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W2076304758 abstract "Two major types of cleavage sites with different sequence preferences have been proposed for the human immunodeficiency virus type 1 (HIV-1) proteinase. To understand the nature of these sequence preferences better, single and multiple amino acid substitutions were introduced into a type 1 cleavage site peptide, thus changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a residue at a particular position in the substrate depends strongly on the neighboring residues, including both those at the same side and at the opposite side of the peptide backbone of the substrate. Based on these results, pseudosymmetric (palindromic) substrates were designed. The retroviral proteinases are symmetrical dimers of two identical subunits; however, the residues of naturally occurring cleavage sites do not show symmetrical arrangements, and no obvious symmetrical substrate preference has been observed for the specificity of HIV proteinase. To examine the role of the asymmetry created by the peptide bonds on the specificity of the respective primed and nonprimed halves of the binding site, amino acid substitutions were introduced into a palindromic sequence. In general, the results suggested that the asymmetry does not result in substantial differences in specificity of the S3 and S3′ subsites, whereas its effect is more pronounced for the S2 and S2′ subsites. Although it was possible to design several good palindromic substrates, asymmetrical arrangements may be preferred by the HIV proteinase. Two major types of cleavage sites with different sequence preferences have been proposed for the human immunodeficiency virus type 1 (HIV-1) proteinase. To understand the nature of these sequence preferences better, single and multiple amino acid substitutions were introduced into a type 1 cleavage site peptide, thus changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a residue at a particular position in the substrate depends strongly on the neighboring residues, including both those at the same side and at the opposite side of the peptide backbone of the substrate. Based on these results, pseudosymmetric (palindromic) substrates were designed. The retroviral proteinases are symmetrical dimers of two identical subunits; however, the residues of naturally occurring cleavage sites do not show symmetrical arrangements, and no obvious symmetrical substrate preference has been observed for the specificity of HIV proteinase. To examine the role of the asymmetry created by the peptide bonds on the specificity of the respective primed and nonprimed halves of the binding site, amino acid substitutions were introduced into a palindromic sequence. In general, the results suggested that the asymmetry does not result in substantial differences in specificity of the S3 and S3′ subsites, whereas its effect is more pronounced for the S2 and S2′ subsites. Although it was possible to design several good palindromic substrates, asymmetrical arrangements may be preferred by the HIV proteinase. The specificity of retroviral proteinases has been studied intensively using both polyproteins and oligopeptides as substrates (for review, see Refs. 1Oroszlan S. Luftig R.B. Curr. Top. Microbiol. Immunol. 1990; 157: 153-185PubMed Google Scholar, 2Dunn B.M. Gustchina A. Wlodawer A. Kay J. Methods Enzymol. 1994; 241: 254-278Crossref PubMed Scopus (68) Google Scholar, 3Tomasselli A.G. Heinrickson R.L. Methods Enzymol. 1994; 241: 279-301Crossref PubMed Scopus (55) Google Scholar). These studies have provided a basis for the rational design of potent, selective inhibitors. Various proteinase inhibitors are now in clinical trials or approved for therapy (for review, see Refs. 4Wlodawer A. Erickson J.W. Annu. Rev. Biochem. 