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Purification and Characterization of a Heterodimeric 23/20-kDa Proteolytic Fragment of Bacterial Glutathione Transferase B1-1

https://doi.org/10.1006/abbi.1996.0177Get rights and content

Abstract

The proteolytic attack of bacterial glutathioneS-transferase (GSTB1-1) by trypsin cleaves and inactivates the enzyme. The polypeptide portion of GSTB1-1 encompassing the cleavage site (Lys35–Lys36) constitutes an exposed and flexible region of the GSTB1-1 G-site. By sequentially using a benzamidine-affinity chromatography and GSH-affinity column, a proteolyzed form of GSTB1-1 (23/20 kDa), in which only one subunit has been cleaved, has been purified and characterized. Gel filtration, sequence analysis of subunits separated by HPLC, and CD experiments indicate that the 23/20-kDa GSTB1-1 form is a dimer and maintains its secondary structure. In addition, kinetic determinations reveal that the proteolytic cleavage of one polypeptide chain inactivates one active site but does not influence the catalytic efficiency of the second one. Previous refolding studies on GSTB1-1 have shown that the formation of a correct dimer precedes the recovery of the full activity of the enzyme, indicating that the dimeric structure is essential for catalytic activity of GSTB1-1. Thus, although GSTB1-1 active sites are catalytically independent and, probably, mainly located on each monomer, interactions deriving from the dimeric arrangement of the molecule appear essential for maintaining each active site in a fully active conformation. The catalytic independence of the two active sites, as well as the importance of dimeric structure for catalytic activity, has already been established for other GSTs. Thus, despite the very low sequence identity and kinetic differences between bacterial and other distant members of the GST superfamily, the results reported here indicate that important properties of the GST active site are conserved.

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    In Proteus mirabilis, three forms of GSTs had been explored [13]. P. mirabilis GST B1-1, the first cGST of beta class, was extensively studied relating to its structure and function [6,14–22]. But little information about the character and function of other forms of GSTs from P. mirabilis was reported.

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