• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of prosciprotein sciencecshl presssubscriptionsetoc alertsthe protein societyjournal home
Protein Sci. Jan 1999; 8(1): 180–195.
PMCID: PMC2144115

Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease.

Abstract

The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.

Full Text

The Full Text of this article is available as a PDF (359K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Alexov EG, Gunner MR. Incorporating protein conformational flexibility into the calculation of pH-dependent protein properties. Biophys J. 1997 May;72(5):2075–2093. [PMC free article] [PubMed]
  • Antosiewicz J, McCammon JA, Gilson MK. Prediction of pH-dependent properties of proteins. J Mol Biol. 1994 May 6;238(3):415–436. [PubMed]
  • Antosiewicz J, McCammon JA, Gilson MK. The determinants of pKas in proteins. Biochemistry. 1996 Jun 18;35(24):7819–7833. [PubMed]
  • Baldwin ET, Bhat TN, Gulnik S, Liu B, Topol IA, Kiso Y, Mimoto T, Mitsuya H, Erickson JW. Structure of HIV-1 protease with KNI-272, a tight-binding transition-state analog containing allophenylnorstatine. Structure. 1995 Jun 15;3(6):581–590. [PubMed]
  • Bashford D, Case DA, Dalvit C, Tennant L, Wright PE. Electrostatic calculations of side-chain pK(a) values in myoglobin and comparison with NMR data for histidines. Biochemistry. 1993 Aug 10;32(31):8045–8056. [PubMed]
  • Bashford D, Gerwert K. Electrostatic calculations of the pKa values of ionizable groups in bacteriorhodopsin. J Mol Biol. 1992 Mar 20;224(2):473–486. [PubMed]
  • Bashford D, Karplus M. pKa's of ionizable groups in proteins: atomic detail from a continuum electrostatic model. Biochemistry. 1990 Nov 6;29(44):10219–10225. [PubMed]
  • Bernstein FC, Koetzle TF, Williams GJ, Meyer EF, Jr, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. [PubMed]
  • Beroza P, Fredkin DR, Okamura MY, Feher G. Protonation of interacting residues in a protein by a Monte Carlo method: application to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5804–5808. [PMC free article] [PubMed]
  • Beveridge AJ, Heywood GC. A quantum mechanical study of the active site of aspartic proteinases. Biochemistry. 1993 Apr 6;32(13):3325–3333. [PubMed]
  • Brocklehurst K. A sound basis for pH-dependent kinetic studies on enzymes. Protein Eng. 1994 Mar;7(3):291–299. [PubMed]
  • Brünger AT, Karplus M. Polar hydrogen positions in proteins: empirical energy placement and neutron diffraction comparison. Proteins. 1988;4(2):148–156. [PubMed]
  • Cheng YS, Yin FH, Foundling S, Blomstrom D, Kettner CA. Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9660–9664. [PMC free article] [PubMed]
  • Cleland WW. Determining the chemical mechanisms of enzyme-catalyzed reactions by kinetic studies. Adv Enzymol Relat Areas Mol Biol. 1977;45:273–387. [PubMed]
  • Debouck C, Gorniak JG, Strickler JE, Meek TD, Metcalf BW, Rosenberg M. Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8903–8906. [PMC free article] [PubMed]
  • Erickson-Viitanen S, Klabe RM, Cawood PG, O'Neal PL, Meek JL. Potency and selectivity of inhibition of human immunodeficiency virus protease by a small nonpeptide cyclic urea, DMP 323. Antimicrob Agents Chemother. 1994 Jul;38(7):1628–1634. [PMC free article] [PubMed]
