Rational reconstruction of the active site of a class mu glutathione S-transferase.
Isoenzymes 3-3 and 4-4 of the mu class glutathione S-transferases share 77% sequence identity but have distinctly different catalytic properties. Analysis of the crystal structure of isoenzyme 3-3 in complex with the diastereomeric products of the addition of GSH to phenanthrene 9,10-oxide (Ji, X., Johnson, W. W., Sesay, M. A., Dickert, L., Prasad, S. M., Ammon, H. L., Armstrong, R. N., and Gilliland, G. L. (1994) Biochemistry 33, 1043-1052) reveals that 3 residues that are in van der Waals contact with the xenobiotic portion of the product are different in the type 4 subunit. The three mutations, V9I, I111A, and S209A, have been introduced into isoenzyme 3-3 individually and in combination in an attempt to minimally reconstruct the active site of the enzyme to mimic the type 4 subunit in structure and function. The results suggest that the V9I mutation is an important determinant in the stereoselectivity of the enzyme toward enones and epoxides. The I111A mutation increases the catalytic efficiency of the enzyme toward para-substituted 4-phenyl-3-buten-2-ones (XPBO) as measured by kcat/KmXPBO but does not affect kcat. The S209A mutation has no effect on catalysis. The double and triple mutants V9I/I111A and V9I/I111A/S209A exhibit both a high stereoselectivity and high kcat/KmXPBO comparable to that of isoenzyme 4-4. Analysis of substituent effects on the kinetics and stereoselectivity of the enzyme toward the enone substrates suggests that the mechanistic bases for the catalytic behavior of the isoenzyme 4-4 and the reconstructed mutants are not identical. The results provide functional evidence for the catalytic importance of specific residues previously identified by x-ray crystallography.
Shan
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Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Armstrong
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D000818:Animals; D001665:Binding Sites; D002074:Butanones / Q000378:metabolism; D004852:Epoxy Compounds / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D010616:Phenanthrenes / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D002074:Butanones; D004852:Epoxy Compounds; D007527:Isoenzymes; D010616:Phenanthrenes; C046665:9,10-epoxy-9,10-dihydrophenanthrene; C025383:benzylideneacetone; D005982:Glutathione Transferase