Advertisement

Enzyme Active Sites

  • Sandra J. Smith-Gill

Abstract

Monoclonal antibodies (MAbs) have provided powerful new tools for analysis of enzyme function. MAbs directed at specific subsites of the enzyme active site allow analysis of catalysis at a new level of resolution even for relatively simple, monomeric enzymes. Because many different MAbs can be prepared to a given enzyme protein or polypeptide complex, and each will be exquisitely specific for a given antigenic determinant or epitope, multiple subunits and associated catalytic functions become potentially separable. In addition, because the binding of MAbs to protein antigens is sensitive to even single residue changes in the antigenic determinant, it is possible to discriminate between very closely related but structurally distinct multiple molecular forms of enzymes.1 This discussion reviews several unique studies in which MAbs have been used to examine catalysis in ways not previously approachable by conventional biochemical methods.

Keywords

Influenza Virus Sialic Acid Antigenic Determinant Catalytic Center Antigenic Variant 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Harris, H., 1983, Applications of monoclonal antibodies in enzyme genetics, Annu. Rev. Genet. 17:279–314.PubMedCrossRefGoogle Scholar
  2. 2.
    Benjamin, D. C., Berzofsy, J. A., East, I. J., Gurd, F. R. N., Hannum, C., Leach, S. J., Margoliash, E., Michael, J. C., Miller, A., Prager, E. M., Reichlin, M., Sercarz, E. E., Smith-Gill, S. J., Todd, P. E., and Wilson, A. C., 1984, The antigenic structure of proteins: A reappraisal, Annu. Rev. Immunol. 2:67–101.PubMedCrossRefGoogle Scholar
  3. 3.
    Webster, R. G., Brown, L. E., and Laver, W. G., 1984, Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies, Virology 135:30–42.PubMedCrossRefGoogle Scholar
  4. 4.
    Frackelton, A. R., Jr., and Rotman, B., 1980, Functional diversity of antibodies elicited by bacterial β-D-galactosidase, J. Immunol. 255:5286–5290.Google Scholar
  5. 5.
    Solomon, B., Moav, N., Pines, G., and Katchalski-Katzir, E., 1984, Interaction of carboxypep-tidase A with monoclonal antibodies, Mol. Immunol. 21:1–11.PubMedCrossRefGoogle Scholar
  6. 6.
    Arnon, R., 1977, Immunochemistry of lysozyme, in: Immunochemistry of Enzymes and Their Antibodies (M. R. J. Salton, ed.), Wiley, New York, pp. 1–28.Google Scholar
  7. 7.
    Carroll, S. B., and Stollar, B. D., 1983, Conservation of a DNA-binding site in the largest subunit of eukaryotic RNA polymerase II, J. Mol. Biol. 170:777–790.PubMedCrossRefGoogle Scholar
  8. 8.
    Colman, P. M., Varghese, J. N., and Laver, W. G., 1983, Structure of the catalytic and antigenic sites in influenza virus neuraminidase, Nature 303:41–44.PubMedCrossRefGoogle Scholar
  9. 9.
    Varghese, J. N., Laver, W. G., and Colman, P. M., 1983, Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 A resolution, Nature 303:35–40.PubMedCrossRefGoogle Scholar
  10. 10.
    Laver, W. G., Colman, P. M., Ward, C. W., Varghese, J. M., Air, G. M., Webster, R. G., Hinshaw, V., Brown, L., and Jackson, D., 1984, Influenza virus neuraminidase: Structure and variation, in: The Molecular Virology and Epidemiology of Influenza (C. Stuart-Harris and C. W. Potter, eds.), Academic Press, New York, pp. 77–91.Google Scholar
  11. 11.
    Jackson, D. C., and Webster, R. G., 1982, A topographic map of the enzyme active center and antigenic sites on the neuraminidase of influenza virus A/Tokyo/3/67 (H2N2), Virology 123:69–77. PubMedCrossRefGoogle Scholar
  12. 12.
    Webster, R. G., Hinshaw, V. S., and Laver, W. G., 1982, Selection and analysis of antigenic variants of the neuraminidase of N2 influenza viruses with monoclonal antibodies, Virology 117:93–104.PubMedCrossRefGoogle Scholar
  13. 13.
    Laver, W. G., Air, G. M., Webster, R. G., and Markoff, L. J., 1982, Amino acid sequence changes in antigenic variants of type A influenza virus N2 neuraminidase, Virology 122:450–460.PubMedCrossRefGoogle Scholar
  14. 14.
    Lentz, M. R., Air, G. M., Laver, W. G., and Webster, R. G., 1984, Sequence of the neuraminidase gene of influenza virus A/Tokyo/3/67 and previously uncharacterized monoclonal variants, Virology 135:257–265.PubMedCrossRefGoogle Scholar
  15. 15.
    Lewis, M. K., and Burgess, R. R., 1982, Eukaryotic RNA polymerases, in: The Enzymes, Volume 15 (P. D. Boyer, ed.) Academic Press, New York, pp. 109–153.Google Scholar
  16. 16.
    Rose, K. M., Maguire, K. A., Wurpel, J. N. D., Stetler, D. A., and Marquez, E. D., 1983, Monoclonal antibodies directed against mammalian RNA polymerase I. Identification of the catalytic center, J. Biol. Chem. 258:12976–12981.PubMedGoogle Scholar
  17. 17.
    Gowda, S., and Sridhara, S., 1983, Monoclonal antibody to RNA polymerase I of the silkworm, J. Biol. Chem. 258:14532–14538.PubMedGoogle Scholar