  • Geller M, Miller M, Swanson SM, Maizel J. Analysis of the structure of HIV-1 protease complexed with a hexapeptide inhibitor. Part II: Molecular dynamic studies of the active site region. Proteins. 1997 Feb;27(2):195–203. [PubMed]
  • Gibas CJ, Subramaniam S. Explicit solvent models in protein pKa calculations. Biophys J. 1996 Jul;71(1):138–147. [PMC free article] [PubMed]
  • Gilson MK. Multiple-site titration and molecular modeling: two rapid methods for computing energies and forces for ionizable groups in proteins. Proteins. 1993 Mar;15(3):266–282. [PubMed]
  • Gilson MK, Honig B. Calculation of the total electrostatic energy of a macromolecular system: solvation energies, binding energies, and conformational analysis. Proteins. 1988;4(1):7–18. [PubMed]
  • Gilson MK, Honig BH. Energetics of charge-charge interactions in proteins. Proteins. 1988;3(1):32–52. [PubMed]
  • Gilson MK, Honig BH. The dielectric constant of a folded protein. Biopolymers. 1986 Nov;25(11):2097–2119. [PubMed]
  • Gilson MK, Rashin A, Fine R, Honig B. On the calculation of electrostatic interactions in proteins. J Mol Biol. 1985 Aug 5;184(3):503–516. [PubMed]
  • Grant SK, Deckman IC, Culp JS, Minnich MD, Brooks IS, Hensley P, Debouck C, Meek TD. Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases. Biochemistry. 1992 Oct 6;31(39):9491–9501. [PubMed]
  • Graves MC, Lim JJ, Heimer EP, Kramer RA. An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2449–2453. [PMC free article] [PubMed]
  • Hansen J, Billich S, Schulze T, Sukrow S, Moelling K. Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody. EMBO J. 1988 Jun;7(6):1785–1791. [PMC free article] [PubMed]
  • Hyland LJ, Tomaszek TA, Jr, Meek TD. Human immunodeficiency virus-1 protease. 2. Use of pH rate studies and solvent kinetic isotope effects to elucidate details of chemical mechanism. Biochemistry. 1991 Aug 27;30(34):8454–8463. [PubMed]
  • Ido E, Han HP, Kezdy FJ, Tang J. Kinetic studies of human immunodeficiency virus type 1 protease and its active-site hydrogen bond mutant A28S. J Biol Chem. 1991 Dec 25;266(36):24359–24366. [PubMed]
  • Jadhav PK, Ala P, Woerner FJ, Chang CH, Garber SS, Anton ED, Bacheler LT. Cyclic urea amides: HIV-1 protease inhibitors with low nanomolar potency against both wild type and protease inhibitor resistant mutants of HIV. J Med Chem. 1997 Jan 17;40(2):181–191. [PubMed]
  • Jaskólski M, Tomasselli AG, Sawyer TK, Staples DG, Heinrikson RL, Schneider J, Kent SB, Wlodawer A. Structure at 2.5-A resolution of chemically synthesized human immunodeficiency virus type 1 protease complexed with a hydroxyethylene-based inhibitor. Biochemistry. 1991 Feb 12;30(6):1600–1609. [PubMed]
  • Kageyama S, Mimoto T, Murakawa Y, Nomizu M, Ford H, Jr, Shirasaka T, Gulnik S, Erickson J, Takada K, Hayashi H, et al. In vitro anti-human immunodeficiency virus (HIV) activities of transition state mimetic HIV protease inhibitors containing allophenylnorstatine. Antimicrob Agents Chemother. 1993 Apr;37(4):810–817. [PMC free article] [PubMed]
  • Kick EK, Roe DC, Skillman AG, Liu G, Ewing TJ, Sun Y, Kuntz ID, Ellman JA. Structure-based design and combinatorial chemistry yield low nanomolar inhibitors of cathepsin D. Chem Biol. 1997 Apr;4(4):297–307. [PubMed]
  • Klapper I, Hagstrom R, Fine R, Sharp K, Honig B. Focusing of electric fields in the active site of Cu-Zn superoxide dismutase: effects of ionic strength and amino-acid modification. Proteins. 1986 Sep;1(1):47–59. [PubMed]
