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J Biol Chem, Vol. 275, Issue 6, 3915-3921, February 11, 2000

Opposite Effects of Ca²⁺ and GTP Binding on Tissue Transglutaminase Tertiary Structure^*

Almerinda Di Venere^§, Antonello Rossi, Fabio De Matteis^¶, Nicola Rosato^§, Alessandro Finazzi Agrò, and Giampiero Mei^§**

From the  Department of Experimental Medicine and Biochemical Sciences and ^§ INFM, ^¶ Department of Physics and INFM, University of Rome "Tor Vergata," Rome 00133, and  AFaR-CRCCS Divisione di Neurologia, Ospedale San Giovanni Calibita Fatebenefratelli, Rome 00100, Italy

Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides. The protein activity is known to be finely tuned by Ca²⁺ and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements. We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure. In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS). Unlike Ca²⁺, the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called "molten globule" state. The effect of GTP binding (but not that of Ca²⁺) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least 0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP. These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.

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