distribution between non-polar and polar residues, but only one basic amino acid residue, an Arg in VW20. The hydropathy index (30Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17279) Google Scholar) is varying widely around zero, suggesting that it does not matter whether the amino acid residue is hydrophobic or not. Although there does not seem to be a preference for any particular amino acid at the C1′ position, a hydrophobic residue is found in more than 70% of the sequences. This tendency is reflected in an average hydropathy index of 1.3. At the C2′ position, on the contrary, there is a preference for basic amino acid residues. Seven sequences, among the first group of substrates, contain an amino acid residue with a basic side chain, whereas the remaining five all have a hydrophobic amino acid residue. The average hydropathy index is −1.4, indicating a tendency to favor polar side chains. There are five lysine residues present at the C2′ position, and the probability that this is a coincidence is less than 1%. The C3′ position is the only one in which amino acids containing acidic side chains are found, but the position is dominated by hydrophobic amino acid residues. However, because of the strongly hydrophilic amino acid residues, aspartate and glutamate, the average hydropathy index is only 1.2. Even though aromatic amino acid residues are present in some of the preferred substrates, it is striking that the two substrates that were not reduced after 3 days of incubation with reduced hPDI, VW30 and VW31, contain a large number of aromatic residues. These two substrates were, however, easily reduced and were fluorescent after treatment with DTT. The most plausible reason for the lack of hPDI-mediated reduction of these substrates is that the amino acid residues, containing fairly bulky side chains, cause steric hindrance, making the disulfide bond between the two peptides inaccessible to hPDI. Screening of the library allowed the identification of good substrates, but it gave only a qualitative impression of substrate specificity. To obtain a more quantitative measure for the activity of hPDI, we synthesized selected substrates in soluble form as described previously (22Spetzler J.C. Westphal V. Winther J.R. Meldal M. J. Pept. Sci. 1998; 4: 128-137Crossref PubMed Google Scholar). These disulfidelinked peptide substrates turned out to be extremely sensitive to reduction by reduced hPDI. The reaction of the substrates with DTT was investigated under pseudofirst-order conditions using various concentrations of DTT (100 - 500 μm) and a fixed concentration of substrate (40 nm). All substrates showed the same second order rate constant in the DTT reduction (k DTT = 5.3 ± 0.6 s−1m−1) (data not shown). Figs. 3 and 4 show the reduction of substrate by reduced hPDI, as increase in fluorescence plotted against time. The time course for reduction of substrate VW6s by 2.5–3.0 nm hPDI at three different substrate concentrations, 15, 75, and 430 nm (Fig. 3, panels A, B, and C, respectively), is shown. When looking at three different substrates, VW1s, VW17s, and VW19s, all at 100 nm, it is clear that the reduction of the substrates by hPDI (3 nm) occurs at different rates (Fig. 4). Since the substrate concentration is in large excess as compared with enzyme concentration, we make the approximation that the substrate concentration is constant throughout the reaction. The reaction is therefore approximated to a pseudofirst-order. Thesolid lines shown in Figs. 3 and 4 represent fits to Equation 1.F(t)=F∞(1−exp(−kobs·t))(Eq. 1) Here, k obs is the observed first-order rate constant, and F(t) is the intensity of fluorescence at time t. F(t) attains the value F ∞ at the end of the reaction.Figure 4Reaction of reduced hPDI with three different substrates, using 100 nm of each substrate. Thecurves represent the increase in fluorescence from reduction of the substrate after addition of 3 nm reduced hPDI. The background from buffer and unreduced substrate was subtracted. The insets show the length of the experiments from which the fitting parameters (using Equation 1) were obtained. The fit is shown by the solid line. A, one of the poorer substrates, VW19s; B, one of the intermediate substrates, VW17s; C, one of the better substrates, VW1s.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since the reaction is approximated as pseudofirst-order, Equation 1holds only for substrate concentrations much higher than enzyme concentrations. Nevertheless, employing Equation 1 to the data set resulted in a reasonable fit even at substrate concentrations only 5 times larger than enzyme concentrations (Fig. 3 A). The usual kinetic parameters of a Michaelis-Menten equation do not apply to this system since hPDI is added in the reduced form and is oxidized upon reaction with substrate. The system is therefore not a classic enzyme-substrate system, but can be described by presteady-state kinetics. The dependence of the first-order rate constant,k obs, on the concentration of the substrate [S]0 obtained by fitting the data to Equation 1 is shown in Fig. 5. The hyperbolic pattern of k obs with [S]0 for most of the substrates is not in agreement with a simple one-step mechanism (Reaction 1), which would have implied a linear dependence of k obs on the substrate concentration,i.e. k obs = k 1[S]0 +k −1 (31Olsen K. Svensson B. Christensen U. Eur. J. Biochem. 1992; 209: 777-784Crossref PubMed Scopus (42) Google Scholar).PDISHSH+AS–SB ⇄k−1k1 PDISHS–S A+BSHREACTION 1In the reaction schemes, the reduced hPDI is written as PDISHSH and the substrate composed of two peptides linked via a disulfide bond (Abz
Referência(s)