Modular mutagenesis of exons 1, 2, and 8 of a glutathione S-transferase from the mu class. Mechanistic and structural consequences for chimeras of isoenzyme 3-3.
Exons 1 and 2 and exon 8 of the mu class GSH transferases from rat encode sequence-variable regions 1 and 4 of mu class isoenzymes, respectively. These two of four variable regions are located at the N- and C-termini of this isoenzyme class and impinge on the active site. In order to assess the influence of these variable regions on the catalytic diversity of the class mu isoenzymes, seven chimeric isoenzymes were constructed by transplantation of the variable regions of the sequence of the type 4 subunit into the corresponding regions of the type 3 subunit. The chimeric isoenzymes exhibit unique catalytic properties. Replacement of all, or part, of variable region 4 of the type 3 subunit with that of the type 4 subunit results in chimeric catalysts with higher turnover numbers in nucleophilic aromatic substitution reactions. Analysis of the crystal structure of isoenzyme 3-3 [Ji, X., Zhang, P., Armstrong, R. N., & Gilliland, G. L. (1992) Biochemistry (preceding paper in this issue)] suggests that interaction of the flexible C-terminal tail with the N-terminal domain helps limit the rate of product release from the active site of isoenzyme 3-3 in this type of reaction. Substitution of all, or part, of the sequence-variable region 1 of subunit 3 with that of subunit 4 results in chimeric isoenzymes that mimic the high stereoselectivity but not the catalytic efficiency of isoenzyme 4-4 toward alpha,beta-unsaturated ketones.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhang
P
P
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Liu
S
S
Shan
S O
SO
Ji
X
X
Gilliland
G L
GL
Armstrong
R N
RN
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D013816:Thermodynamics
D007527:Isoenzymes; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase