Site-directed mutagenesis of human tissue transglutaminase: Cys-277 is essential for transglutaminase activity but not for GTPase activity

Abstract

Transglutaminases (EC 2.3.2.13) catalyze an acyl-transfer reaction between peptide-bound glutamine residues and primary amines, including the ϵ-amino group of lysine residues in protein. Purified human erythrocyte transglutaminase was found to have another activity, i.e., GTP hydrolysis. Treatment of the enzyme with iodoacetamide, a cysteine-directed reagent, caused a 94% loss of TGase activity within 8 min, but no significant loss of GTPase activity. Cys-277, a known residue which is selectively modified by iodoacetamide, was replaced with Ser by site-directed mutagenesis to assess the role of the Cys-277 in the transglutaminase/GTPase activities. Wild-type cDNA, coding for human endothelial cell transglutaminase, and its C277S-mutated cDNA were cloned into a plasmid vector that contained a promotor from phage T7, and then expressed in Escherichia coli. The wild-type recombinant enzyme was indistinguishable from human erythrocyte transglutaminase in mobility on a SDS-polyacrylamide gel, immunoreactivity and catalytic activities for transglutaminase and GTPase. However, the recombinant enzyme was not blocked at the N-terminal alanine residue, as is the case in the naturally occurring erythrocyte enzyme. The C277S mutant enzyme showed no transglutaminase activity, but had K m and k cat values for GTPase activity that were comparable to those of wild-type recombinant and natural erythrocyte enzymes. These results demonstrate that Cys-277 is essential for transglutaminase activity but not for GTPase activity, and that N-terminal blocking of tissue-type transglutaminase is not critical for either transglutaminase or GTPase activities.