Abstract
The location of an enzyme’s active site is usually known prior to the determination of its three-dimensional structure. Analysis of sequence homology may indicate residues that are near an enzyme’s active site. Catalytic residues are often identified when site-directed mutagenesis experiments or chemical modification of residues lead to an alteration in enzyme activity. The enzyme’s three-dimensional (3D) structure is then used to supplement and interpret preceding experimental data. Occasionally, a new protein structure is solved before the active site has been located through conventional methods. Using the extracellular endonuclease from Serratia marcescens as an example, we show here how the active site of an enzyme can sometimes be determined from electrostatic analysis of its 3D structure (1).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Miller, M., Tanner, J., Alpaugh, M., Benedik, M., and Krause, K. (1994) 2.1 Å structure of Serratia endonuclease suggests a mechanism for binding to double-stranded DNA. Natural Struct. Biol. 1, 461–468.
Benedik, M. J. and Strych, U. (1998) Serratia marcescens and its extracellular nuclease. FEMS Microbiol. Lett. 165, 1–13.
Eisenstein, B. (1995) Enterobacteriaceae, in Principles and Practice of Infectious Diseases 4th ed. (Mandell, G. L., Bennett, J. E., and Dolin, R., eds.), Churchill Livingstone, New York, pp. 1964–1980.
Friedhoff, P., Kolmes, B., Gimadutdinow, O., Wende, W., Krause, K., and Pingoud, A. (1996) Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis. Nucleic Acids Res. 24, 2632–2639.
Filimonova, M. N., Balaban, N. P., Sharipova, F. P., and Leshchinskaia, I. B. (1980) Isolation and physico-chemical properties of homogenous nuclease from Serratia marcescens. Biokhimiia 45, 2096–2104.
Benzon (1993) Product Specifications for Benzonase, the first industrial endonuclease. Benzon Pharma A/S, Helseholmen 1, P.O. Box 1185, DK-2650, Hvidovre, Denmark.
Feynman, R. P., Leighton, R. B., and Sands, M. (1963) The Feynman Lectures on Physics. Addison-Wesley, Reading, MA.
Lipscomb, W. N. (1982) Acceleration of reactions by enzymes. Accts. Chem. Res. 15, 232–238.
Purcell, E. M. (1965) Electricity and Magnetism. McGraw Hill, New York.
Honig, B. and Nicholls, A. (1995) Classical electrostatics in biology and chemistry. Science 268, 1144–1149.
Nicholls, A., Sharp, K. A., and Honig, B. (1991) Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296.
Madura, J. D., Briggs, J. M., Wade, R. C., Davis, M. E., Luty, B. A., Ilin, A., Antosiewicz, J., Gilson, M. K., Bagheri, B., Scott, L. R., and McCammon, J. A. (1995) Electrostatics and diffusion of molecules in solution: simulations with the University of Houston Brownian Dynamics program. Comput. Phys. Commun. 91, 57–95.
Davis, M. E., Madura, J. D., Luty, B. A., and McCammon, J. A. (1991) Electrostatics and diffusion of molecules in solution: simulations with the University of Houston Brownian Dynamics program. Comput. Phys. Commun. 62, 187–197.
Davis, M. E. and McCammon, J. A. (1990) Electrostatics in biomolecular structure and dynamics. Chem. Rev. 90, 509–521.
Gilson, M. K. (1995) Theory of electrostatic interactions in macromolecules. Curr. Opin. Struct. Biol. 5, 216–223.
Warshel, A. and Papazyan, A. (1998) Electrostatic effects in macromolecules: fundamental concepts and practical modeling. Curr. Opin. Struct. Biol. 8, 211–217.
Miller, M. D., Cai, J., and Krause, K. L. (1999) The active site of Serratia endonuclease contains a conserved magnesium-water cluster. J. Mol. Biol. 288, 975–987.
Jones, T. A. (1978) A graphics model building and refinement system for macromolecules. J. Appl. Crystallogr. 11, 268–272.
Friedhoff, P., Gimadutdinow, O., and Pingoud, A. (1994) Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis. Nucleic Acids Res. 22, 3280–3287.
Abola, E. E., Bernstein, F. C., Bryant, S. H., Koetzle, T. F., and Weng, J. (1987) Protein Data Bank, in Crystallographic Databases-Information Content, Software Systems, Scientific Applications (Allen, F. H., Bergerhoff, G., and Sievers, R., eds.), Data Commission of the International Union of Crystallography, Bonn/Cambridge/Chester, pp. 107–132.
Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977) The protein data bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535–542.
Koradi, R., Billeter, M., and Wüthrich, K. (1996) MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graphics 14, 51–55.
Nicholls, A. and Honig, B. (1991) A rapid finite difference algorithm, utilizing successive over-relaxation to solve the Poisson-Bolzmann equation. J. Comp. Chem. 12, 435–445.
Jones, T. A., Zou, J.-Y., and Cowan, S. W. (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallographica A47, 110–119.
Nayal, M., Hitz, B., and Honig, B. (1999) GRASS Browser: Graphical Representation and Analysis of Structure Server. Columbia University, New York. http://trantor.bioc. columbia.edu/GRASS/surfserv_enter.cgi.
MDL (1999) MDL Worldwide Services: Chime Free Support. MDL Information Systems, San Leandro, CA. http://www.mdli.com/support/chime/chimefree.htm.
Hartman, J. and Wernecke, J. (1996) The VRML 2.0 Handbook: Building Moving Worlds on the Web. Addison-Wesley, Reading, MA.
SGI (1999) SGI-Cosmo Products References. Silicon Graphics, Inc., Mountain View, CA. http://www.sgi.com/software/cosmo/redirect.html.
Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., and Karplus, M. (1983) CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–217.
Jorgensen, W. L. and Tirado-Rives, J. (1988) The OPLS potential function for proteins. Energy minimizations for crystals of cyclic peptides and crambin. J. Am. Chem. Soc. 110, 1657–1666.
Weiner, S. J., Kollman, P. A., Nguyen, D. T., and Case, D. A. (1986) An all atom force field for simulations of proteins and nucleic acids. J. Comput. Chem. 7, 230.
Sitkoff, D., Sharp, K. A., and Honig, B. (1994) Accurate calculation of hydration free energies using macroscopic solvent models. J. Phys. Chem. 98, 1978–1988.
Miller, M. and Krause, K. (1996) Identification of the Serratia endonuclease dimer: structural basis and implications for catalysis. Protein Sci. 5, 24–33.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Krause, K.L., Miller, M.D. (2001). Using Electrostatics to Define the Active Site of Serratia Endonuclease. In: Schein, C.H. (eds) Nuclease Methods and Protocols. Methods in Molecular Biology™, vol 160. Humana Press. https://doi.org/10.1385/1-59259-233-3:249
Download citation
DOI: https://doi.org/10.1385/1-59259-233-3:249
Publisher Name: Humana Press
Print ISBN: 978-0-89603-679-6
Online ISBN: 978-1-59259-233-3
eBook Packages: Springer Protocols