W0158

Roles of Calcium Ions in the Activation and Activity of the Transglutaminase 3 Enzyme. Karen M. Boeshans^1,2, Peter M. Steinert¹, Bijan Ahvazi^1,2, ¹Laboratory of Skin Biology, and ²Laboratory of X-ray Crystallography/Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892-8023, U.S.A.

The transglutaminase 3 enzyme is widely expressed in many tissues including epithelia. We have shown previously that it can bind three Ca²⁺ ions. That in site one is constitutively bound, while those in sites two and three are acquired during activation and are required for activity. In particular, binding at site three opens a channel through the enzyme and exposes two tryptophan residues near the active site that are thought to be important for enzyme reaction. In this study, we have solved the structures of three more forms of this enzyme by X-ray crystallography in the presence of Ca²⁺ and/or Mg²⁺which provide new insights on the precise contribution of each Ca²⁺ ion to activation and activity. First, we found that Ca²⁺ ion in site one can be exchanged with difficulty and it has a binding affinity of K_d= 0.3 μM (-6.70 ± 0.52 kcal/mol), which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca²⁺ of the Group 2 family of alkali earth metals, and its occupancy is required for activity. Site three can be occupied by some lanthanides, Ca²⁺ or Mg²; however, when Mg²⁺ is present, the enzyme is inactive, and one side of the channel is closed. Thus Ca²⁺ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site.