Distal Substrate Interactions Enhance Plasmepsin Activity
2004; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês
10.1074/jbc.m412086200
ISSN1083-351X
AutoresEva S. Istvan, Daniel E. Goldberg,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoPlasmepsin II (PM II) is an aspartic protease active in hemoglobin (Hb) degradation in the protozoan parasite Plasmodium falciparum. A fluorescence-quenched octapeptide substrate based on the initial hemoglobin cleavage site is recognized well by PM II. C-terminal extension of this peptide has little effect, but N-terminal extension results in higher maximal velocity and dramatic concentration-dependent substrate inhibition. This inhibition, but not the rate stimulation, depends on the presence of a DABCYL group on the peptide N terminus. Using site-directed mutagenesis, we have identified PM II residues that interact with N-terminal amino acids of peptide substrates. The same residues influence degradation of Hb by PM II. Cathepsin E (CatE), a related mammalian aspartic protease, is also stimulated by N-terminally extended substrates. This suggests that distal substrate interactions as far out as P6 may be a general property of aspartic proteases and may be important in substrate and inhibitor recognition. Although PM II and CatE are similar in their ability to cleave Hb-based peptides and Hb α-chains, CatE is not able to degrade native Hb, which is a substrate for PM II. Based on these results, we propose that PM II may have the special feature of being a Hb denaturase. Plasmepsin II (PM II) is an aspartic protease active in hemoglobin (Hb) degradation in the protozoan parasite Plasmodium falciparum. A fluorescence-quenched octapeptide substrate based on the initial hemoglobin cleavage site is recognized well by PM II. C-terminal extension of this peptide has little effect, but N-terminal extension results in higher maximal velocity and dramatic concentration-dependent substrate inhibition. This inhibition, but not the rate stimulation, depends on the presence of a DABCYL group on the peptide N terminus. Using site-directed mutagenesis, we have identified PM II residues that interact with N-terminal amino acids of peptide substrates. The same residues influence degradation of Hb by PM II. Cathepsin E (CatE), a related mammalian aspartic protease, is also stimulated by N-terminally extended substrates. This suggests that distal substrate interactions as far out as P6 may be a general property of aspartic proteases and may be important in substrate and inhibitor recognition. Although PM II and CatE are similar in their ability to cleave Hb-based peptides and Hb α-chains, CatE is not able to degrade native Hb, which is a substrate for PM II. Based on these results, we propose that PM II may have the special feature of being a Hb denaturase. Malaria is a large burden on society, causing at least 300 million acute illnesses and 1–3 million deaths annually (1Breman J.G. Alilio M.S. Mills A. Am. J. Trop. Med. Hyg. 2004; 71: 1-15Crossref PubMed Scopus (481) Google Scholar). Malaria is spread by Anopheles mosquitoes, and the protozoan parasite grows and multiplies within erythrocytes in its asexual cycle. Of the four types of malaria infecting humans, the most severe form is caused by Plasmodium falciparum. Increasing drug resistance to P. falciparum has created an urgent need for new drugs. To mature inside erythrocytes, parasites degrade vast amounts of Hb in an enormous catabolic effort (2Banerjee R. Goldberg D.E. Rosenthal P.J. Antimalarial Chemotherapy: Mechanisms of Action, Resistance, and New Directions in Drug Discovery. Humana Press, Totowa, NJ2000: 43-63Google Scholar). Hb amino acids are incorporated into parasite proteins (3Sherman I.W. Ruble J.A. Tanigoshi L. Mil. Med. 1969; 134: 954-961Crossref PubMed Scopus (19) Google Scholar), and parasites are able to grow, albeit poorly, in amino acid-limiting media, emphasizing the ability of parasites to rely on Hb 1The abbreviations used are: Hb, hemoglobin; PM, plasmepsin; m-PM II, mature PM II; CatE, cathepsin E; CatD, cathepsin D; GABA, γ-aminobutyric acid; EDANS, 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; MES, 4-morpholineethanesulfonic acid. 1The abbreviations used are: Hb, hemoglobin; PM, plasmepsin; m-PM II, mature PM II; CatE, cathepsin E; CatD, cathepsin D; GABA, γ-aminobutyric acid; EDANS, 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; MES, 4-morpholineethanesulfonic acid. degradation (4Divo A.A. Geary T.G. Davis N.L. Jensen J.B. J. Protozool. 