  • Knowles JR. The intrinsic pKa-values of functional groups in enzymes: improper deductions from the pH-dependence of steady-state parameters. CRC Crit Rev Biochem. 1976 Nov;4(2):165–173. [PubMed]
  • Kohl NE, Emini EA, Schleif WA, Davis LJ, Heimbach JC, Dixon RA, Scolnick EM, Sigal IS. Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4686–4690. [PMC free article] [PubMed]
  • Lam PY, Ru Y, Jadhav PK, Aldrich PE, DeLucca GV, Eyermann CJ, Chang CH, Emmett G, Holler ER, Daneker WF, et al. Cyclic HIV protease inhibitors: synthesis, conformational analysis, P2/P2' structure-activity relationship, and molecular recognition of cyclic ureas. J Med Chem. 1996 Aug 30;39(18):3514–3525. [PubMed]
  • Lam PY, Jadhav PK, Eyermann CJ, Hodge CN, Ru Y, Bacheler LT, Meek JL, Otto MJ, Rayner MM, Wong YN, et al. Rational design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors. Science. 1994 Jan 21;263(5145):380–384. [PubMed]
  • Liu H, Müller-Plathe F, van Gunsteren WF. A combined quantum/classical molecular dynamics study of the catalytic mechanism of HIV protease. J Mol Biol. 1996 Aug 23;261(3):454–469. [PubMed]
  • Matthew JB, Gurd FR, Garcia-Moreno B, Flanagan MA, March KL, Shire SJ. pH-dependent processes in proteins. CRC Crit Rev Biochem. 1985;18(2):91–197. [PubMed]
  • McIntosh LP, Hand G, Johnson PE, Joshi MD, Körner M, Plesniak LA, Ziser L, Wakarchuk WW, Withers SG. The pKa of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of bacillus circulans xylanase. Biochemistry. 1996 Aug 6;35(31):9958–9966. [PubMed]
  • Mildner AM, Rothrock DJ, Leone JW, Bannow CA, Lull JM, Reardon IM, Sarcich JL, Howe WJ, Tomich CS, Smith CW, et al. The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties. Biochemistry. 1994 Aug 16;33(32):9405–9413. [PubMed]
  • Miller M, Geller M, Gribskov M, Kent SB. Analysis of the structure of chemically synthesized HIV-1 protease complexed with a hexapeptide inhibitor. Part I: Crystallographic refinement of 2 A data. Proteins. 1997 Feb;27(2):184–194. [PubMed]
  • Miller M, Schneider J, Sathyanarayana BK, Toth MV, Marshall GR, Clawson L, Selk L, Kent SB, Wlodawer A. Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution. Science. 1989 Dec 1;246(4934):1149–1152. [PubMed]
  • Moore ML, Bryan WM, Fakhoury SA, Magaard VW, Huffman WF, Dayton BD, Meek TD, Hyland L, Dreyer GB, Metcalf BW, et al. Peptide substrates and inhibitors of the HIV-1 protease. Biochem Biophys Res Commun. 1989 Mar 15;159(2):420–425. [PubMed]
  • Muir TW, Williams MJ, Kent SB. Detection of synthetic protein isomers and conformers by electrospray mass spectrometry. Anal Biochem. 1995 Jan 1;224(1):100–109. [PubMed]
  • Oberoi H, Allewell NM. Multigrid solution of the nonlinear Poisson-Boltzmann equation and calculation of titration curves. Biophys J. 1993 Jul;65(1):48–55. [PMC free article] [PubMed]
  • Oda Y, Yamazaki T, Nagayama K, Kanaya S, Kuroda Y, Nakamura H. Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR. Biochemistry. 1994 May 3;33(17):5275–5284. [PubMed]
  • Rich DH, Green J, Toth MV, Marshall GR, Kent SB. Hydroxyethylamine analogues of the p17/p24 substrate cleavage site are tight-binding inhibitors of HIV protease. J Med Chem. 1990 May;33(5):1285–1288. [PubMed]
  • Schock HB, Garsky VM, Kuo LC. Mutational anatomy of an HIV-1 protease variant conferring cross-resistance to protease inhibitors in clinical trials. Compensatory modulations of binding and activity. J Biol Chem. 1996 Dec 13;271(50):31957–31963. [PubMed]