1993; 62: 543-585Crossref PubMed Scopus (804) Google Scholar, 5Winslow, D. L., and Otto, M. J. (1995) AIDS, 9, Suppl. A, S183–S192.Google Scholar, 6Mellors J.W. Nat. Med. 1996; 2: 274-275Crossref PubMed Scopus (26) Google Scholar). Comparison of cleavage site sequences of human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are: HIV-1 and HIV-2, human immunodeficiency virus type 1 and type 2; MA, matrix protein; CA, capsid protein; NC, nucleocapsid protein. The nomenclature of viral proteins is according to Leis et al. (36Leis J. Baltimore D. Bishop J.M. Coffin J. Fleissner E. Goff S.P. Oroszlan S. Robinson H. Skalka A.M. Temin H.M. Vogt V. J. Virol. 1988; 62: 1808-1809Crossref PubMed Google Scholar). and type 2 (HIV-2) suggested that the enzyme had a broad specificity and lacked consensus substrate sequence (7Poorman R.A. Tomasselli A. Heinrikson R.L. Kézdy F.J. J. Biol. Chem. 1991; 266: 14554-14561Abstract Full Text PDF PubMed Google Scholar). Initially three types of cleavage sites were proposed for HIV-1, HIV-2, and simian immunodeficiency virus (8Henderson L.E. Benveniste R.E. Sowder R. Copeland T.D. Schultz A.M. Oroszlan S. J. Virol. 1988; 62: 2587-2595Crossref PubMed Google Scholar). Subsequently, two major types of cleavage sites were proposed for retroviral proteinases, type 1 having -Tyr(Phe)*Pro- and type 2 having hydrophobic residues (excluding Pro) at the site of cleavage (9Pettit S.C. Simsic J. Loeb D.D. Everitt L. Hutchison III, C.A. Swanstrom R. J. Biol. Chem. 1991; 266: 14539-14547Abstract Full Text PDF PubMed Google Scholar, 10Tözsér J. Weber I.T. Gustchina A. Bláha I. Copeland T.D. Louis J.M. Oroszlan S. Biochemistry. 1992; 31: 4793-4800Crossref PubMed Scopus (99) Google Scholar, 11Griffith J.T. Phylip L.H. Konvalinka J. Strop P. Gustchina A. Wlodawer A. Davenport R.J. Briggs R. Dunn B.M. Kay J. Biochemistry. 1992; 31: 5193-5200Crossref PubMed Scopus (99) Google Scholar). These two types of cleavage sites were proposed to have different preferences for the P2 and P2′ positions, where the peptide bond between P1 and P1′ is cleaved (notation is according to Ref. 12Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4766) Google Scholar). Our studies with type 1 substrates indicated a preference for small residues like Cys or Asn at the P2 position and a preference for β-branched Val or Ile at the P2′ position (10Tözsér J. Weber I.T. Gustchina A. Bláha I. Copeland T.D. Louis J.M. Oroszlan S. Biochemistry. 1992; 31: 4793-4800Crossref PubMed Scopus (99) Google Scholar). The lower catalytic constants with P2 β-branched residues were predicted to be due to steric collision with P1′ Pro (10Tözsér J. Weber I.T. Gustchina A. Bláha I. Copeland T.D. Louis J.M. Oroszlan S. Biochemistry. 1992; 31: 4793-4800Crossref PubMed Scopus (99) Google Scholar). On the other hand, using a series of peptides based on a type 2 cleavage site, β-branched residues, especially Val, were found to be favorable at P2, whereas Glu was preferred at P2′ (11Griffith J.T. Phylip L.H. Konvalinka J. Strop P. Gustchina A. Wlodawer A. Davenport R.J. Briggs R. Dunn B.M. Kay J. Biochemistry. 1992; 31: 5193-5200Crossref PubMed Scopus (99) Google Scholar). Interestingly, Griffith et al. (11Griffith J.T. Phylip L.H. Konvalinka J. Strop P. Gustchina A. Wlodawer A. Davenport R.J. Briggs R. Dunn B.M. Kay J. Biochemistry. 