1985; 32: 59-64Crossref PubMed Scopus (171) Google Scholar, 5Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (249) Google Scholar, 6Liu J. Gluzman I.Y. Drew M.E. Goldberg D.E. J. Biol. Chem. 2005; 280: 1432-1437Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Additionally, Hb degradation may create space for the growing parasite and contribute to osmolar maintenance (7Lew V.L. Tiffert T. Ginsburg H. Blood. 2003; 101: 4189-4194Crossref PubMed Scopus (192) Google Scholar). Thus, hemoglobinases are considered an important chemotherapeutic target. Aspartic proteases may be especially important for the survival of the parasite because inhibitors specific for this class of enzymes kill parasites early in their erythrocytic life cycle (5Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (249) Google Scholar, 8Bailly E. Jambou R. Savel J. Jaureguiberry G. J. Protozool. 1992; 39: 593-599Crossref PubMed Scopus (77) Google Scholar). Three aspartic proteases, called plasmepsins (PMs), and one closely related protease (histo-aspartic protease) containing a histidine in place of an active site aspartate, reside in the food vacuole, the site of Hb degradation (9Banerjee R. Liu J. Beatty W. Pelosof L. Klemba M. Goldberg D.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 990-995Crossref PubMed Scopus (369) Google Scholar). Each of the food vacuole PMs cleaves Hb in the α-chain between Phe-33 and Leu-34, (10Gluzman I.Y. Francis S.E. Oksman A. Smith C.E. Duffin K.L. Goldberg D.E. J. Clin. Investig. 1994; 93: 1602-1608Crossref PubMed Scopus (248) Google Scholar, 11Goldberg D.E. Slater A.F. Beavis R. Chait B. Cerami A. Henderson G.B. J. Exp. Med. 1991; 173: 961-969Crossref PubMed Scopus (231) Google Scholar, 12Wyatt D.M. Berry C. FEBS Lett. 2002; 513: 159-162Crossref PubMed Scopus (66) Google Scholar). Cleavage at this site is speculated to lead to Hb denaturation and, thus, generation of additional protease-accessible sites. To better characterize the molecular mechanism underlying Hb degradation by PMs, we studied the catalytic properties of PM II with peptide substrates and Hb. Using fluorogenic peptide substrates, we discovered that PM II is subject to dramatic substrate inhibition. In addition, peptides extending far into unprimed subsites stimulate protease activity. Neither substrate inhibition nor rate enhancement is unique to PM II, but both are also present in distant relatives and may be common features of aspartic protease catalysis. The ability to degrade native Hb, on the other hand, is not shared by relatives of PM, as CatE cleaves peptides based on the primary Hb cleavage site well but does not act on Hb. Protein Expression and Purification—Mature PM II (m-PM II) was generated by amplifying a construct encoding the proform of PM II (13Luker K.E. Francis S.E. Gluzman I.Y. Goldberg D.E. Mol. Biochem. Parasitol. 1996; 79: 71-78Crossref PubMed Scopus (75) Google Scholar) with the primers 5′-GCCATGGGTAGTTCAAATGAT-3′ and 5′-CCAACAATTGGATCTACTGAA-3′. The PCR product was subcloned into pCR4, its sequence was confirmed, and it was digested with XbaI and MunI. The fragment was subcloned into an expression construct containing the C-terminal portion of PM II in the pET-3d vector. A construct containing 46 amino acids of the pro-domain (p77-p122) was amplified with the primers 5′-CCATATGTCCGAACATTTAACTATT-3′ and 5′-GGATCCCTAATAATTTGTTTTAGTAAG-3′ and, using the NdeI and BamHI sites, subcloned as a C-terminal glutathione S-transferase fusion protein into a pGEX expression vector. The proform-PM II construct contains an alteration in codon 238, with a change from Lys to Arg (13Luker K.E. Francis S.E. Gluzman I.Y. Goldberg D.E. Mol. Biochem. Parasitol. 