Subunit Interface Residues of Glutathione S-Transferase A1-1 that Are Important in the Monomer-Dimer Equilibrium

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• 作者：Melissa A. Vargo ; Lucia Nguyen ; and Roberta F. Colman
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：March 30, 2004
• 年：2004
• 卷：43
• 期：12
• 页码：3327 - 3335
• 全文大小：536K
• 年卷期：v.43,no.12(March 30, 2004)
• ISSN：1520-4995

文摘

Alpha class glutathione S-transferase, isozyme A1-1, is a dimer (51 kDa) of identical subunits.Using the crystal structure, two main areas of subunit interaction were chosen for study: (1) the hydrophobicball and socket comprised of Phe52 from one subunit fitting into a socket formed on the other subunit byMet94, Phe136, and Val139 and (2) the Arg/Glu region consisting of Arg69 and Glu97 from both subunits.We introduced substitutions of these residues, by site-directed mutagenesis, to evaluate the importance ofeach at the subunit interface and to determine if monomeric enzymes could be generated using singlemutations. Mutating each residue of the socket region to alanine results in little change in the kineticparameters, and all are dimeric enzymes. In contrast, when Phe52, the ball residue, is replaced with alanine,the enzyme has very low activity and a weight average molecular mass of 31.9 kDa, as determined bysedimentation equilibrium experiments. Substitutions for Glu97 which eliminate the charge cause noappreciable changes in the kinetic parameters or molecular mass. Eliminating the charge on Arg69 (as inR69Q) results in a dimeric enzyme; however, when the charge is reversed (as in R69E), the weight averagemolecular mass is greatly shifted toward that of the monomer (33 kDa) and the changes in kinetic parametersare reasonably small. We determined the molecular masses in the presence of glutathione for F52A andR69E to ascertain whether the monomeric species retains activity. For R69E, it appears that the monomeris active, albeit less so than the dimer, while for F52A, the monomer and dimer both appear to exhibitvery low activity. The dimeric species is needed to obtain high specific activity. We conclude that, of theresidues that were studied, Phe52 and Arg69 are the major determinants of dimer formation and a singlemutation at either position substantially hinders dimerization. The use of a mutant glutathione S-transferasewhich retains activity yet has a greatly weakened tendency to dimerize (such as R69E) may be advantageousfor certain applications of GST fusion proteins.