European Biophysics Journal

, Volume 41, Issue 5, pp 449–460 | Cite as

Ionizable side chains at catalytic active sites of enzymes

Original Paper


Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.


Computational geometry Active site Charge density 


  1. Antosiewicz J, McCammon JA, Gilson MK (1996) The determinants of pKas in proteins. Biochemistry 35(24):7819–7833. doi:10.1021/bi9601565 PubMedCrossRefGoogle Scholar
  2. Barthel J, Krienke H, Kunz W (1998) Physical chemistry of electrolyte solutions: modern aspects. Springer, New YorkGoogle Scholar
  3. Berman HM, Battistuz T, Bhat TN, Bluhm WF, Bourne PE, Burkhardt K, Feng Z, Gilliland GL, Iype L, Jain S, Fagan P, Marvin J, Padilla D, Ravichandran V, Schneider B, Thanki N, Weissig H, Westbrook JD, Zardecki C (2002) The Protein Data Bank. Acta Crystallogr D Biol Crystallogr 58:899–907. doi:10.1107/S0907444902003451 Google Scholar
  4. Boda D, Giri J, Henderson D, Eisenberg B, Gillespie D (2011) Analyzing the components of the free-energy landscape in a calcium selective ion channel by Widom’s particle insertion method. J Chem Phys 134:055102–055114PubMedCrossRefGoogle Scholar
  5. Boda D, Nonner W, Henderson D, Eisenberg B, Gillespie D (2008) Volume exclusion in calcium selective channels. Biophys J 94(9):3486–3496. doi:10.1529/biophysj.107.122796 PubMedCrossRefGoogle Scholar
  6. Boda D, Nonner W, Valisko M, Henderson D, Eisenberg B, Gillespie D (2007) Steric selectivity in Na channels arising from protein polarization and mobile side chains. Biophys J 93(6):1960–1980. doi:10.1529/biophysj.107.105478 PubMedCrossRefGoogle Scholar
  7. Boda D, Valisko M, Henderson D, Eisenberg B, Gillespie D, Nonner W (2009) Ionic selectivity in L-type calcium channels by electrostatics and hard-core repulsion. J Gen Physiol 133(5):497–509. doi:10.1085/jgp.200910211 PubMedCrossRefGoogle Scholar
  8. Bostick DL, Brooks CL 3rd (2009) Statistical determinants of selective ionic complexation: ions in solvent, transport proteins, and other “hosts”. Biophys J 96(11):4470–4492. doi:10.1016/j.bpj.2009.03.001 PubMedCrossRefGoogle Scholar
  9. Cannon JJ, Tang D, Hur N, Kim D (2010) Competitive entry of sodium and potassium into nanoscale pores. J Phys Chem B 114(38):12252–12256. doi:10.1021/jp104609d PubMedCrossRefGoogle Scholar
  10. Cohen EJ, Edsall J (1943) Proteins, amino acids, and peptides. Reinhold, New YorkGoogle Scholar
  11. Cojocaru V, Balali-Mood K, Sansom MS, Wade RC (2011) Structure and dynamics of the membrane-bound cytochrome P450 2C9. PLoS Comput Biol 7(8):e1002152. doi:10.1371/journal.pcbi.1002152 PubMedCrossRefGoogle Scholar
  12. Connolly ML (1985) Computation of molecular volume. J Am Chem Soc 107(5):1118–1124. doi:10.1021/ja00291a006 CrossRefGoogle Scholar
  13. Davis ME, McCammon JA (1990) Electrostatics in biomolecular structure and dynamics. Chem Rev 90:509–521CrossRefGoogle Scholar
  14. Dixon M, Webb EC (1979) Enzymes. Academic Press, New YorkGoogle Scholar
  15. Doi M (2009) Gel dynamics. J Phys Soc Jpn 78:052001CrossRefGoogle Scholar
  16. Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, Liang J (2006) CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res 34(suppl 2):W116–W118PubMedCrossRefGoogle Scholar