1996; 79: 71-78Crossref PubMed Scopus (75) Google Scholar). This conservative change does not affect peptide cleavage kinetics (data not shown). m-PM II mutant constructs were prepared using QuikChange with the primers (complement primers are not listed) 5′-GATGTTATCAGATGCCCATTCTTACCTAAATATGTAACTCTTTGTAACAACAGAAAATT-3′ (for m-F244K), 5′-ATGGGTTCAGAGTCCCATTCTGACCTGAATATGTAACTCTTTGTAACAACAGCAAAT-3′ (for m-F244E), 5′-GTTGCAGAATTTAGATGTTATCAAAGTCCCATTCTTACCTTTCTAT-3′ (for m-R238K), and 5′-TTAGATGTTATCAGAGTCCCATTACCTTTCTATGTAACTCTTTGTACC-3′ (for the deletion of Phe-241, m-d241). Expression of recombinant proteins was in Escherichia coli BL21(DE3) cells with 200 μm isopropyl 1-thio-β-d-galactopyranoside at 37 °C for 4–6 h in TB media (Qbiogene, Carlsbad, CA). After cell lysis by sonication, inclusion bodies were resuspended in buffer A (20 mm Tris-HCl, pH 8.5, 1 mm EDTA, 150 mm NaCl) and washed 2 times each in buffer B (20 mm Tris-HCl, pH 8.5, 1 mm EDTA, 150 mm NaCl, 1% Triton X-100) and buffer C (20 mm Tris-HCl, pH 8.5, 1 mm EDTA). Inclusion bodies were solubilized in buffer D (6 m urea, 20 mm Tris-HCl, pH 8.5, 1 mm EDTA) and applied to a heparin-Sepharose FF column equilibrated in buffer D. The proteins were collected in the flow-through and applied to a Mono Q 10/10 column equilibrated in buffer D. Proteins were eluted from the Mono Q column with a linear gradient of 0–1 m NaCl. Refolding was achieved by rapid 20–30-fold dilution into buffer E (20 mm Tris-HCl, pH 8.5, 1 mm EDTA, 10% glycerol) at room temperature and stirring for at least 14 h. To autoactivate the proform of PM II, sodium citrate, pH 4.7, at a final concentration of 0.1 m was added. The activated enzyme was then dialyzed into buffer E. Proteins were concentrated on a Mono Q 10/10 column, preequilibrated in buffer E, and eluted with a linear gradient of 0–1 m NaCl. Final purification and removal of aggregates were performed with two gel filtrations on Superdex 75. Gel filtration 1 was carried out in buffer F (20 mm Tris-HCl, pH 8.5, 1 mm EDTA, 10% glycerol, 0.25 m NaCl) on proteins that had been treated with 5 mm dithiothreitol. Gel filtration 2 was done in buffer G (20 mm MES, pH 6.0, 0.25 m NaCl). To prevent autolysis proteins were stored in buffer G containing 50 mm Tris-HCl, pH 8.5, at 4 °C. To estimate the molecular masses of proteins, the Superdex 75 column was calibrated with low molecular weight standards in buffer G. Enzyme Kinetics—Kinetic assays were performed with internally quenched fluorescent peptide substrates in real time using a Cary Eclipse fluorimeter (Varian) with excitation at 336 nm (slit width 5 nm) and emission at 490 nm (slit width 10 nm). The appearance of cleavage products was measured at 37 °C for 40–200s. Progress curves were followed to verify that linear initial rates were being measured, and different enzyme concentrations were used to ensure that activity increased linearly with increasing enzyme. Sequences of synthetic peptides were based on the primary site of cleavage within Hb, and numbering of the peptides corresponds to the residues within the α-chain of Hb. The sequences of synthetic peptides were as follows: 3037a, DABCYL-GABA-ERMFLSFP-EDANS; 2837a, DABCYL-GABA-ALERMFLSFP-EDANS; 3037b, EDANS-CO-CH2-CH2-CO-ERMFLSFP-diaminopimelate-(DABCYL)OH; 2837b, EDANS-CO-CH2-CH2-CO-ALERMFLSFP-diaminopimelate-(DABCYL)OH. Peptides varying in length and/or identity of the residue at position 28 (Figs. 2 and 3) contained N-terminal DABCYL with a GABA linker and C-terminal EDANS groups. Assays were performed in 0.1 m sodium acetate, pH 5.0, containing 10% glycerol (for peptides 3037b and 2837b) or 20% glycerol for all peptides containing N-terminal DABCYL groups. All peptides were determined to be ≥95% pure. They were dissolved in Me2SO, and a constant 2% Me2SO was used in the assays. Protease concentrations ranged between 0.5 and 3 nm. Catalytic parameters were determined only for substrates that were free of substrate inhibition. Averages of three to four measurements were used at each substrate concentration. Km and Vmax values were calculated by direct fitting of initial rates using the Michaelis-Menten equation and Kaleidograph software. Concentrations of enzymes were determined by active-site titration with pepstatin, and the kcat was calculated from kcat = Vmax/[E] (14Copeland R.A. Enzymes: a Practical Introduction to Structure, Mechanism, and Data Analysis. 