Substrate-induced inactivation of argininosuccinate lyase by monofluorofumarate and difluorofumarate

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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.degree. C and 10 mM arginine are (monofluorofumarate) 1.4 mM and 5% and (difluorofumarate) 46 .mu.M and 0.5%. As inhibitors, both of these compounds inactivated 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 > 75% of its original activity with half-lives of 6 and 24 min for the monofluorofumarate- and difluorofumarate-inhibited enzyme. The time-dependent inactivation may be 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.