  17. Durand-Vidal S, Simonin J-P, Turq P (2000) Electrolytes at interfaces. Kluwer, BostonGoogle Scholar
  18. Durand-Vidal S, Turq P, Bernard O, Treiner C, Blum L (1996) New perspectives in transport phenomena in electrolytes. Phys A 231:123–143CrossRefGoogle Scholar
  19. Edelsbrunner H, Facello M, Fu P, Liang J (1995) Measuring proteins and voids in proteins. Syst Sci Proc Twenty-Eighth Hawaii Int Conf Syst Sci 5:256–264. doi:10.1109/HICSS.1995.375331 Google Scholar
  20. Edelsbrunner H, Facello M, Liang J (1998) On the definition and the construction of pockets in macromolecules. Discret Appl Math 88:83–102CrossRefGoogle Scholar
  21. Edsall J, Wyman J (1958) Biophysical chemistry. Academic Press, NYGoogle Scholar
  22. Eisenberg B (2005) Living transistors: a physicist’s view of ion channels. Available on p 24 as q-bio/0506016v2
  23. Eisenberg B (2010) Multiple scales in the simulation of ion channels and proteins. J Phys Chem C 114(48):20719–20733. doi:10.1021/jp106760t CrossRefGoogle Scholar
  24. Eisenberg B (2011a) Crowded charges in ion channels. In: Advances in chemical physics. Wiley, New York, pp 77–223 also available at http://arXiv.orgas arXiv 1009.1786v1001 doi:10.1002/9781118158715.ch2
  25. Eisenberg B (2011b) Life’s solutions are not ideal. Posted on arXivorg with Paper ID arXiv:11050184v1Google Scholar
  26. Eisenberg B (2011c) Mass action in ionic solutions. Chem Phys Lett 511:1–6. doi:10.1016/j.cplett.2011.05.037 PubMedCrossRefGoogle Scholar
  27. Eisenberg B (2012) Ions in fluctuating channels: transistors alive. fluctuations and noise letters (in the press): Earlier version ‘Living transistors: a physicist’s view of ion channels’. Available on as q-bio/0506016v0506012
  28. Eisenberg B, Hyon Y, Liu C (2010) Energy variational analysis EnVarA of ions in water and channels: field theory for primitive models of complex ionic fluids. J Chem Phys 133:104104PubMedCrossRefGoogle Scholar
  29. Eisenberg RS (1990) Channels as enzymes: oxymoron and tautology. J Membr Biol 115:1–12. Available on arXiv as Google Scholar
  30. Eisenberg RS (1996a) Atomic biology, electrostatics and ionic channels. In: Elber R (ed) New developments and theoretical studies of proteins, vol 7. World Scientific, Philadelphia, pp 269–357. Published in the physics ArXiv as arXiv:0807.0715Google Scholar
  31. Eisenberg RS (1996b) Computing the field in proteins and channels. J Membr Biol 150:1–25. Also available on as arXiv 1009.2857Google Scholar
  32. Ellinor PT, Yang J, Sather WA, Zhang J-F, Tsien R (1995) Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions. Neuron 15:1121–1132PubMedCrossRefGoogle Scholar
  33. Fawcett WR (2004) Liquids, solutions, and interfaces: from classical macroscopic descriptions to modern microscopic details. Oxford University Press, New YorkGoogle Scholar
  34. Fischer E (1894a) Einfluss der Configuration auf die Wirkung der Enzyme. Berichte der deutschen chemischen Gesellschaft 27:2985–2993Google Scholar
  35. Fischer E (1894b) Einfluss der Configuration auf die Wirkung der Enzyme. II. Berichte der deutschen chemischen Gesellschaft 27:3479–3483Google Scholar
