Glutamic acid-65 is an essential residue for catalysis in Proteus mirabilis glutathione S-transferase B1-1

The Biochemical Journal
Nerino AllocatiCarmine Di Ilio

Abstract

The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu(65), Ser(103) and Glu(104) are in hydrogen-bonding distance of the N-terminal amino group of the gamma-glutamyl moiety of the co-substrate, GSH. Glu(65) was mutated to either aspartic acid or leucine, and Ser(103) and Glu(104) were both mutated to alanine. Glu(65) mutants (Glu(65)-->Asp and Glu(65)-->Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser(103)-->Ala and Glu(104)-->Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser(103) and Glu(104) are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu(65) is crucial for catalysis.

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Jan 1, 1988·CRC Critical Reviews in Biochemistry·B Mannervik, U H Danielson
Mar 16, 1994·Biochimica Et Biophysica Acta·M C Wilce, M W Parker
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Apr 28, 2000·The Journal of Biological Chemistry·P G BoardJ Pandit

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Citations

Jul 4, 2012·Proceedings of the National Academy of Sciences of the United States of America·Paul K FyfeWilliam N Hunter
Oct 10, 2013·Applied Biochemistry and Biotechnology·Evangelia ChronopoulouNikolaos E Labrou
Nov 20, 2008·The FEBS Journal·Nerino AllocatiCarmine Di Ilio

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