1992; 31: 5193-5200Crossref PubMed Scopus (99) Google Scholar) found Glu as the preferred P2′ residue in a peptide series, when the P2-P1′ sequence of a type 1 cleavage site (-Asn-Tyr*Pro-) was substituted into a type 2 substrate. Although some of the differences of the subsite specificity in type 1 and type 2 cleavage sites were explained by molecular modeling, most of the dependence of the specificity of proteinases on the sequence context is unexplored. The HIV-1 proteinase is a dimer of two identical subunits. It exhibits an exact crystallographic, 2-fold rotational (C2) symmetry in the structure without inhibitor (for review, see Ref. 4Wlodawer A. Erickson J.W. Annu. Rev. Biochem. 1993; 62: 543-585Crossref PubMed Scopus (804) Google Scholar). Based on this symmetry, the potential advantages of C2 symmetric HIV-1 proteinase inhibitors including high selectivity, potency, and stability were proposed, and structurally symmetric HIV-1 proteinase inhibitors were designed containing two amino-terminal halves of a putative substrate (13Erickson J. Neidhart D.J. VanDrie J. Kempf D.J. Wand X.C. Norbeck D.W. Plattner J.J. Rittenhouse J.W. Turon M. Wideburg N. Kohlbrenner W.E. Simmer R. Helfrich R. Paul D.A. Knigge M. Science. 1990; 249: 527-533Crossref PubMed Scopus (507) Google Scholar). Crystal structures of HIV-1 proteinase with inhibitors can be either symmetric or asymmetric (4Wlodawer A. Erickson J.W. Annu. Rev. Biochem. 1993; 62: 543-585Crossref PubMed Scopus (804) Google Scholar). The symmetry or asymmetry was initially thought to arise from the symmetry or asymmetry of the inhibitor, but even crystal structures of HIV proteinase with symmetric inhibitors can have asymmetric proteinase subunits (14Dreyer G.B. Boehm J.C. Chenera B. DesJarlais R.L. Hassell A.M. Meek T.D. Tomaszek Jr., T.A. Biochemistry. 1993; 32: 937-947Crossref PubMed Scopus (76) Google Scholar). Considering the symmetry of the HIV proteinase, a symmetrical preference for substrate residues would be expected for naturally occurring cleavage sites, since the high mutation rate could readily evolve such sequences. However, there is no obvious preference for symmetrical sequences in HIV proteinase cleavage sites (listed in Ref.15Tözsér J. Bláha I. Copeland T.D. Wondrak E.M. Oroszlan S. FEBS Lett. 1991; 281: 77-80Crossref PubMed Scopus (148) Google Scholar) or in other retroviral proteinase cleavage sites (see Ref. 1Oroszlan S. Luftig R.B. Curr. Top. Microbiol. Immunol. 1990; 157: 153-185PubMed Google Scholar). Using a series of oligopeptides containing single amino acid substitutions in a naturally occurring type 1 cleavage site peptide, the respective P and P′ positions (for example, P2 and P2′) appeared to be similar, but substantial differences were also found. For example, the peptide containing P2 Asn was a very good substrate; however, Asn at P2′ resulted in a poor substrate (10Tözsér J. Weber I.T. Gustchina A. Bláha I. Copeland T.D. Louis J.M. Oroszlan S. Biochemistry. 1992; 31: 4793-4800Crossref PubMed Scopus (99) Google Scholar). It was not clear whether these differences were because of the asymmetrical interactions of the peptide amides and carbonyl oxygens in the substrate or intramolecular interactions of the substrate side chains (10Tözsér J. Weber I.T. Gustchina A. Bláha I. Copeland T.D. Louis J.M. Oroszlan S. Biochemistry. 1992; 31: 4793-4800Crossref PubMed Scopus (99) Google Scholar). To explore further the dependence of HIV proteinase specificity on the sequence context of its substrates we introduced single or multiple substitutions into a type 1 cleavage site peptide and changed it to a naturally occurring type 2 cleavage site sequence. Based on the results obtained, we designed a pseudosymmetric (palindromic) substrate and introduced amino acid substitutions into this sequence to explore the effect of asymmetry created by the peptide backbone on the different specificities of the respective primed and