  36. Fraenkel D (2010a) Monoprotic mineral acids analyzed by the smaller-ion shell model of strong electrolyte solutions. J Phys Chem B 115(3):557–568. doi:10.1021/jp108997f PubMedCrossRefGoogle Scholar
  37. Fraenkel D (2010b) Simplified electrostatic model for the thermodynamic excess potentials of binary strong electrolyte solutions with size-dissimilar ions. Mol Phys 108(11):1435–1466CrossRefGoogle Scholar
  38. Friedman HL (1981) Electrolyte solutions at equilibrium. Annu Rev Phys Chem 32(1):179–204. doi:10.1146/annurev.pc.32.100181.001143 CrossRefGoogle Scholar
  39. Fuoss RM, Accascina F (1959) Electrolytic conductance. Interscience, New YorkGoogle Scholar
  40. Fuoss RM, Onsager L (1955) Conductance of strong electrolytes at finite dilutions. Proc Nat Acad Sci USA 41(5):274–283PubMedCrossRefGoogle Scholar
  41. Gillespie D, Giri J, Fill M (2009) Reinterpreting the anomalous mole fraction effect. the ryanodine receptor case study. Biophys J 97(8):2212–2221PubMedCrossRefGoogle Scholar
  42. Gutteridge A, Thornton JM (2005) Understanding nature’s catalytic toolkit. Trends Biochem Sci 30(11):622–629. doi:10.1016/j.tibs.2005.09.006 PubMedCrossRefGoogle Scholar
  43. Hansen J-P, McDonald IR (2006) Theory of simple liquids, 3rd edn. Academic Press, New YorkGoogle Scholar
  44. Helfferich F (1962 (1995 reprint)) Ion exchange. McGraw Hill reprinted by Dover, New YorkGoogle Scholar
  45. Hille B (2001) Ionic channels of excitable membranes, 3rd edn. Sinauer Associates Inc., SunderlandGoogle Scholar
  46. Honig B, Nichols A (1995) Classical electrostatics in biology and chemistry. Science 268:1144–1149PubMedCrossRefGoogle Scholar
  47. Hovarth AL (1985) Handbook of aqueous electrolyte solutions: physical properties, estimation, and correlation methods. Ellis Horwood, New YorkGoogle Scholar
  48. Howard JJ, Perkyns JS, Pettitt BM (2010) The behavior of ions near a charged wall-dependence on ion size, concentration, and surface charge. J Phys Chem B 114(18):6074–6083. doi:10.1021/jp9108865 PubMedCrossRefGoogle Scholar
  49. Howe RT, Sodini CG (1997) Microelectronics: an integrated approach. Prentice Hall, Upper Saddle RiverGoogle Scholar
  50. Hünenberger PH, Reif M (2011) Single-ion solvation. RSC Publishing, CambridgeGoogle Scholar
  51. Hyon Y, Kwak DY, Liu C (2010) Energetic variational approach in complex fluids: maximum dissipation principle. Available at as IMA Preprint Series # 2228 26 (4: April):1291–1304. Available at as IMA Preprint Series # 2228
  52. Justice J-C (1983) Conductance of electrolyte solutions. In: Conway BE, Bockris JOM, Yaeger E (eds) Comprehensive treatise of electrochemistry. Thermondynbamic and transport properties of aqueous and molten electrolytes, vol 5. Plenum, New York, pp 223–338Google Scholar
  53. Kalyuzhnyi YV, Vlachy V, Dill KA (2010) Aqueous alkali halide solutions: can osmotic coefficients be explained on the basis of the ionic sizes alone? Phys Chem Chem Phys 12(23):6260–6266PubMedCrossRefGoogle Scholar
  54. Koch SE, Bodi I, Schwartz A, Varadi G (2000) Architecture of Ca(2+) channel pore-lining segments revealed by covalent modification of substituted cysteines. J Biol Chem 275(44):34493–34500. doi:10.1074/jbc.M005569200 PubMedCrossRefGoogle Scholar