2nd Ed. John Wiley & Sons, Inc., New York2000Crossref Google Scholar). For all substrates displaying substrate inhibition, rates were measured for equal time periods. In these cases, the reported rates may not represent initial rates, but they are consistent throughout the experiment. Because substrate inhibition depended on protein concentration, the same concentration of PM II was used throughout. Fluorescence units were converted to molarity by measuring fluorescence intensities of complete digests at three peptide concentrations for each peptide.Fig. 3Side chains at P6 contribute to substrate inhibition and rate enhancement. m-PM II was prepared and rates were analyzed as in Fig. 2. A, side chains contribute to substrate inhibition. Altering P6 in the decapeptide from Ala (magenta) to Gly (green) results in a peptide whose properties resemble the P5-P4′ nonapeptide (orange). Changing Ala to the small polar Ser side chain (blue) does not significantly alter the peptide cleavage characteristics. B, bulky side chains enhance substrate inhibition. Changing P6 from Ala to larger hydrophobic groups gives more substantial substrate inhibition. Altering Ala to Trp (dark red) results in a peptide with extreme substrate inhibition. C, Asp at P6 eliminates substrate inhibition (green), whereas the peptide with the longer E side chain (blue) shows substrate inhibition and rate enhancement. D, basic side chains either behave like the 2837a peptide (Arg, green) or decrease the rate enhancement (Lys, blue). Positive charges located at the end of long side chains at P6 may not influence the peptides. Instead, the aliphatic character of the side chains may determine cleavage characteristics.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mapping of Cleavage Sites by Mass Spectrometry—Peptides 3037b and 2837b were dissolved in 30% acetonitrile, 0.01% trifluoroacetic acid and diluted to a concentration of 1 μm for use. Digests were set up at pH 5.0 for 30 min to 4 h with either PM II, CatE, cathepsin D (CatD), or pepsin in 0.1 m ammonium acetate. Digested peptides were purified using ZipTips (C18 matrix) and prepared for matrix-assisted laser desorption ionization mass spectroscopy using α-cyano-4-hydroxycinnamic acid as the matrix. Spectra were acquired on an Applied Bioscience 4700 Explorer Proteomics Analyzer in positive ion mode using the reflector. Sequence information was obtained using peptide fragmentation in tandem mass spectroscopy mode. For 3037b, a product with a mass of 930.4 was assigned to the N-terminal peptide in the cleavage reaction Phe-33–Leu-34, whereas the same cleavage resulted in a mass of 1114.5 with the 2837b peptide. A product with a mass of 967.4 from the parent 2837b peptide was mapped to the M32-F33 cleavage site. Hemoglobin Degradation—Purified Hb and α-chains were gifts of the laboratory of Gary Ackers (Washington University School of Medicine, St. Louis, MO). The chains of Hb were isolated from one another with p-mercuribenzoate (15Bucci E. Methods Enzymol. 1981; 76: 97-106Crossref PubMed Scopus (49) Google Scholar). Purified α-chains contain heme but are not folded as tightly as native Hb (16Hargrove M.S. Whitaker T. Olson J.S. Vali R.J. Mathews A.J. J. Biol. Chem. 1997; 272: 17385-17389Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Degradation experiments were performed by incubation at 37 °C for 30 min to 18 h in 0.1 m sodium acetate, pH 5.0. Reactions were stopped by 2-fold dilution into SDS-PAGE-loading buffer and boiling for 3 min. Hb concentrations were estimated by densitometry using ImageJ software. Typical substrate concentrations were 0.2–10 μm heme, and protease concentrations were 0.1–0.3 μm. The stability of PM II mutants in the reaction mixtures was tested with fluorogenic peptides, and they were not different from the wild-type protease. Reagents—Synthetic peptides were obtained from Anaspec (San Jose, CA). High performance liquid chromatography columns and gel filtration standards were from Amersham Biosciences. Pepstatin A and isopropyl 1-thio-β-d-galactopyranoside were from Roche Applied Science. CatD (bovine), CatE (mouse recombinant), pepsin (porcine), and pET-3d were from EMD Biosciences (San Diego, CA). QuikChange mutagenesis kit was from