nonprimed proteinase subsites. To study whether the enzyme prefers pseudosymmetric (palindromic) or asymmetric arrangements of the substrate residues, we have also studied the doubly substituted (also palindromic) versions of the starting pseudosymmetric substrate. Oligopeptides were synthesized by standard tert-butoxycarbonyl or 9-fluorenylmethyloxycarbonyl chemistry on a model 430A automated peptide synthesizer (Applied Biosystems, Inc.) or a semiautomatic Vega peptide synthesizer (Vega-Fox Biochemicals). All peptides were synthesized with an amide end. Amino acid composition of the peptides was determined with either a Durrum D-500 or a Waters Pico-Tag amino acid analyzer. Stock solutions and dilutions were made in distilled water (or in 10 mm dithiothreitol for peptides containing Cys residues), and the peptide concentrations were determined by amino acid analysis. Purified HIV-1 proteinase was prepared as described previously (16Louis J. McDonald R. Nashed N. Wondrak E.M. Jerina D. Oroszlan S. Mora P. Eur. J. Biochem. 1991; 199: 361-369Crossref PubMed Scopus (61) Google Scholar). Active site titration for the HIV-1 proteinase was performed with compound 3 (17Grobelny D. Wondrak E.M. Galardy R.E. Oroszlan S. Biochem. Biophys. Res. Commun. 1990; 169: 1111-1116Crossref PubMed Scopus (93) Google Scholar). The proteinase assays were performed in 0.25 m potassium phosphate buffer, pH 5.6, containing 7.5% glycerol, 1 mm EDTA, 2.5 mm dithiothreitol, 0.1% Nonidet P-40, 2 m NaCl in the presence of 8–140 nm enzyme. The reaction mixture was incubated at 37 °C for 1 h, and the reaction was stopped by the addition of guanidine HCl (6 m final concentration). The solution was acidified by the addition of trifluoroacetic acid, and an aliquot was injected onto a Nova-Pak C18 reversed-phase chromatography column (3.9 × 150 mm, Waters Associates, Inc.) using an automatic injector. Substrates and the cleavage products were separated using an increasing water-acetonitrile gradient (0–100%) in the presence of 0.05% trifluoroacetic acid. Cleavage products of proteinase-catalyzed hydrolysis for these peptides were identified by amino acid analysis and/or by NH2-terminal sequencing. Kinetic parameters were determined by fitting the data obtained at less than 20% substrate hydrolysis to the Michaelis-Menten equation by using the Fig. P program (Fig. P Software Corp.). The substrate concentration was 0.01–5.0 mm depending on the approximateK m values. The structures were examined on Silicon Graphics computers running the program Sybyl (Tripos Inc., St. Louis, MO) or CHAIN (18Sack J.S. J. Mol. Graph. 1988; 6: 224-225Crossref Google Scholar). The starting model for the HIV-1 proteinase with the substrate Val-Ser-Gln-Asn-Tyr*Pro-Ile-Val-Gln (asterisk indicates the site of cleavage) was described previously (19Bagossi P. Cheng E.Y.S. Oroszlan S. Tözsér J. Protein Eng. 1996; 9: 997-1003Crossref PubMed Scopus (12) Google Scholar). All the other enzyme-substrate structures were built from this model by altering the side chain(s) of the appropriate residue(s). Each of the side chain torsion angles for substituted residues in the peptide substrate was rotated through 360° in steps of 15° to find the conformation with the smallest nonbonded energy as described (20Weber I.T. Harrison R.W. Protein Eng. 1996; 9: 679-690Crossref PubMed Scopus (33) Google Scholar). Energy minimization and molecular dynamics of the modified substrates were run using the program AMMP (21Harrison R.W. J. Comp. Chem. 1993; 14: 1112-1122Crossref Scopus (81) Google Scholar), as described previously (19Bagossi P. Cheng E.Y.S. Oroszlan S. Tözsér J. Protein Eng. 1996; 9: 997-1003Crossref PubMed Scopus (12) Google Scholar). Finally, the model structures were examined in the computer graphics system. Previously, we performed extensive comparisons of the specificities of HIV-1 and HIV-2 proteinases using oligopeptides representing naturally occurring cleavage sites in their Gag and Gag-Pol polyproteins (15Tözsér J. Bláha I. Copeland T.D. Wondrak E.M. Oroszlan S. FEBS Lett. 1991; 281: 77-80Crossref PubMed Scopus (148) Google Scholar). These cleavage sites have been classified as type 1, which contains an aromatic amino acid and Pro at P1and P1′, respectively, and type 2, which has mainly hydrophobic residues but not Pro at the site of cleavage. We showed that an oligopeptide (peptide 1 in Table I) representing the cleavage site in p66 of HIV-1 for generating the p51 subunit of the heterodimeric reverse transcriptase of HIV-1 and another peptide (peptide 2 in Table I) representing the homologous sequence in p68 of HIV-2, and therefore proposed to be the cleavage site (22Le Grice S.F.J. Ette R. Mills J. Mous J. J. Biol. Chem. 1989; 264: 14902-14908Abstract Full Text PDF PubMed Google Scholar), were substrates of the HIV proteinases (15Tözsér J. Bláha I. Copeland T.D. Wondrak E.M. Oroszlan S. FEBS Lett. 1991; 281: 77-80Crossref PubMed Scopus (148) Google Scholar). These peptides, which match type 2 cleavage site sequences, were the starting points for our design of palindromic substrates since they are partly symmetric with aromatic amino acids at the P1, P1′, and they contain negatively charged residues at the P3 and P3′ positions. In addition, peptide 2 also contains Thr at both P2 and P2′ positions. Furthermore, peptides 1 and 2 with the exception of the P2′ residues share the same sequence in the P4-P3′ region, which is the major determinant for specificity (23Tözsér J. Gustchina A. Weber I.T. Bláha I. Wondrak E.M. Oroszlan S. FEBS Lett. 1991; 279: 356-360Crossref PubMed Scopus (66) Google Scholar). However, peptide 2 was found to be a much poorer substrate of the HIV proteinases than peptide 1 (see Table I and Ref. 15Tözsér J. Bláha I. Copeland T.D. Wondrak E.M. Oroszlan S. FEBS Lett. 1991; 281: 77-80Crossref PubMed Scopus (148) Google Scholar).Table VAssay of P 2 and P 2 ′ substituted peptides based on a palindromic sequence by HIV-1 proteinaseNumberSequence5-aAmino acids substituted in sequence 17 are underlined.K mk catk cat/K mmms −1mm −1 s −117SDTY*YTDS0.21 ± 0.040.34 ± 0.021.6230SDGY*YTDS1.41 ± 0.280.06 ± 0.010.0431SDTY*YGDS1.82 ± 0.280.09 ± 0.010.0532SDGY*YGDSNot hydrolyzed5-bNot hydrolyzed when incubated with 140 nm HIV-1 proteinase for 1 h at 37 °C.33SDEY*YTDS4.86 ± 1.043.66 ± 0.530.7534SDTY*YEDS0.91 ± 0.160.34 ± 0.040.3635SDEY*YEDS5.14 ± 0.860.79 ± 0.090.1536SDCY*YTDS0.09 ± 0.0060.44 ± 0.014.8937SDTY*YCDS0.12 ± 0.021.04 ± 0.068.6738SDCY*YCDS1.00 ± 0.202.78 ± 0.202.7839SDAY*YTDS0.098 ± 0.0041.30 ± 0.0213.2740SDTY*YADS0.17 ± 0.040.82 ± 0.034.8241SDAY*YADS0.24 ± 0.033.16 ± 0.1713.1742SDIY*YTDS0.10 ± 0.021.02 ± 0.0810.2043SDTY*YIDS0.03 ± 0.0010.55 ± 0.0318.3344SDIY*YIDSNot determined5-cLow solubility of the substrate did not allow the kinetic measurements.45SDLY*YTDS0.51 ± 0.050.48 ± 0.030.9446SDTY*YLDS0.06 ± 0.020.57 ± 0.099.5047SDLY*YLDSNot determined5-cLow solubility of the substrate did not allow the kinetic measurements.5-a Amino acids substituted in sequence 17 are underlined.5-b Not hydrolyzed when incubated with 140 nm HIV-1 proteinase for 1 h at 37 °C.5-c Low solubility of the substrate did not allow the kinetic measurements. Open table in a new tab Table IIIAssay of P 3 and P 3 ′ substituted peptides based on a palindromic sequence by HIV-1 proteinaseNumberSequence3-aAmino acids substituted in sequence 17 are underlined.K mk catk