  55. Kokubo H, Pettitt BM (2007) Preferential solvation in urea solutions at different concentrations: properties from simulation studies. J Phys Chem B 111(19):5233–5242. doi:10.1021/jp067659x PubMedCrossRefGoogle Scholar
  56. Kokubo H, Rosgen J, Bolen DW, Pettitt BM (2007) Molecular basis of the apparent near ideality of urea solutions. Biophys J 93(10):3392–3407. doi:10.1529/biophysj.107.114181 PubMedCrossRefGoogle Scholar
  57. Kontogeorgis GM, Folas GK (2009) Thermodynamic models for industrial applications: from classical and advanced mixing rules to association theories. Wiley, New York. doi:10.1002/9780470747537.ch15
  58. Kornyshev AA (2007) Double-layer in ionic liquids: paradigm change? J Phys Chem B 111(20):5545–5557PubMedCrossRefGoogle Scholar
  59. Kunz W (2009) Specific ion effects. World Scientific Singapore, SingaporeCrossRefGoogle Scholar
  60. Kyte J (1995) Mechanism in protein chemistry. Garland, New YorkGoogle Scholar
  61. Lee LL (2008) Molecular thermodynamics of electrolyte solutions. World Scientific Singapore, SingaporeGoogle Scholar
  62. Li B (2009) Continuum electrostatics for ionic solutions with non-uniform ionic sizes. Nonlinearity 22(4):811CrossRefGoogle Scholar
  63. Liang J, Dill KA (2001) Are proteins well-packed? Biophys J 81(2):751–766PubMedCrossRefGoogle Scholar
  64. Liang J, Edelsbrunner H, Woodward C (1998) Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design. Protein Sci 7:1884–1897PubMedCrossRefGoogle Scholar
  65. Linderstrom-Lang K (1924) On the ionisation of proteins. Compt Rend Trav Lab Carlsberg (ser chimie) 15(7):1–29Google Scholar
  66. Liu C (2009) An introduction of elastic complex fluids: an energetic variational approach. In: Hou TY, Liu C, Liu JG (eds) Multi-scale phenomena in complex fluids: modeling, analysis and numerical simulations. World Scientific Publishing Company, SingaporeGoogle Scholar
  67. Ludemann SK, Lounnas V, Wade RC (2000a) How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms. J Mol Biol 303(5):797–811. doi:10.1006/jmbi.2000.4154 PubMedCrossRefGoogle Scholar
  68. Ludemann SK, Lounnas V, Wade RC (2000b) How do substrates enter and products exit the buried active site of cytochrome P450cam? 2. Steered molecular dynamics and adiabatic mapping of substrate pathways. J Mol Biol 303(5):813–830. doi:10.1006/jmbi.2000.4155 PubMedCrossRefGoogle Scholar
  69. Markowich PA, Ringhofer CA, Schmeiser C (1990) Semiconductor equations. Springer-Verlag, New YorkCrossRefGoogle Scholar
  70. McCleskey EW (2000) Ion channel selectivity using an electric stew. Biophys J 79(4):1691–1692PubMedCrossRefGoogle Scholar
  71. Miedema H, Meter-Arkema A, Wierenga J, Tang J, Eisenberg B, Nonner W, Hektor H, Gillespie D, Meijberg W (2004) Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca2+ channels. Biophys J 87(5):3137–3147PubMedCrossRefGoogle Scholar
  72. Miedema H, Vrouenraets M, Wierenga J, Gillespie D, Eisenberg B, Meijberg W, Nonner W (2006) Ca2+ selectivity of a chemically modified OmpF with reduced pore volume. Biophys J 91(12):4392–4400. doi:10.1529/biophysj.106.087114 PubMedCrossRefGoogle Scholar
  73. Nonner W, Gillespie D, Henderson D, Eisenberg B (2001) Ion accumulation in a biological calcium channel: effects of solvent and confining pressure. J Phys Chem B 105:6427–6436CrossRefGoogle Scholar
