Affiliation:
1. John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, ACT 2601, Australia
2. Centre for Bioinformation Science, JCSMR and Mathematical Sciences Institute, Australian National University, P.O. Box 334, Canberra City, ACT 2601, Australia
Abstract
The Kappa class of GSTs (glutathione transferases) comprises soluble enzymes originally isolated from the mitochondrial matrix of rats. We have characterized a Kappa class cDNA from human breast. The cDNA is derived from a single gene comprising eight exons and seven introns located on chromosome 7q34–35. Recombinant hGSTK1-1 was expressed in Escherichia coli as a homodimer (subunit molecular mass ~25.5 kDa). Significant glutathione-conjugating activity was found only with the model substrate CDNB (1-chloro-2,4-ditnitrobenzene). Hyperbolic kinetics were obtained for GSH (parameters: Kmapp, 3.3±0.95 mM; Vmaxapp, 21.4±1.8 µmol/min per mg of enzyme), while sigmoidal kinetics were obtained for CDNB (parameters: S0.5app, 1.5±1.0 mM; Vmaxapp, 40.3±0.3 µmol/min per mg of enzyme; Hill coefficient, 1.3), reflecting low affinities for both substrates. Sequence analyses, homology modelling and secondary structure predictions show that hGSTK1 has (a) most similarity to bacterial HCCA (2-hydroxychromene-2-carboxylate) isomerases and (b) a predicted C-terminal domain structure that is almost identical to that of bacterial disulphide-bond-forming DsbA oxidoreductase (root mean square deviation 0.5–0.6 Å). The structures of hGSTK1 and HCCA isomerase are predicted to possess a thioredoxin fold with a polyhelical domain (αx) embedded between the β-strands (βαβαxββα, where the underlined elements represent the N and C motifs of the thioredoxin fold), as occurs in the bacterial disulphide-bond-forming oxidoreductases. This is in contrast with the cytosolic GSTs, where the helical domain occurs exclusively at the C-terminus (βαβαββααx). Although hGSTK1-1 catalyses some typical GST reactions, we propose that it is structurally distinct from other classes of cytosolic GSTs. The present study suggests that the Kappa class may have arisen in prokaryotes well before the divergence of the cytosolic GSTs.
Subject
Cell Biology,Molecular Biology,Biochemistry
Cited by
87 articles.
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