cat/K mmms −1mm −1 s −117SDTY*YTDS0.21 ± 0.040.34 ± 0.021.6218SGTY*YTDS0.50 ± 0.060.46 ± 0.020.9219SDTY*YTGS0.35 ± 0.030.29 ± 0.010.8320SGTY*YTGS2.88 ± 0.440.62 ± 0.040.2221SQTY*YTDS0.62 ± 0.090.43 ± 0.040.6922SDTY*YTQS0.36 ± 0.060.21 ± 0.020.5823SQTY*YTQS0.22 ± 0.030.060 ± 0.0030.2724SFTY*YTDS0.052 ± 0.0170.08 ± 0.011.5425SDTY*YTFS0.24 ± 0.050.22 ± 0.020.9226SFTY*YTFSNot determined3-bLow solubility of the substrate did not allow the kinetic measurements.27SLTY*YTDS0.250 ± 0.0650.010 ± 0.0020.0428SDTY*YTLS0.082 ± 0.0150.014 ± 0.0010.1729SLTY*YTLSNot determined3-bLow solubility of the substrate did not allow the kinetic measurements.3-a Amino acids substituted in sequence 17 are underlined.3-b Low solubility of the substrate did not allow the kinetic measurements. Open table in a new tab Table IAssay of substrates having the sequence of naturally occurring cleavage sites and those containing single or multiple substitutions in the cleavage site sequence between the matrix and capsid proteins of HIV-1 by HIV-1 proteinaseNumberSequence1-aAmino acids substituted in the sequence of peptide 3 are underlined.K mk catk cat/K mmms −1mm −1 s −11 AETF*YVDGAA1-bPeptide representing the determined cleavage site in reverse transcriptase of HIV-1. Kinetic parameters for this peptide were also reported in Ref. 15.0.046 ± 0.0060.42 ± 0.029.12GAETF*YTDGS1-cPeptide representing a proposed cleavage site in reverse transcriptase of HIV-2 (22).0.33 ± 0.060.21 ± 0.030.643VSQNY*PIVQ1-dPeptide representing the MA/CA cleavage site of HIV-1 (peptide 3, previously designated as SP-211). Kinetic parameters for this peptide were also reported in Ref. 15.0.15 ± 0.036.8 ± 0.0745.34VSQNY*YIVQ0.020 ± 0.0030.010 ± 0.0010.505VSQTY*PIVQ0.41 ± 0.050.70 ± 0.021.76VSQNY*PTVQ0.85 ± 0.202.07 ± 0.372.437VSDNY*PIVQ1.77 ± 0.322.62 ± 0.151.488VSQNY*PIDQ6.84 ± 0.983.09 ± 0.310.459VSQTY*YIVQ0.035 ± 0.0050.020 ± 0.0010.5710VSQNY*YTVQ0.026 ± 0.0070.18 ± 0.016.9211VSQTY*YTVQ0.021 ± 0.0030.050 ± 0.0012.3812VSQNY*YIDQ<0.02ND1-eND, not determined.0.091-fDetermined as competitive substrate with peptide 45 (TableV).13VSQTY*YIDQ0.006 ± 0.0010.10 ± 0.0116.6714VSQNY*YTDQ0.20 ± 0.030.20 ± 0.021.0015VSQTY*YTDQ0.17 ± 0.030.10 ± 0.010.5916VSDTY*YTDQ0.19 ± 0.030.030 ± 0.0030.161-a Amino acids substituted in the sequence of peptide 3 are underlined.1-b Peptide representing the determined cleavage site in reverse transcriptase of HIV-1. Kinetic parameters for this peptide were also reported in Ref. 15Tözsér J. Bláha I. Copeland T.D. Wondrak E.M. Oroszlan S. FEBS Lett. 1991; 281: 77-80Crossref PubMed Scopus (148) Google Scholar.1-c Peptide representing a proposed cleavage site in reverse transcriptase of HIV-2 (22Le Grice S.F.J. Ette R. Mills J. Mous J. J. Biol. Chem. 1989; 264: 14902-14908Abstract Full Text PDF PubMed Google Scholar).1-d Peptide representing the MA/CA cleavage site of HIV-1 (peptide 3, previously designated as SP-211). Kinetic parameters for this peptide were also reported in Ref. 15Tözsér J. Bláha I. Copeland T.D. Wondrak E.M. Oroszlan S. FEBS Lett. 1991; 281: 77-80Crossref PubMed Scopus (148) Google Scholar.1-e ND, not determined.1-f Determined as competitive substrate with peptide 45 (TableV). Open table in a new tab Subsequently Fan et al. (24Fan N. Rank K.B. Leone J.W. Heinrikson R.L. Bannow C.A. Smith C.W. Evans D.B. Poppe S.M. Tarpley W.G. Rothrock D.J. Tomasselli A.G. Sharma S.K. J. Biol. Chem. 1995; 270: 13573-13579Abstract Full Text Full Text PDF PubMed Google Scholar) demonstrated that in fact the sequence of peptide 2 does not represent the actual cleavage site required to be cleaved to produce the smaller subunit of the HIV-2 reverse transcriptase. They found that the real cleavage