Site-directed mutagenesis of human tissue transglutaminase: Cys-277 is essential for transglutaminase activity but not for GTPase activity.
Transglutaminases (EC 2.3.2.13) catalyze an acyl-transfer reaction between peptide-bound glutamine residues and primary amines, including the epsilon-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 promoter 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 Km and kcat 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.
Lee
K N
KN
Samuel Roberts Noble Foundation, Inc., Biomedical Division, Ardmore, OK.
Arnold
S A
SA
Birckbichler
P J
PJ
Patterson
M K
MK
Fraij
B M
BM
Takeuchi
Y
Y
Carter
H A
HA
D001665:Binding Sites; D003545:Cysteine; D004229:Dithiothreitol; D004912:Erythrocytes / Q000201:enzymology*; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics; D012694:Serine; D011503:Transglutaminases / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D012694:Serine; D011503:Transglutaminases; D020558:GTP Phosphohydrolases; D003545:Cysteine; D004229:Dithiothreitol; D007460:Iodoacetamide