Maleylacetone cis-trans-isomerase: affinity chromatography on glutathione-bound sepharose. Two-substrate-binding sequence from inhibition patterns

Abstract

Maleylacetone cis-trans-isomerase isolated from Vibrio 01 binds glutathione strongly; Km = 1.4 .times. 10-4 M. Oxidized glutathione and S-methylglutathione are competitive inhibitors, Ki [inhibition constant] = 9.4 .times. 10-4 and 1.2 .times. 10-3 M, respectively. Based on these interactions, 3 glutathione-bound agarose affinity adsorbents were synthesized and tested. Affinity chromatography of the isomerase with one of these affords 70- to 100-fold purifications. In separate syntheses, portions of the affinity arm were prepared and examined as to their inhibitory properties in the enzyme-catalyzed reaction. The fragment, containing glutathione bound through its S to the C chain, is a powerful competitive inhibitor for glutathione (Ki = 6 .times. 10-5 M). The isomerase binds glutathione through the backbone of the tripeptide, and the thiol group is required for activity. The initial velocity patterns of the enzyme-catalyzed reaction resulting from simultaneous variation of glutathione and maleylacetone concentrations were examined in the absence and presence of inhibitors resembling glutathione. The observed kinetic patterns suggest an ordered sequence of binding: maleylacetone first followed by glutathione.