site has the sequence of AFAM*ALTD and is downstream from the one proposed by Le Grice et al. (22Le Grice S.F.J. Ette R. Mills J. Mous J. J. Biol. Chem. 1989; 264: 14902-14908Abstract Full Text PDF PubMed Google Scholar). Nevertheless, we in this study and Fanet al. (24Fan N. Rank K.B. Leone J.W. Heinrikson R.L. Bannow C.A. Smith C.W. Evans D.B. Poppe S.M. Tarpley W.G. Rothrock D.J. Tomasselli A.G. Sharma S.K. J. Biol. Chem. 1995; 270: 13573-13579Abstract Full Text Full Text PDF PubMed Google Scholar) have confirmed our initial finding that the HIV-2-derived peptide 2 or its shorter octapeptide homolog is a substrate of the HIV-1 proteinase. We have also compared the specificity of the HIV-1 and HIV-2 proteinases using a series of oligopeptide substrates containing single amino acid substitutions in the sequence of SP-211 (see peptide 3 in Table I), a peptide that corresponds to the type 1 MA/CA cleavage site in HIV-1 (10Tözsér J. Weber I.T. Gustchina A. Bláha I. Copeland T.D. Louis J.M. Oroszlan S. Biochemistry. 1992; 31: 4793-4800Crossref PubMed Scopus (99) Google Scholar, 23Tözsér J. Gustchina A. Weber I.T. Bláha I. Wondrak E.M. Oroszlan S. FEBS Lett. 1991; 279: 356-360Crossref PubMed Scopus (66) Google Scholar). In these studies it was found that substitution of Pro at the P1′ position to any other amino acid tested, including Tyr, formed nonhydrolyzable or very poor substrates of HIV proteinases. These P1′-substituted peptides inhibited the hydrolysis of SP-211 by HIV proteinase, which suggested that they were able to bind to the enzyme (10Tözsér J. Weber I.T. Gustchina A. Bláha I. Copeland T.D. Louis J.M. Oroszlan S. Biochemistry. 1992; 31: 4793-4800Crossref PubMed Scopus (99) Google Scholar). The best inhibition was obtained with the P1′ Tyr-substituted peptide, 2J. Tözsér and S. Oroszlan, unpublished results. suggesting its high affinity for the HIV-1 proteinase. In good agreement with these preliminary findings, both the K m andk cat values determined in the present study were substantially lower for the P1′ Tyr-substituted peptide compared with the unmodified one (compare peptides 3 and 4 in Table I). Substitution of P1′ Pro of SP-211 with Tyr converts a type 1 substrate to a type 2 substrate. However, the P1′ Tyr-substituted peptide is a poor substrate of HIV-1 proteinase (peptide 4 in Table I). To understand better the specificity of HIV-1 proteinase and the differences of the subsite preference in type 1 and type 2 cleavage sites, further single and multiple substitutions were carried out in the type 1 MA/CA cleavage site substrate (peptide 3), introducing residues characteristic of the type 2 cleavage site peptides 1 and 2 (Table I). The same substitutions were also introduced in the P1′ Tyr-modified (type 2) substrate. This peptide series allowed us to compare the preference for P2 Asn over Thr as well as P2′ Ile over Thr in different sequence contexts. The ln (k cat/K m ) values are directly related to the free energy of the binding of the transition state by the enzyme (25Fersht A. Enzyme Structure and Mechanism. 2nd Ed. W. H. Freeman and Co., New York1985: 311-317Google Scholar). If the subsite interactions are mostly independent of each other, thek cat/K m ratios (equal toe −ΔΔG/RT) for substrate pairs havingX and Y residues at the same subsite, should be similar and independent of the surrounding sequence, as has been found for e.g. trypsin (26Pozsgay M. Cs-Szabó G. Bajusz S. Simmonson R. Gáspár R. Elödi P. Eur. J. Biochem. 