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Limited proteolysis of glutathione transferase P1-1 (GSTP1-1) by chymotrypsin performed at 20°C and 30°C mainly generates two complementary peptides of 17 kDa and 6 kDa molecular mass with concomitant loss of catalytic capacity. Sequence analysis of these peptides showed that the peptide bond between Tyr47 and Gly48 was cleaved. The analysis of the recently resolved three-dimensional structure of GSTP1-1 [Reinemer, P., Dirr. H. W., Ladenstein, R., Huber, R., Lo Bello. M., Federici, G. & Parker, M. W. (1992) J. Mol. Biol. 227, 214–2261 suggests that the proteolytically cleaved bond results located in a portion of the polypeptide chain lining the G-site which has been demonstrated to be part of an exposed and flexible region of the N-terminal domain (structural elements αB′ and αB2 [Aceto. A., Caccuri, A. M., Sacchetta, P., Bucciarelli, T., Dragani, B., Rosato, N., Fedcrici, G. & Di Ilio, C. (1992) Biochem. J. 285, 241–245]. The fragments which are generated by proteolysis at 20°C, remain linked by noncovalent interaction in a complex (nicked GSTP1-1) which is dissociated by incubation at higher temperatures. As shown by circular dichroic analysis, although inactive, nicked GSTP1-1 retains an overall secondary structure closely resembling that of the parent enzyme. However, the fluorescence data of the nicked GSTP1-1 indicate that the Trp38, which is near the chymotrypsin-cleavable bond, becomes exposed in a more polar environment. This indicates that, in the nicked enzyme, the polypeptide portion containing the structural elements αB′ and αB2 has more freedom of fluctuation. The fact that this polypeptide chain portion contains two essential amino acid residues of the G-site (Trp38 and Lys42) might account for the loss of ability to bind glutathione by the nicked enzyme which is consequently catalytically inactive. Proteolysis performed at 30°C generated a homodimeric 17-kDa fragment. The structural analysis of this fragment suggests that the GSTP1-1 αC helix, which is located in the domain I and is thought to be involved in the inter-domain interaction, could exert a critical role in maintaining the native folding of domain II. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of glutathione transferase from Proteus mirabilis was determined. The sequence was reconstructed by analysis of peptides obtained after cleavage by trypsin, Glu-C and Asp-N endoproteinases. The enzyme subunit of 203 amino acid residues corresponding to a molecular mass of 22856 Da. Comparison of this sequence with other known primary structures of the corresponding enzyme from different sources shows a low level of identity (17–26%) with only seven conserved residues in all the sequences considered. This novel glutathione transferase could represent the prototype of a new class, possibly including other bacterial enzymes. [ABSTRACT FROM AUTHOR]