Amino Acids

, Volume 39, Issue 1, pp 297–304

Thermodynamics of binding of regulatory ligands to tissue transglutaminase

  • Carlo M. Bergamini
  • Alessia Dondi
  • Vincenzo Lanzara
  • Monica Squerzanti
  • Carlo Cervellati
  • Katy Montin
  • Carlo Mischiati
  • Gianluca Tasco
  • Russel Collighan
  • Martin Griffin
  • Rita Casadio
Original Article

DOI: 10.1007/s00726-009-0442-5

Cite this article as:
Bergamini, C.M., Dondi, A., Lanzara, V. et al. Amino Acids (2010) 39: 297. doi:10.1007/s00726-009-0442-5

Abstract

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (KD ≈ 1 μM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of “apparent binding sites” in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (KD ≈ 0.15 μM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.

Keywords

TransglutaminaseBinding of ligandsCalciumGTPIsothermal titration calorimetry

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Carlo M. Bergamini
    • 1
    • 2
  • Alessia Dondi
    • 1
  • Vincenzo Lanzara
    • 1
  • Monica Squerzanti
    • 1
  • Carlo Cervellati
    • 1
  • Katy Montin
    • 1
  • Carlo Mischiati
    • 1
  • Gianluca Tasco
    • 3
  • Russel Collighan
    • 4
  • Martin Griffin
    • 4
  • Rita Casadio
    • 3
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of FerraraFerraraItaly
  2. 2.Interdisciplinary Centre for the Study of Inflammation (ICSI)University of FerraraFerraraItaly
  3. 3.Department of Biology, Biocomputing CentreUniversity of BolognaBolognaItaly
  4. 4.School of Life and Health SciencesAston UniversityBirminghamUK