1981; 115: 497-502Crossref PubMed Scopus (28) Google Scholar) and chymotrypsin (27Tözsér J. Cs-Szabó G. Pozsgay M. Aurell L. Elödi P. Acta Biochim. Biophys. Hung. 1986; 21: 335-348PubMed Google Scholar). Single substitutions of P2 Asn and P2′ Ile with Thr (peptides 5 and 6 in Table I, respectively) resulted in substantial increases in K m and decreases in catalytic constants compared with the unmodified peptide 3. Comparison of kinetic parameters of peptides 1 and 2 as well as of peptides 3 and 6 suggests that β-branched hydrophobic residues such as Val or Ile at P2′ positions are much more favorable than the β-branched but more hydrophilic Thr in these two different sequence contexts. Furthermore, a similar preference was found in two other series of peptides (11Griffith J.T. Phylip L.H. Konvalinka J. Strop P. Gustchina A. Wlodawer A. Davenport R.J. Briggs R. Dunn B.M. Kay J. Biochemistry. 1992; 31: 5193-5200Crossref PubMed Scopus (99) Google Scholar). Single substitutions of P3 Gln and P3′ Val to Asp (peptide 7 and 8 of Table I, respectively) resulted in dramatic increases in K m values but only moderate decreases in k cat values, as was found previously for cleavage with HIV-2 proteinase (10Tözsér J. Weber I.T. Gustchina A. Bláha I. Copeland T.D. Louis J.M. Oroszlan S. Biochemistry. 1992; 31: 4793-4800Crossref PubMed Scopus (99) Google Scholar). Substituting Thr at P2 residue for Asn in the P1′ Tyr analog of SP-211 (peptide 9 in Table I) did not yield substantial changes in the kinetic parameters, whereas substitution of P2′ Thr for Ile in the same sequence context (peptide 10) yielded an approximately 10-fold increase in (k cat/K m ). The same substitution was very unfavorable in the P1′ Pro-containing peptides (compare peptides 3 and 6 in Table I), suggesting a strong influence of the P1′ residue on the preference for the P2′ residue. Substitution of Thr at both the P2and P2′ positions of peptide 4 was less effective than the single P2′ substitution (compare peptides 10 and 11 of Table I). Interestingly, whereas the P3′ Asp substitution of peptide 4 yielded a substrate that was even less susceptible to hydrolysis (peptide 12 in Table I), a further substitution for P2 Thr (peptide 13) yielded a substrate withK m andk cat/K m values better than those of the naturally occurring type 2 cleavage site peptide 1. As expected from the comparison of peptides 1 and 2, further substitution of P2′ Ile for Thr yielded a substrate with much lowerk cat/K m value because of the substantial increase of the K m (peptide 14 of TableI). Enzyme-substrate models were built and energy minimized to explore the possible interactions of the enzyme and the substrates at the molecular level. The substrate lies in an extended β-conformation in the substrate binding site, which puts P4, P2, P1′, and P3′ residues at one side and P3, P1, and P2′ on the other side (Fig. 1). Adjacent substrate binding sites (like S2 and S1′) partially overlap (Fig. 1). Molecular modeling suggested that the P2 Thr-substituted SP-211 analog could not fit well in the S2 subsite (not shown) because of the predicted interaction with P1′ Pro, as described previously for another β-branched amino acid, Val (10Tözsér J. Weber I.T. Gustchina A. Bláha I. Copeland T.D. Louis J.M. Oroszlan S. Biochemistry. 1992; 31: 4793-4800Crossref PubMed Scopus (99) Google Scholar). When the P1′ Pro is changed to Tyr, this restraint is removed. However, comparison of peptides 4 and 9 suggested that this substitution alone is not sufficient to make Thr preferable over Asn in the SP-211 sequence context, but the additional P3′ Val to Asp exchange is required (peptides 12 and 13). However, a further substitution" @default.
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W2076304758 title "Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity" @default.
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