Site-directed mutation in conserved anionic regions of guinea pig liver transglutaminase.
Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl) lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein-bound glutamine residues. There are conserved anionic regions in transglutaminases, some of which are thought to be possible calcium-binding sites. By site-directed mutagenesis, three mutant forms of recombinant guinea-pig liver transglutaminase, in which some acidic amino acid residues in two conserved regions became nonionic, were expressed in Escherichia coli: TGM1, with Asp-231 and -232 changed to Asn; TGM2, with Glu-445, -448, -449, -450, and -452 changed to Gln; and TGM3, with the mutations of both TGM1 and TGM2. The size and level of synthesis of the mutant proteins were unchanged when monitored by immunoblotting. All mutants retained enzyme activity, and their apparent Km values for substrates during histamine incorporation into acetyl alpha s1-casein were similar to those of the wild-type enzyme, but their Vmax values were smaller. The deamidation rate of glutamine residues in the acetyl alpha s1-casein was unaffected, but the rate of protein cross-linking catalyzed by these mutants was very low. All mutations caused with the enzyme a decrease in the sensitivity to activation by calcium and an increase in the sensitivity to inhibition by GTP. These results indicated that the negative charges of some acidic amino acid residues in the two conserved anionic regions of transglutaminase are not essential for its activity but the loss of their negative charges affects some catalytic properties.
Ikura
K
K
Department of Chemistry and Materials Technology, Faculty of Engineering and Design, Kyoto Institute of Technology, Japan.
Yu
C
C
Nagao
M
M
Sasaki
R
R
Furuyoshi
S
S
Kawabata
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002645:Chickens; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004789:Enzyme Activation; D004926:Escherichia coli; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D011503:Transglutaminases / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D011503:Transglutaminases; D002118:Calcium