Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

Abstract

Human glutathione transferase (GST ) has been crystallized as a homodimer, with a subunit molecular mass of ϳ23 kDa; however, in solution the average molecular mass depends on protein concentration, approaching that of monomer at <0.03 mg/ml, concentrations typically used to measure catalytic activity of the enzyme. Electrostatic interaction at the subunit interface greatly influences the dimer-monomer equilibrium of the enzyme and is an important force for holding subunits together. Arg-70, Arg-74, Asp-90, Asp-94, and Thr-67 were selected as target sites for mutagenesis, because they are at the subunit interface. R70Q, R74Q, D90N, D94N, and T67A mutant enzymes were constructed, expressed in Escherichia coli, and purified. The construct of N-terminal His tag enzyme facilitates the purification of GST , resulting in a high yield of enzyme, but does not alter the kinetic parameters or secondary structure of the enzyme. Our results indicate that these mutant enzymes show no appreciable changes in K m for 1-chloro-2,4-dinitrobenzene and have similar CD spectra to that of wild-type enzyme. However, elimination of the charges of either Arg-70, Arg-74, Asp-90, or Asp-94 shifts the dimer-monomer equilibrium toward monomer. In addition, replacement of Asp-94 or Arg-70 causes a large increase in the K m GSH , whereas substitution for Asp-90 or Arg-74 primarily results in a marked decrease in V max . The GST retains substantial catalytic activity as a monomer probably because the glutathione and electrophilic substrate sites (such as for 1-chloro-2,4-dinitrobenzene) are predominantly located within each subunit.

Glutathione and Glutathione‐S‐Transferase in Detoxification Mechanisms

“…While cytosolic GSTs are crystallized as a homodimer or heterodimer, at least some of them, if not all, exist as an equilibrium mixture of monomers and dimers in solution (Hearne and Colman, 2006;Huang et al, 2008;Vargo et al, 2004). The fact that the monomeric form of several human GST enzymes possesses at least partial activity as compared with the dimer (Hearne and Colman, 2006;Huang et al, 2008;Vargo et al, 2004) demonstrates that each subunit of a dimeric enzyme represents an independent catalytic unit. In other words, all the catalytic reactions between the common substrate GSH and various hydrophobic substrates take place at the subunit level.…”

“…In other words, all the catalytic reactions between the common substrate GSH and various hydrophobic substrates take place at the subunit level. Nonetheless, dimerization is thought to stabilize the tertiary structure and the catalytic site of each subunit (Erhardt and Dirr, 1995;Huang et al, 2008) and to increase the efficiency of the enzyme (Hegazy et al, 2004).…”

GSTpi modulates JNK activity through a direct interaction with JNK substrate, ATF2

“…6 GSTpi exists both as a monomer of 23.5 kDa (at low protein concentrations) and as a dimer (at higher protein concentrations); each monomer contains one active site. [6][7][8][9] Human GSTpi is polymorphic, with differences in residues 105 and 114. Four haplotypes involving variation between these two residues have been determined and characterized, as follows: GSTpi haplotype A (WT), with Ile105 and Ala114; haplotype B, with Val-105 and Ala-114; haplotype C, with Val-105 and Val-114; and haplotype D, with Ile-105 and Val-114.…”

Understanding HIV-1 Protease Autoprocessing for Novel Therapeutic Development

“…GST is an example of the fusion tags. GST is a well-characterized homodimer with the dissociation constants for dimeric GST ranging from the low micromolar to the low nanomolar, depending on assay conditions [89,90]. Owing to the tight dimer association and slow exchange kinetics [91], the GST moiety is expected to facilitate dimerization of the fusion precursor and subsequent autoprocessing.…”