Substrate-Induced Inactivation of Argininosuccinate Lyase by Monofluorofumarate and Difluorofumarate

Lisa J. Garrard, J. Michael Mathis, Frank M. Raushel

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Monofluorofumarate and difluorofumarate were tested as alternate substrates and inhibitors of the reverse reaction of bovine liver argininosuccinate lyase. Km and Vmax values relative to fumarate at pH 7.5, 25 °C., and 10 mM arginine are (monofluorofumarate) 1.4 mM and 5% and (difluorofumarate) 46 µM and 0.5%. As inhibitors, both of these compounds were shown to inactivate the enzyme activity in a pseudo-first-order process that is dependent on the presence of arginine. The rate of inactivation at saturating monofluorofumarate and difluorofumarate is 13 and 1.3 min−1, respectively. After removal of excess inhibitor, the inactivated enzyme can be restored to greater than 75% of its original activity with half-lives of 6 and 24 min for the monofluorofumarate- and difluorofumarate-inhibited enzyme. Evidence is presented to suggest that the time-dependent inactivation is caused by covalent addition of an enzyme nucleophile with an electrophilic reaction intermediate. In the inhibition by monofluorofumarate, the postulated intermediate is proposed to occur by the spontaneous loss of HF from 2-fluoroargininosuccinate.

Original languageEnglish
Pages (from-to)3729-3735
Number of pages7
JournalBiochemistry
Volume22
Issue number16
DOIs
StatePublished - 1 Jan 1983

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Argininosuccinate Lyase
Substrates
Arginine
Enzymes
Reaction intermediates
Fumarates
Nucleophiles
Enzyme activity
Enzyme Inhibitors
Liver
2-fluorofumarate

Cite this

@article{ac08a6d368754813b306b04730b2ab28,
title = "Substrate-Induced Inactivation of Argininosuccinate Lyase by Monofluorofumarate and Difluorofumarate",
abstract = "Monofluorofumarate and difluorofumarate were tested as alternate substrates and inhibitors of the reverse reaction of bovine liver argininosuccinate lyase. Km and Vmax values relative to fumarate at pH 7.5, 25 °C., and 10 mM arginine are (monofluorofumarate) 1.4 mM and 5{\%} and (difluorofumarate) 46 µM and 0.5{\%}. As inhibitors, both of these compounds were shown to inactivate the enzyme activity in a pseudo-first-order process that is dependent on the presence of arginine. The rate of inactivation at saturating monofluorofumarate and difluorofumarate is 13 and 1.3 min−1, respectively. After removal of excess inhibitor, the inactivated enzyme can be restored to greater than 75{\%} of its original activity with half-lives of 6 and 24 min for the monofluorofumarate- and difluorofumarate-inhibited enzyme. Evidence is presented to suggest that the time-dependent inactivation is caused by covalent addition of an enzyme nucleophile with an electrophilic reaction intermediate. In the inhibition by monofluorofumarate, the postulated intermediate is proposed to occur by the spontaneous loss of HF from 2-fluoroargininosuccinate.",
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Substrate-Induced Inactivation of Argininosuccinate Lyase by Monofluorofumarate and Difluorofumarate. / Garrard, Lisa J.; Mathis, J. Michael; Raushel, Frank M.

In: Biochemistry, Vol. 22, No. 16, 01.01.1983, p. 3729-3735.

Research output: Contribution to journalArticle

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AB - Monofluorofumarate and difluorofumarate were tested as alternate substrates and inhibitors of the reverse reaction of bovine liver argininosuccinate lyase. Km and Vmax values relative to fumarate at pH 7.5, 25 °C., and 10 mM arginine are (monofluorofumarate) 1.4 mM and 5% and (difluorofumarate) 46 µM and 0.5%. As inhibitors, both of these compounds were shown to inactivate the enzyme activity in a pseudo-first-order process that is dependent on the presence of arginine. The rate of inactivation at saturating monofluorofumarate and difluorofumarate is 13 and 1.3 min−1, respectively. After removal of excess inhibitor, the inactivated enzyme can be restored to greater than 75% of its original activity with half-lives of 6 and 24 min for the monofluorofumarate- and difluorofumarate-inhibited enzyme. Evidence is presented to suggest that the time-dependent inactivation is caused by covalent addition of an enzyme nucleophile with an electrophilic reaction intermediate. In the inhibition by monofluorofumarate, the postulated intermediate is proposed to occur by the spontaneous loss of HF from 2-fluoroargininosuccinate.

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