Design of two chimaeric human-rat class alpha glutathione transferases for probing the contribution of C-terminal segments of protein structure to the catalytic properties.
Two chimaeric human-rat class Alpha glutathione transferases were constructed by fusion of DNA segments derived from the plasmids pTGT2-AT and pGTB38 and expression of the corresponding proteins in Escherichia coli. The recombinant proteins H1R1/1 and H1R1/2 encoded by plasmids pH1R1/1 and pH1R1/2 are composed of a segment of the human class Alpha subunit 1 from the N-terminus to His-143 and Pro-207 respectively, followed by the complementary C-terminal portion of the rat class Alpha subunit 1 sequence. Compared with the parental human enzyme, H1R1/1 is altered in 20 positions due to the introduction of 79 residues from the rat enzyme, while H1R1/2 is altered in five positions out of 15 in the C-terminal region. The design of mutant H1R1/1 is equivalent to introduction of exons 6 and 7 of the rat subunit 1 gene in place of the homologous human nucleotide sequence. The two chimaeric proteins are enzymatically active with several substrates, even though the activity in most cases is somewhat decreased in comparison with the wild-type human enzyme. Inhibition studies show that the kinetic properties mimic those of the human enzyme, indicating that the N-terminal two-thirds of the primary structure plays the major role in governing the catalytic properties. The results of this study demonstrate that recombination of segments of primary structure between homologous enzymes may serve as a useful cassette technique for design of novel catalytically active proteins.
Björnestedt
R
R
Department of Biochemistry, Uppsala University, Sweden.
Widersten
M
M
Board
P G
PG
Mannervik
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D013448:Sulfobromophthalein / Q000737:chemistry; D014220:Trialkyltin Compounds / Q000737:chemistry
D011993:Recombinant Fusion Proteins; D014220:Trialkyltin Compounds; D013448:Sulfobromophthalein; C011559:tributyltin; D005982:Glutathione Transferase