  74. Otyepka M, Skopalik J, Anzenbacherova E, Anzenbacher P (2007a) What common structural features and variations of mammalian P450 s are known to date? Biochim Biophys Acta 1770(3):376–389. doi:10.1016/j.bbagen.2006.09.013 PubMedCrossRefGoogle Scholar
  75. Otyepka M, Skopalík J, Anzenbacherová E, Anzenbacher P (2007b) What common structural features and variations of mammalian P450s are known to date? Biochimica et Biophysica Acta (BBA)—Gen Subj 1770(3):376–389. doi:10.1016/j.bbagen.2006.09.013 CrossRefGoogle Scholar
  76. Patwardhan VS, Kumar A (1993) Thermodynamic properties of aqueous solutions of mixed electrolytes: A new mixing rule. AIChE J 39(4):711–714CrossRefGoogle Scholar
  77. Pierret RF (1996) Semiconductor device fundamentals. Addison Wesley, New YorkGoogle Scholar
  78. Pitzer KS (1995) Thermodynamics, 3rd edn. McGraw Hill, New YorkGoogle Scholar
  79. Pohl HA (1978) Dielectrophoresis: The behavior of neutral matter in nonuniform electric fields. Cambridge University Press, New YorkGoogle Scholar
  80. Porter CT, Bartlett GJ, Thornton JM (2004) The Catalytic Site Atlas: a resource of catalytic sites and residues identified in enzymes using structural data. Nucleic Acids Res 32(suppl 1):D129–D133PubMedCrossRefGoogle Scholar
  81. Pytkowicz RM (1979) Activity coefficients in electrolyte solutions, vol 1. CRC, Boca RatonGoogle Scholar
  82. Saranya N, Selvaraj S (2009) Variation of protein binding cavity volume and ligand volume in protein-ligand complexes. Bioorg Med Chem Lett 19(19):5769–5772. doi:10.1016/j.bmcl.2009.07.140 PubMedCrossRefGoogle Scholar
  83. Sather WA, McCleskey EW (2003) Permeation and selectivity in calcium channels. Annu Rev Physiol 65:133–159PubMedCrossRefGoogle Scholar
  84. Segel IH (1993) Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme, Systems edn. Wiley, Interscience, New YorkGoogle Scholar
  85. Sheng P, Zhang J, Liu C (2008) Onsager principle and electrorheological fluid dynamics. Prog Theoret Phys 175:131–143. doi:10.1143/PTPS.175.131 CrossRefGoogle Scholar
  86. Siegler WC, Crank JA, Armstrong DW, Synovec RE (2010) Increasing selectivity in comprehensive three-dimensional gas chromatography via an ionic liquid stationary phase column in one dimension. J Chromatogr A 1217(18):3144–3149PubMedCrossRefGoogle Scholar
  87. Spohr HV, Patey GN (2010) Structural and dynamical properties of ionic liquids: competing influences of molecular properties. J Chem Phys 132(15):154504–154512. doi:10.1063/1.3380830 PubMedCrossRefGoogle Scholar
  88. Sze SM (1981) Physics of semiconductor devices. Wiley, New YorkGoogle Scholar
  89. Tanford C (1957) Theory of protein titration curves. II. Calculations for simple models at low ionic strength. J Am Chem Soc 79(20):5340–5347. doi:10.1021/ja01577a002 CrossRefGoogle Scholar
  90. Tanford C, Kirkwood JG (1957) Theory of protein titration curves. I. General equations for impenetrable spheres. J Am Chem Soc 79:5333–5339CrossRefGoogle Scholar
  91. Tanford C, Roxby R (1972) Interpretation of protein titration curves. application to lysozyme. Biochemistry 11(11):2192–2198. doi:10.1021/bi00761a029 PubMedCrossRefGoogle Scholar
