Increase in ADP-ribosyltransferase activity of rat T lymphocyte alloantigen RT6.1 by a single amino acid mutation.
A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various mammalian cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini. Although the first Glu in the common motif is replaced by Gln (Q207EEVLIP) in rat T lymphocyte alloantigens RT6.1 and RT6.2, the two RT6s appear to have both activities of NAD+ glycohydrolase and ADP-ribosyltransferase to a lesser extent. To investigate the significance of the Glu-rich motif in the two enzyme activities, we produced a mutant RT6.1 (Q207E), in which Gln207 was replaced by Glu, together with wild-type RT6s, in Escherichia coli. Kinetic analysis revealed that there were no marked differences in the Vmax and Km values of NAD+ glycohydrolases among the three recombinant proteins. The recombinant RT6.1 and RT6.2 displayed extremely low auto-ADP-ribosylation, although the latter modification was somewhat higher than the former. In contrast, much greater auto-modification was observed for the Q207E mutant. Moreover, the mutant could effectively ADP-ribosylate agmatine as a substrate. Thus, the single amino acid mutation of RT6.1 caused a marked increase in its ADP-ribosyltransferase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the enzyme activity.
Maehama
T
T
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Hoshino
S
S
Katada
T
T
D036002:ADP Ribose Transferases; D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte; D001483:Base Sequence; D017931:DNA Primers; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006649:Histocompatibility Antigens / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D017354:Point Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology*
D000945:Antigens, Differentiation, T-Lymphocyte; D017931:DNA Primers; D006649:Histocompatibility Antigens; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C090163:Art2b protein, rat