Site-directed mutation in conserved anionic regions of guinea pig liver transglutaminase.

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Ikura K, Yu C, Nagao M, Sasaki R, Furuyoshi S, Kawabata N

Site-directed mutation in conserved anionic regions of guinea pig liver transglutaminase.

Arch Biochem Biophys. 1995 Apr 20;318(2):307-13.

PubMed ID

7733658 [ View in PubMed

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Abstract

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.

DrugBank Data that Cites this Article

Drug Enzymes

Drug	Enzyme	Kind	Organism	Pharmacological Action	Actions
L-Glutamine	Protein-glutamine gamma-glutamyltransferase E	Protein	Humans	Unknown	Substrate	Details