  92. Tipton KF (1994) Enzyme nomenclature. Recommendations 1992. Eur J Biochem 223(1):1–5. doi:10.1111/j.1432-1033.1994.tb18960.x PubMedCrossRefGoogle Scholar
  93. Torrie GM, Valleau A (1982) Electrical double layers: 4. limitations of the Gouy-Chapman Theory. J Phys Chem 86:3251–3257CrossRefGoogle Scholar
  94. Tosteson D (1989) Membrane transport: people and ideas. American Physiological Society, BethesdaGoogle Scholar
  95. Varma S, Rempe SB (2010) Multibody effects in ion binding and selectivity. Biophys J 99(10):3394–3401. doi:10.1016/j.bpj.2010.09.019 PubMedCrossRefGoogle Scholar
  96. Varma S, Rogers DM, Pratt LR, Rempe SB (2011) Perspectives on: ion selectivity: design principles for K+ selectivity in membrane transport. J Gen Physiol 137(6):479–488. doi:10.1085/jgp.201010579 PubMedCrossRefGoogle Scholar
  97. Vincze J, Valisko M, Boda D (2010) The nonmonotonic concentration dependence of the mean activity coefficient of electrolytes is a result of a balance between solvation and ion–ion correlations. J Chem Phys 133(15):154507–154508. doi:10.1063/1.3489418 PubMedCrossRefGoogle Scholar
  98. Voet D, Voet J (2004) Biochemistry, 3rd edn. Wiley, HobokenGoogle Scholar
  99. Vrouenraets M, Wierenga J, Meijberg W, Miedema H (2006) Chemical modification of the bacterial porin OmpF: gain of selectivity by volume reduction. Biophys J 90(4):1202–1211PubMedCrossRefGoogle Scholar
  100. Warshel A (1981) Calculations of enzymatic reactions: calculations of pKa, proton transfer reactions, and general acid catalysis reactions in enzymes. Biochemistry 20(11):3167–3177PubMedCrossRefGoogle Scholar
  101. Warshel A, Russell ST (1984) Calculations of electrostatic interactions in biological systems and in solutions. Q Rev Biophys 17:283–422PubMedCrossRefGoogle Scholar
  102. Warshel A, Sharma PK, Kato M, Xiang Y, Liu H, Olsson MHM (2006) Electrostatic basis for enzyme catalysis. Chem Rev 106(8):3210–3235. doi:10.1021/cr0503106 PubMedCrossRefGoogle Scholar
  103. Wu XS, Edwards HD, Sather WA (2000) Side chain orientation in the selectivity filter of a voltage-gated Ca2+ channel. J Biol Chem 275:31778–31785PubMedCrossRefGoogle Scholar
  104. Yang J, Ellinor PT, Sather WA, Zhang JF, Tsien R (1993) Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels. Nature 366:158–161PubMedCrossRefGoogle Scholar
  105. Yu H, Noskov SY, Roux B (2009) Hydration number, topological control, and ion selectivity. J Phys Chem. doi:10.1021/jp901233v Google Scholar
  106. Zemaitis JF Jr, Clark DM, Rafal M, Scrivner NC (1986) Handbook of aqueous electrolyte thermodynamics. Design institute for physical property data. American Institute of Chemical Engineers, New YorkCrossRefGoogle Scholar
  107. Zhang C, Raugei S, Eisenberg B, Carloni P (2010) Molecular dynamics in physiological solutions: force fields, alkali metal ions, and ionic strength. J Chem Theory Comput 6(7):2167–2175. doi:10.1021/ct9006579 CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2012

Authors and Affiliations

  • David Jimenez-Morales
    • 1
  • Jie Liang
    • 1
  • Bob Eisenberg
    • 1
    • 2
  1. 1.Department of BioengineeringUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Department of Molecular Biophysics and PhysiologyRush UniversityChicagoUSA

Personalised recommendations