Thermodynamics of binding of regulatory ligands to tissue transglutaminase.
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 (K (D) approximately 1 microM), 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 (K (D) approximately 0.15 microM) 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.
Bergamini
Carlo M
CM
Department of Biochemistry and Molecular Biology, University of Ferrara, Via Borsari 46, 44100, Ferrara, Italy. bgc@unife.it
Dondi
Alessia
A
Lanzara
Vincenzo
V
Squerzanti
Monica
M
Cervellati
Carlo
C
Montin
Katy
K
Mischiati
Carlo
C
Tasco
Gianluca
G
Collighan
Russel
R
Griffin
Martin
M
Casadio
Rita
R
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002151:Calorimetry; D019295:Computational Biology; D019204:GTP-Binding Proteins; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013816:Thermodynamics; D011503:Transglutaminases / Q000737:chemistry* / Q000378:metabolism
D008024:Ligands; D006160:Guanosine Triphosphate; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D002118:Calcium