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Inactivation of succinate dehydrogenase by 3-nitropropionate.

C. J. Coles,D. Edmondson,T. Singer

Published 1979 in Journal of Biological Chemistry

ABSTRACT

This paper examines proposals in the literature that 3-nitropropionate is a suicide inactivator of succinate dehydrogenase and that it acts by nucleophilic addition to N-5 of the covalently bound flavin component of the enzyme. With purified, soluble preparations of the enzyme, the inhibition developed slowly, and nearly complete inactivation occurred with a stoichiometric amount of 3-nitropropionate dianion. These facts and the very slow oxidation of 3-nitropropionate by the enzyme (about 0.1% of the rate of succinate oxidation) seem to fit the usual criteria for an active site-directed suicide inhibitor. In accord with this, 3-nitroacrylate, the expected product of dehydrogenation by the enzyme, inactivates it extremely rapidly and irreversibly. Inactivation of the enzyme by 3-nitropropionate is accompanied by changes in the absorption spectrum of the enzyme and a loss of its slight flavin-type fluorescence. The spectral changes are not those expected from alkylation of N-5 of the flavin but are similar to the perturbations caused by binding of oxalacetate at the substrate site. Moreover, on denaturation of the enzyme or proteolytic digestion, even in anaerobic conditions, the absorption and fluorescence spectra of the oxidized flavin are observed. This is contrary to the behavior expected of an N-5 alkylated flavin. Several lines of evidence suggest that the oxidation product, 3nitroacrylic acid, reacts with an essential -SH group at the substrate site. Thus, prior treatment with 3nitropropionate prevents the binding of 14C!-labeled oxalacetate at the substrate site, and, conversely, prior binding of oxalacetate to the enzyme prevented the irreversible inactivation by a 2-fold excess of 3-nitropropionate. Inactivation of the enzyme by 3-nitropropionate also prevented the alkylation of one -SH group by N-ethyl[‘4C]maleimide. The -SH group in question is located in the 70,000-dalton subunit and is known from prior studies to be the combining site of succinate and of oxalacetate. It is suggested that the inactivation step involves a nucleophilic attack by this essential -SH group on the double bond of 3-nitroacrylate.

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