Studies on the activity and activation of rat liver microsomal glutathione transferase, in particular with a substrate analogue series.

Morgenstern, Ralf; Lundqvist, Gerd; Hancock, Victorine; DePierre, Joseph W.

doi:10.1016/s0021-9258(18)68694-6

Cited by 61 publications

(24 citation statements)

References 17 publications

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“…The Hammett plot data obtained here are very different from those obtained previously at 30 °C . At the higher temperature, k cat values were limited by GSH thiolate formation or chemical reactivity in a logical fashion, allowing only activation of the more reactive substrates.…”

Section: Resultscontrasting

confidence: 99%

“…In addition, we have derived the steady-state rate equation and used it to calculate predicted K M , k cat , and k cat / K M values, which we in turn have compared to the experimentally derived constants for all substrate combinations. Studying MGST1, we take advantage of being able to control the rate of the chemical catalytic step by using a series of substrates with defined reactivity (Figure ). We can also modulate the rate of the GSH thiolate anion formation step by pretreating the enzyme with a sulfhydryl reagent leading to activation …”

Section: Resultsmentioning

confidence: 99%

“…In principle, the microscopic rate and equilibrium constants obtained for MGST1 should also fit the steady-state behavior of the enzyme. However, for practical reasons (the activated MGST1 is unstable without GSH at 30 °C), the pre-steady-state kinetic characterization was performed at 5 °C, whereas earlier published steady-state analysis of the same protein was performed at 30 °C. , Thus, direct comparison between pre-steady-state and steady-state data has not been possible, and a global mechanism could not be tested. To fill this gap, we have performed a steady-state analysis of MGST1 at 5 °C to determine whether the microscopic rate constants obtained for MGST1 accurately predict the steady-state kinetic parameters.…”

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confidence: 99%

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Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation

et al. 2017

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Microsomal glutathione transferase 1 (MGST1) has a unique ability to be activated, up to 30-fold, by modification with sulfhydryl reagents. MGST1 exhibits one-third-of-the-sites-reactivity towards glutathione and hence heterogeneous binding to different active sites in the homo-trimer. Limited turnover stopped flow kinetic measurements of the activated enzyme allowed us to more accurately determine KD for the “third” low affinity GSH-binding site (1.4 ± 0.3 mM). The rate of thiolate formation, k2 (0.77 ± 0.06 s−1), relevant to turnover, could also be determined. By deriving the steady-state rate equation for a random sequential mechanism for MGST1 we can predict KM, kcat and kcat/KM values from these and previously determined pre steady-state rate constants (all determined at 5°C). To assess whether the pre steady-state behavior can account for the steady-state kinetic behavior we have determined experimental values for kinetic parameters at 5°C. For reactive substrates and activated enzyme, data for the microscopic steps account for the global mechanism of MGST1. For unactivated enzyme and more reactive electrophilic substrates, pre steady-state and steady-state data can be reconciled only if a more active subpopulation of MGST1 is assumed. We suggest that unactivated MGST1 can be partially activated in its unmodified form. The existence of an activated subpopulation (approximately 10%) could be demonstrated in limited turnover experiments. We therefore suggest that MSGT1 displays a pre-existing dynamic equilibrium between high and low activity forms.

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Section: Resultscontrasting

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation

et al. 2017

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“…A correlation has been reported previously for the GST4-4 and MGST1 catalyzed reactions of the 4-substituted CDNB analogs. 22,25,26 Clearly, GST catalyzed reactions can be inuenced a Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako-Shi, Saitama 351-0198, Japan. E-mail: y-ito@riken.jp; Fax: +81-48-462-9300; Tel: +81-48-467-4979 b Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo-Shi, Hokkaido 060-0812, Japan.…”

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confidence: 99%

Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions

Shibata

Nakano

Ito

et al. 2013

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We have synthesized a series of 4-substituted-2-nitrobenzene-sulfonyl compounds for caged fluorogenic probes and conducted a Hammett plot analysis using the steady-state kinetic parameters. The results revealed that the glutathione transferase (GST) alpha catalyzed reaction was dependent on the σ value in the same way as the non-enzymatic reaction, whereas the dependence of the σ value of the GST mu and pi was not as pronounced as that of GST alpha.

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“…Furthermore, we expanded the prodrug concept toward etoposide. Tailored modification might thus produce prodrugs that can be used to target specific types of cancers depending on their GST levels and composition as well as those displaying drug resistance. , Designing different prodrug-types for different patient populations could contribute to a more personalized cancer therapy approach, were the therapeutic window is increased. Notably, TLK-286, a previously developed prodrug that targets GSTP1 overexpressing cancer cells went through to Phase III clinical trials showing the potential of this strategy. , …”

Section: Discussionmentioning

confidence: 99%

Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells

et al. 2016

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Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS–etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS–DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS–DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS–DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS–DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS–DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance mechanisms in cells. Here we propose that GST-dependent prodrugs require a conversion rate “window” in order to selectively target GST overexpressing cells, while limiting their effects on normal cells. Prodrugs are furthermore a suitable system to specifically target GSTs and to overcome various drug resistance mechanisms that apply to the parental drug.

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Studies on the activity and activation of rat liver microsomal glutathione transferase, in particular with a substrate analogue series.

Cited by 61 publications

References 17 publications

Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation

Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation

Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions

Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells

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