Stratagene (La Jolla, CA), and the pCR4 vector was from Invitrogen. All other reagents were from Sigma. Preparation of Recombinant Mature Plasmepsin II—In the parasite, PM II is translated as an inactive zymogen containing a 124-amino acid-long N-terminal pro-sequence that has a membrane-spanning domain. Within the food vacuole the pro-sequence is removed by a calpain-like maturase, and native PM II is released (17Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar). In vitro, a PM II construct with a partial pro-sequence that is missing the transmembrane domain (N terminus at M70p) efficiently inhibits activity by stabilizing an open and catalytically incompetent conformation of the protease active site (18Bernstein N.K. Cherney M.M. Yowell C.A. Dame J.B. James M.N. J. Mol. Biol. 2003; 329: 505-524Crossref PubMed Scopus (42) Google Scholar). At acidic pH, this partial pro-PM II auto-activates to form auto-PM II containing 14 N-terminal amino acids that are not present in mature, native PM II (Fig. 1A). Auto-PM II and native PM II have been shown to exhibit similar kinetic parameters with a synthetic peptide substrate (13Luker K.E. Francis S.E. Gluzman I.Y. Goldberg D.E. Mol. Biochem. Parasitol. 1996; 79: 71-78Crossref PubMed Scopus (75) Google Scholar). We sought to establish an expression system that bypasses the need for auto-activation. PM II expressed well from a pET3d construct that contained G124p after an initiator Met (Fig. 1A), and inclusion bodies of the protein (referred to as mature or m-PM II) were purified. We thought that the pro-domain of PM II might be required for proper folding of the protease and hoped that providing this domain in trans would suffice. We prepared and purified a soluble glutathione S-transferase fusion protein containing residues p77-p122 of the pro-domain and added this glutathione S-transferase-pro-PM II at a molar ratio of 1:1 to m-PM II during refolding. As a control reaction m-PM II was prepared without the addition of glutathione S-transferase-pro-PM II. To judge the success of refolding, we measured the protease activity of both fractions and determined that it was equal (data not shown), indicating that in vitro, the residues encoding m-PM II are sufficient for proper folding. Consequently, m-PM II was purified and refolded similarly to auto-PM II (Fig. 1B). Gel filtration gave an estimated Mr of 37,400 for auto-PM II and an Mr of 36,300 for m-PM II, compared with calculated Mr values of 38,200 for auto-PM II and 37,000 for m-PM II (data not shown), and suggested that both proteins were monomeric under the conditions used. The yield of purified inclusion bodies was ∼2-fold higher, and that of the refolded, active protein was up to 3-fold higher with m-PM II compared with auto-PM II. Using a fluorescence-quenched octapeptide (3037b), auto-PM II and m-PM II showed similar kinetic parameters (Table I).Table IKinetic parameters of PM II and CatE with internally quenched fluorescent octapeptide or decapeptide Initial rates of peptide cleavage used 2 nm PM II, 1.6 nm CatE (3037b), or 0.37–0.76 nm CatE (2837b). For PM II, substrate concentrations were 0.1–8 μm with 3037b and 0.1–6 μm with 2837b. For CatE, substrate concentrations were 0.1–10 μm with 3037b and 0.1–8 μm with 2837b.Peptide3037b2837b(kcat/Km2837b)/(kcat/Km3037b)Kmkcatkcat/KmKmkcatkcat/Kmμms-1s-1 μm-1μms-1s-1 μm-1auto-PM II3.05 ± 0.411.100.3601.05 ± 0.112.522.406.67m-PM II3.86 ± 0.220.8750.2271.00 ± 0.102.332.3410.3m-F244E3.02 ± 0.230.4310.1431.38 ± 0.110.9520.6704.82m-F244K6.11 ± 0.410.8450.1382.33 ± 0.391.510.6474.70m-F244A4.33 ± 0.641.110.2561.27 ± 0.122.511.987.73m-d2412.94 ± 0.190.5960.2031.00 ± 0.101.051.065.21CatE8.12 ± 0.612.500.3085.01 ± 0.6712.12.427.85 Open table in a new tab N-terminally Extended Substrate Peptides Enhance Plasmepsin II Activity—In attempting to model peptide substrates of PM II in the enzyme active site (co-crystal of PM II with inhibitors; Protein Data Bank codes 1SME, 1LF2, and 1LEE) (19Silva A.M. Lee A.Y. Gulnik S.V. Maier P. Collins J. Bhat T.N. Collins P.J. Cachau R.E. Luker K.E. Gluzman I.Y. Francis S.E. Oksman A. Goldberg D.E. Erickson J.