  • Shire SJ, Hanania GI, Gurd FR. Electrostatic effects in myoglobin. Hydrogen ion equilibria in sperm whale ferrimyoglobin. Biochemistry. 1974 Jul 2;13(14):2967–2974. [PubMed]
  • Silva AM, Cachau RE, Sham HL, Erickson JW. Inhibition and catalytic mechanism of HIV-1 aspartic protease. J Mol Biol. 1996 Jan 19;255(2):321–346. [PubMed]
  • Simonson T, Perahia D. Internal and interfacial dielectric properties of cytochrome c from molecular dynamics in aqueous solution. Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1082–1086. [PMC free article] [PubMed]
  • Simonson T, Perahia D, Brünger AT. Microscopic theory of the dielectric properties of proteins. Biophys J. 1991 Mar;59(3):670–690. [PMC free article] [PubMed]
  • Smith R, Brereton IM, Chai RY, Kent SB. Ionization states of the catalytic residues in HIV-1 protease. Nat Struct Biol. 1996 Nov;3(11):946–950. [PubMed]
  • Spinelli S, Liu QZ, Alzari PM, Hirel PH, Poljak RJ. The three-dimensional structure of the aspartyl protease from the HIV-1 isolate BRU. Biochimie. 1991 Nov;73(11):1391–1396. [PubMed]
  • Stivers JT, Abeygunawardana C, Mildvan AS, Hajipour G, Whitman CP. 4-Oxalocrotonate tautomerase: pH dependence of catalysis and pKa values of active site residues. Biochemistry. 1996 Jan 23;35(3):814–823. [PubMed]
  • Tipton KF, Dixon HB. Effects of pH on enzymes. Methods Enzymol. 1979;63:183–234. [PubMed]
  • Todd MJ, Semo N, Freire E. The structural stability of the HIV-1 protease. J Mol Biol. 1998 Oct 23;283(2):475–488. [PubMed]
  • Tomasselli AG, Mildner AM, Rothrock DJ, Sarcich JL, Lull J, Leone J, Heinrikson RL. Mutants of HIV-1 protease with enhanced stability to autodegradation. Adv Exp Med Biol. 1995;362:387–398. [PubMed]
  • Wang YX, Freedberg DI, Yamazaki T, Wingfield PT, Stahl SJ, Kaufman JD, Kiso Y, Torchia DA. Solution NMR evidence that the HIV-1 protease catalytic aspartyl groups have different ionization states in the complex formed with the asymmetric drug KNI-272. Biochemistry. 1996 Aug 6;35(31):9945–9950. [PubMed]
  • Warwicker J, Watson HC. Calculation of the electric potential in the active site cleft due to alpha-helix dipoles. J Mol Biol. 1982 Jun 5;157(4):671–679. [PubMed]
  • Wlodawer A, Miller M, Jaskólski M, Sathyanarayana BK, Baldwin E, Weber IT, Selk LM, Clawson L, Schneider J, Kent SB. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science. 1989 Aug 11;245(4918):616–621. [PubMed]
  • WYMAN J. THE BINDING POTENTIAL, A NEGLECTED LINKAGE CONCEPT. J Mol Biol. 1965 Mar;11:631–644. [PubMed]
  • Xie D, Gulnik S, Collins L, Gustchina E, Suvorov L, Erickson JW. Dissection of the pH dependence of inhibitor binding energetics for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues. Biochemistry. 1997 Dec 23;36(51):16166–16172. [PubMed]
  • Yang AS, Gunner MR, Sampogna R, Sharp K, Honig B. On the calculation of pKas in proteins. Proteins. 1993 Mar;15(3):252–265. [PubMed]
  • Yang AS, Honig B. On the pH dependence of protein stability. J Mol Biol. 1993 May 20;231(2):459–474. [PubMed]
  • Zhang ZY, Poorman RA, Maggiora LL, Heinrikson RL, Kézdy FJ. Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide. J Biol Chem. 1991 Aug 25;266(24):15591–15594. [PubMed]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...