Transglutaminases are a family of Ca2+-dependent protein transamidating and cross-linking enzymes involved in variety of biological processes. The Transglutaminase 2 (TG2) is an unique member of the transglutaminase family with several enzymatic, non-enzymatic activities and interacting partners and has been implicated in multiple disease states. In this study, novel amino acid clusters in human TG2 were identified and computational predictions revealed that these peptide sequences contribute to increasing stability of human TG2 and could potentially regulate vital functions. Based on the information from exome databases, TG2 non-synonymous single nucleotide variants were rare and under selective evolutionary constraint compared to other members of transglutaminase family. The damaging non-synonymous single nucleotide variants destabilize the protein structure and can influence vital functions. The transamidase and isopeptidase activities of TG2 were successfully separated by site-directed mutagenesis. Moreover, TG2 transamidase activity was shown to be involved in the formation of covalently cross-linked protein polymers and the potential role of isopeptidase activity in reversing the protein crosslinks was also demonstrated. Finally, a kinetic real-time protein based method to monitor the isopeptidase activity of TG2 was successfully developed.