Residue 234 in glutathione transferase T1-1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates

Abstract

GST (glutathione transferase) T1-1 plays an important role in the biotransformation of halogenated alkanes, which are used in large quantities as solvents and occur as environmental pollutants. Many reactions that are catalysed by GST T1-1 qualify as detoxification processes, but some reactions with dihalogenated alkanes lead to reactive products more toxic than the substrates. Murine GST T1-1 is particularly active with dichloromethane, which may explain the high carcinogenicity of dichloromethane in the mouse. Human GST T1-1 activity is considerably lower with halogenated hydrocarbons and some related substrates. Human GST T1-1 is polymorphic with a frequent null phenotype, suggesting that it is advantageous, under some circumstances, to lack the functional enzyme, which catalyses GSH conjugations that may cause bioactivation. The present study shows that amino acid residue 234 is a determinant of the differences in catalytic efficiency between the human and the rodent enzymes. The replacement of Trp234 in human GST T1-1 by arginine, found in the rodent enzyme, enhanced the alkyltransferase activity by an order of magnitude with a series of homologous iodoalkanes and some typical GST substrates. The specific activity of the alternative mutant Trp234-->Lys was lower than for the parental human GST T1-1 with many substrates, showing that a positive charge is not sufficient for increased activity. The enhanced activity of Trp234-->Arg with alkylating agents was dependent on the substrate tested, whereas no increase of the peroxidase activity with cumene hydroperoxide was noted. Residue 234 therefore is also involved in the control of the substrate selectivity of GST T1-1.

Minor Modifications of the C-terminal Helix Reschedule the Favored Chemical Reactions Catalyzed by Theta Class Glutathione Transferase T1-1

“…The epoxide EPNP, the alkylhalide iodomethane, and NPB have often been regarded as signature substrates of GST T1-1 (16,21). Indeed, the R234W activities with these substrates were still the highest in absolute numbers but just Ͻ3% of the wild type values.…”

“…In position 234, the human enzyme has Trp, whereas the mouse enzyme has Arg. We have previously shown that mutation of Trp into Arg in the human enzyme induces significant elevation of catalytic activity with a number of substrates (16). To test if the reverse mutation, Arg to Trp, in a homologous enzyme is sufficient to suppress catalytic activities, this substitution was introduced in mouse GST T1-1.…”

“…Alternative substrates included the phenethyl isothiocyanate (phenethyl-ITC), benzyl isothiocyanate (benzyl-ITC), allyl isothiocyanate (allyl-ITC), propyl isothiocyanate (propyl-ITC) (17), hydrogen peroxide (H 2 O 2 ), tertiary-butyl hydroperoxide (tert-BuOOH), and cumene hydroperoxide (CuOOH) (18) as well as different monoiodoalkanes and diiodoalkanes (16). The assay conditions are summarized in supplemental Table S1.…”

“…Assay of Enzymatic Activities of Variants of GST T1-1-The activities of purified wild type mouse GST T1-1 and four variants (M232A, M232A/R234W, R234W, and ⌬R234W) were measured at 30°C as published previously using the signature substrates 1,2-epoxy-3-(4-nitrophenoxy)-propane (EPNP), 4-nitrophenethyl bromide (NPB) (15), and iodomethane (16). Alternative substrates included the phenethyl isothiocyanate (phenethyl-ITC), benzyl isothiocyanate (benzyl-ITC), allyl isothiocyanate (allyl-ITC), propyl isothiocyanate (propyl-ITC) (17), hydrogen peroxide (H 2 O 2 ), tertiary-butyl hydroperoxide (tert-BuOOH), and cumene hydroperoxide (CuOOH) (18) as well as different monoiodoalkanes and diiodoalkanes (16).…”

Evolution of highly active enzymes by homology-independent recombination

“…3C). SCR23 also contained two point mutations, L113P and W234R, the latter of which is known to play a role in substrate selectivity (28). It is noteworthy that SCR23 contains a lowhomology C͞O near the middle of the electrophile-determining H site, indicating that exchange of parental C-terminal subdomains can give rise to enzymes with high catalytic activity.…”