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10034-10039Crossref PubMed Scopus (269) Google Scholar, 20Asojo O.A. Afonina E. Gulnik S.V. Yu B. Erickson J.W. Randad R. Medjahed D. Silva A.M. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 2001-2008Crossref PubMed Scopus (79) Google Scholar), we observed that only a small portion of the active site is solvent-accessible. Consequently, P′ residues could be accommodated, but extended P residues clashed with the protein. To investigate whether lengthening peptides N-terminally might have an effect on catalysis, we synthesized a series of peptides based on the sequence of the primary cleavage site within Hb. Both P and P′ were extended beyond the original octapeptide (ERMFLSFP) (13Luker K.E. Francis S.E. Gluzman I.Y. Goldberg D.E. Mol. Biochem. Parasitol. 1996; 79: 71-78Crossref PubMed Scopus (75) Google Scholar), and rates of peptide cleavage were measured (Fig. 2, A and B). As illustrated in Fig. 2A, an extension of two N-terminal amino acids was critical for maximal rate enhancement. Some, but not all of the P-extended peptides, showed strong substrate inhibition. Substrate inhibition without rate stimulation was observed in the nonapeptide P5-P4′, whereas the decapeptide P6-P4′ exhibited rate enhancement as well as substrate inhibition. This suggested that rate enhancement and substrate inhibition are separate phenomena. Peptides that extended beyond the P6 position did not lead to additional rate stimulation. Peptides with a negatively charged N-terminal amino acids (P4-P4′, P4-P6′, P7-P4′) showed attenuation of substrate inhibition. Extending peptides beyond P4′ did not result in either rate stimulation or substrate inhibition (Fig. 2B). Instead, a modest rate decrease for all C-terminally extended peptides was observed. The origin of this rate decrease is unclear. Importantly, C-terminal extension of substrates did not inhibit the rate enhancement of the N-terminal extensions. Thus, rate stimulation is determined by distal P-sites, particularly by P6. Because the substrates were based on the sequence of Hb, extending the peptides not only changed their length but also their character. To analyze the contribution of amino acid side chains in detail, we synthesized a series of modified P6-P4′ substrates in which the P6 residue was changed, whereas all other amino acids remained constant. As illustrated in Fig. 3A, side chains contributed to substrate inhibition and rate enhancement. Truncation of the P6 side chain by altering the residue from Ala to Gly resulted in a peptide with characteristics similar to the P5-P4′ peptide. Replacing P6 with Ser, on the other hand, had little effect. Bulky side chains enhanced substrate inhibition (Fig. 3B), with the most bulky and hydrophobic peptide (P6 = Trp) being the most potent inhibitor. Aspartate at P6 released substrate inhibition (Fig. 3C). Interestingly, an acidic peptide with a longer side chain (P6 = Glu), showed rate enhancement beyond that observed with the ALERMFLSFP peptide. This additional stimulation of the catalysis may result from the longer aliphatic side chain of the glutamate. Basic side chains at P6 had substrate inhibition similar to the ALERMF-LSFP peptide, whereas the extent of rate enhancement was variable and possibly depended on the hydrophobicity and/or orientation of the long side chains (Fig. 3D). To investigate whether the reduction in the rate of product generation at high substrate concentration was due to substrate or product inhibition, we tested whether product peptides could act as inhibitors. Neither the unmodified P1′-P4′ peptide nor unmodified P-peptides of different length or P-peptides containing an N-terminal DABCYL inhibited substrate cleavage under any of the tested conditions. We examined whether the N-terminal DABCYL moiety of the synthetic peptides contributes to substrate inhibition by synthesizing a P4-P4′ octapeptide and a P6-P4′ decapeptide with an N-terminal EDANS and a C-terminal DABCYL group (peptides 3037b and 2837b). These modified substrates showed no substrate inhibition up to a concentration of 10 μm with the octapeptide and only minimal substrate inhibition with the decapeptide (Fig. 4B). The rate stimulation observed previously with the N-terminally extended substrates, however, was still present (Fig. 4 and Table I). This confirmed that rate enhancement and substrate inhibition are independent phenomena and that distal P sites are critical for the stimulation of catalysis. The order of magnitude increase in catalytic efficiency with the longer peptide could be attributed to both better initial binding (decreased Km) and better turnover (higher kcat). Localization of Distal S Sites—To determine which region of PM II interacts with the extended P-sites, we examined the PM II-pepstatin co-crystal structure and introduced a number of site-directed mutations. A loop (residues 240–243 and neighboring amino acids) was of particular interest, since it is highly conserved among PMs and is stabilized by inhibitor binding (see "Discussion"). Replacing the wild-type Phe-244 with Glu or Lys resulted in m-PM II mutant proteins that had reduced catalytic efficiencies with either peptide (Table I). Stimulation by the P-extended peptide was attenuated in the mutant enzymes. However, the reduced catalytic efficiencies of the two mutants were due to different mechanisms; m-F244E had a reduced kcat, whereas m-F244K had an increased Km. Replacing Phe-244 with Ala did not alter the kinetic parameters significantly. This suggests that the Phe-244 side chain does not participate in specific interactions with the peptide but that it is important for the proper formation of the structure and/or flexibility of the extended active site of the enzyme. A deletion of Phe-241 (m-d241) was similar to the m-F244E construct in that primarily the kcat was affected (Table I). Degradation of Hemoglobin by Wild-type and Mutant Plasmepsins—To see whether distal S-sites are only important for peptide cleavage or for protein cleavage as well, we studied the ability of PMs to degrade Hb. As shown in Table II, m-F244E and m-d241 had reduced Hb-cleavage ability, whereas m-F244K and m-F244A degraded Hb as well as the wild-type protein. In the peptide kinetic analysis, m-F244E and m-d241 showed decreased kcat values (Table I). For m-F244E we titrated the concentration of Hb in the assay, and the mutant activity differed from that of wild type only at saturating Hb concentrations above 1 μm (data not shown), thereby demonstrating a kcat effect on protein as well as peptide.Table IIHemoglobin degradation by wild-type and mutant plasmepsin II Hb (heme concentration of 9 μm) was incubated with 0.29 μm PM II in 0.1 m sodium acetate, pH 5.0, at 37 °C. Control reactions contained no enzyme. Aliquots were removed after 0–5 h of incubation, and reactions were stopped by diluting with SDS-PAGE loading buffer and boiling for 3 min. The time course experiment indicated that rates of degradation were linear. Gel electrophoresis was performed on 15% SDS-PAGE with Coomassie staining, and Hb degradation was quantified with densitometry. Reactions were performed in quadruplicate and for three time points.Enzymem-PM IIm-F244Am-F244Em-F244Km-d241pmol of Hb degraded/haMean of 12 measurements4.844.963.554.703.63S.E.±0.10±0.17±0.15±0.14±0.54p valuebThe p value is from Student's t test vs. m-PM II0.519<0.00010.771<0.0001a Mean of 12 measurementsb The p value is from Student's t test vs. m-PM II Open table in a new tab Distal Unprimed Sites Are Important Determinants of Aspartic Protease Catalytic Efficiency—We wondered whether the rate stimulation with increased N-terminal peptide length was unique to PM II or a general feature of aspartic proteases and studied the catalytic properties of mammalian enzymes related to PM II (CatD, CatE, and pepsin). With the exception of pepsin all enzymes exhibited a rate enhancement. Substrate inhibition with peptides containing an N-terminal DABCYL was observed with CatD but not with CatE or pepsin (data not shown). Rate enhancement could either be a consequence of increased binding interactions as seen in PM II or the result from additional cleavage sites available in the longer substrate. To distinguish these possibilities, we used mass spectrometry and analyzed peptide fragments after protein digestion. Of the enzymes studied, PM II and CatE cleaved both 3037b and 2837b at Phe-33–Leu-34. CatD cleaved 3037b at Phe-33–Leu-34 and 2
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