  18. 18.
    Carroll, S. B., and Stollar, B. D., 1982, Inhibitory monoclonal antibody to calf thymus RNA polymerase II blocks formation of enzyme-DNA complexes, Proc. Natl. Acad. Sci. USA 79:7233–7237.PubMedCrossRefGoogle Scholar
  19. 19.
    Dahmus, M. E., and Kedinger, C., 1983, Transcription of adenovirus-2 major late promoter inhibited by monoclonal antibody directed against RNA polymerases II0 and IIA, J. Biol. Chem. 258:2303–2307.PubMedGoogle Scholar
  20. 20.
    Christmann, J. L., and Dahmus, M. E., 1981, Monoclonal antibody specific for calf thymus RNA polymerases IIO and IIA, J. Biol. Chem. 256:11798–11803.PubMedGoogle Scholar
  21. 21.
    Weissbach, A., 1982, Cellular and viral-induced eukaryotic polymerases, in: The Enzymes, Volume 14 (P. D. Boyer, ed.), Academic Press, New York, pp. 67–86.Google Scholar
  22. 22.
    Wang, T. S.-F., Hu, S.-Z., Korn, D., 1984, DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA polymerase-a, J. Biol. Chem. 259:1854–1865.PubMedGoogle Scholar
  23. 23.
    Tanaka, S., Hu, S.-Z., Wang, T. S.-F., and Korn, D., 1982, Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase-a, J. Biol. Chem. 257:8386–8390.PubMedGoogle Scholar
  24. 24.
    Hu, S.-Z., Wang, T. S.-F., and Korn, D., 1984, DNA primase from KB cells. Evidence for a novel model of primase catalysis by a highly purified primase/polymerase-a complex, J. Biol. Chem. 259:2602–2609.PubMedGoogle Scholar
  25. 25.
    Plevani, P., Badaracco, C., Augl, C., and Chang, L. M. S., 1984, DNA polymerase I and DNA primase complex in yeast, J. Biol. Chem. 259:7532–7539.PubMedGoogle Scholar
  26. 26.
    Klein, J., 1982, Immunology. The Science of Self-Nonself Discrimination, Wiley, New York.Google Scholar
  27. 27.
    Golan, M. D., Burger, R., and Loos, M., 1982, Conformational changes in C1q after binding to immune complexes: Detection of neoantigens with monoclonal antibodies, J. Immunol. 129:445–447.PubMedGoogle Scholar
  28. 28.
    Heinz, H.-P., Burger, R., Golan, M. D., and Loos, M., 1984, Activation of the first component of complement, C1, by a monoclonal antibody recognizing the C chain of C1q, J. Immunol. 132:804–808.PubMedGoogle Scholar
  29. 29.
    Celada, F., and Rotman, B., 1984, Monoclonal antibodies in enzymology, in: Handbook of Monoclonal Antibodies. Applications in Biology and Medicine (S. Ferrone and M. P. Dierich, eds.), Noyes Publications, New Jersey (in press).Google Scholar
  30. 30.
    Smith-Gill, S. J., Wilson, A. C., Potter, M., Feldmann, R. J., and Mainhart, C. R., 1982, Mapping the antigenic epitope for a monoclonal antibody against avian lysozyme, J. Immunol. 128:314–322.PubMedGoogle Scholar
  31. 31.
    Smith-Gill, S. J., Lavoie, T. B., and Mainhart, C., 1984, Antigenic regions defined by monoclonal antibodies correspond to structural domains of avian lysozyme, J. Immunol. 133:384–393.PubMedGoogle Scholar
  32. 32.
    Blake, C. C. F., Johnson, L. N., Mair, G. A., North, T. A., Phillips, D. C., and Sarma, V. R., 1967, Crystallographic studies of the activity of hen egg white lysozyme, Proc. R. Soc. Lond. B 167:378–388.PubMedCrossRefGoogle Scholar
  33. 33.
    Phillips, D. C., 1974, Crystallographic studies of lysozyme and its interactions with inhibitors and substrates, in: Lysozyme (E. F. Osserman, R. E. Canfield, and S. Beychok, eds.), Academic Press, New York, pp. 9–30.Google Scholar
  34. 34.
    Rupley, J. A., 1967, The binding and cleavage by lysozyme of N-acetylglucosamine oligosaccharides, Proc. R. Soc. Lond. B 167:416–428.PubMedCrossRefGoogle Scholar
  35. 35.
    Rupley, J. A., and Gates, V., 1967, Studies on the enzymic activity of lysozyme II. The hydrolysis and transfer reactions of N-acetylglucosamine oligosaccharides, Proc. Natl. Acad. Sci. USA 57:496–510.CrossRefGoogle Scholar
  36. 36.
    Pincus, M. R., and Sheraga, H. A., 1979, Conformation energy calculations of enzyme-substrate and enzyme-inhibitor complexes of lysozyme. 2. Calculation of the structures of complexes with a flexible enzyme, Macromolecules 12:633–644.CrossRefGoogle Scholar
  37. 37.
    Smith-Gill, S. J., Rupley, J. A., Pincus, M. R., Carty, R. P., and Sheraga, H. A., 1984, Experimental identification of a theoretically predicted “left-sided” binding mode for (GlcNac)6 in the active site of lysozyme, Biochemistry 23:993–997.PubMedCrossRefGoogle Scholar
  38. 38.
    Holler, E., Rupley, J. A., and Hess, G. P., 1975, Productive and unproductive lysozyme chi-rosaccharide complexes. Equilibrium measurements, Biochemistry 14:1088–1094.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1985

Authors and Affiliations

  • Sandra J. Smith-Gill
    • 1
    • 2
  1. 1.Laboratory of Genetics, National Cancer InstituteNational Institutes of HealthBethesdaUSA
  2. 2.Department of ZoologyUniversity of MarylandCollege ParkUSA

Personalised recommendations