TY - JOUR
T1 - Structural and functional aspects of rat microsomal glutathione transferase
T2 - The roles of cysteine 49, arginine 107, lysine 67, histidine, and tyrosine residues
AU - Weinander, Rolf
AU - Ekström, Lena
AU - Andersson, Claes
AU - Raza, Haider
AU - Bergman, Tomas
AU - Morgenstern, Ralf
PY - 1997/4/4
Y1 - 1997/4/4
N2 - Rat liver microsomal glutathione transferase is rapidly inactivated upon treatment with the arginine-selective reagent phenylglyoxal or the lysine- selective 1,3,5-trinitrobenzenesulfonate. Glutathione sulfonate, an inhibitor of the enzyme, gives nearly complete protection against inactivation and prevents modification, indicating that these residues form part of or reside close to the active site. Sequence analysis of peptides from peptic and tryptic digests of [7-14C]phenylglyoxal- and 1,3,5- trinitrobenzenesulfonate-treated microsomal glutathione transferase indicated arginine 107 and lysine 67 as the sites of modification. A set of mutant forms of microsomal glutathione transferase was constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli BL21(DE3). Arginine 107 was exchanged for alanine and lysine residues. The alanine mutant (R107A) exhibited an activity and inhibition profile similar to that of the wild type enzyme but displayed a decreased thermostability. Thus, arginine 107 does not appear to participate in catalysis or substrate binding; instead, an important structural role is suggested for this residue. Lysine 67 was mutated to alanine and arginine with no effect on activity. All three histidines were replaced by glutamine, and the resulting mutant proteins had activities comparable with that of the wild type. It can thus be concluded that the chemical modification experiments indicating that arginine 107, lysine 67, and one of the histidines partake in catalysis can be disproved. However, protection from modification by a competitive inhibitor indicates that these residues could be close to the glutathione binding site. All tyrosine to phenylalanine substitutions resulted in mutants with activities similar to that of the wild type. Interestingly, the exchange of tyrosine 137 appears to result in activation of the enzyme. Thus, the microsomal glutathione transferase must display an alternate stabilization of the thiolate anion of glutathione other than through interaction with the phenolic hydroxyl group of a tyrosine residue. Substitution of cysteine 49 with alanine resulted in a semiactivated mutant enzyme with enzymatic properties partly resembling the activated form of microsomal glutathione transferase. The function of this mutant was not altered upon reaction with N-ethylmaleimide, and cysteine 40 is thus demonstrated as the site of modification that results in activation of microsomal glutathione transferase.
AB - Rat liver microsomal glutathione transferase is rapidly inactivated upon treatment with the arginine-selective reagent phenylglyoxal or the lysine- selective 1,3,5-trinitrobenzenesulfonate. Glutathione sulfonate, an inhibitor of the enzyme, gives nearly complete protection against inactivation and prevents modification, indicating that these residues form part of or reside close to the active site. Sequence analysis of peptides from peptic and tryptic digests of [7-14C]phenylglyoxal- and 1,3,5- trinitrobenzenesulfonate-treated microsomal glutathione transferase indicated arginine 107 and lysine 67 as the sites of modification. A set of mutant forms of microsomal glutathione transferase was constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli BL21(DE3). Arginine 107 was exchanged for alanine and lysine residues. The alanine mutant (R107A) exhibited an activity and inhibition profile similar to that of the wild type enzyme but displayed a decreased thermostability. Thus, arginine 107 does not appear to participate in catalysis or substrate binding; instead, an important structural role is suggested for this residue. Lysine 67 was mutated to alanine and arginine with no effect on activity. All three histidines were replaced by glutamine, and the resulting mutant proteins had activities comparable with that of the wild type. It can thus be concluded that the chemical modification experiments indicating that arginine 107, lysine 67, and one of the histidines partake in catalysis can be disproved. However, protection from modification by a competitive inhibitor indicates that these residues could be close to the glutathione binding site. All tyrosine to phenylalanine substitutions resulted in mutants with activities similar to that of the wild type. Interestingly, the exchange of tyrosine 137 appears to result in activation of the enzyme. Thus, the microsomal glutathione transferase must display an alternate stabilization of the thiolate anion of glutathione other than through interaction with the phenolic hydroxyl group of a tyrosine residue. Substitution of cysteine 49 with alanine resulted in a semiactivated mutant enzyme with enzymatic properties partly resembling the activated form of microsomal glutathione transferase. The function of this mutant was not altered upon reaction with N-ethylmaleimide, and cysteine 40 is thus demonstrated as the site of modification that results in activation of microsomal glutathione transferase.
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U2 - 10.1074/jbc.272.14.8871
DO - 10.1074/jbc.272.14.8871
M3 - Article
C2 - 9083005
AN - SCOPUS:0030911870
SN - 0021-9258
VL - 272
SP - 8871
EP - 8877
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -