Ligand-induced asymmetry between active sites of cytoplasmic malate dehydrogenase: a chemical modification study

Abstract

The iodoacetate-dependent and iodoacetamide-dependent inhibition of cytoplasmic malate dehydrogenase (s-MDH) has been examined. We have confirmed previous reports that iodoacetate inhibits this dimeric enzyme by modifying a single active site methionine per s-MDH subunit. Time courses for the inactivation of the solution-state enzyme with both reagents indicate each s-MDH subunit is modified with equal rapidity in the absence of substrate or cofactor. However, the subunits react with distinctly different rates in the presence of cofactor or cofactor/substrate combinations, indicating some conformational asymmetry between subunits occurs when these ligands are bound. This is consistent with solution-state s-MDH behaving as a cooperative enzyme. Apo and holo crystalline s-MDH are also inhibited by iodoacetic acid. However, subunits of the crystalline enzyme are inhibited with different rates in the presence or absence of active site ligands. This suggests subunit conformations of the dimeric enzyme are not identical in crystalline s-MDH preparations regardless of ligand binding. Furthermore, by the criterion of inhibition rate constants, subunit conformations of the crystalline enzyme are not rigid but are perturbed by ligand binding. Comparisons of inactivation time courses for solution- and crystalline-state s-MDH suggest crystalline s-MDH exhibits at least some of the subunit asymmetry associated with the solution-state enzyme.

Energy Substrate Requirements of Rat Retinal Pigmented Epithelial Cells in Culture: Relative Importance of Glucose, Amino Acids, and Monocarboxylates

“…CCB, for example, is known to act as an inhibitor of microtubule formation as well as a glucose transport inhibitor. 25 It is also pertinent to note that IOA can inhibit malate dehydrogenase, 46 which would consequently inhibit oxidative reactions fueled by glutamine. In the case of IOA, any remaining MTTreducing activity would therefore have to be generated by nonoxidative and nonglycolytic pathways.…”

Cooperativity in the mechanism of malate dehydrogenase

“…Detailed kinetic studies were also performed on sMDH preparations that were partially inhibited by iodoacetate, a reagent which binds covalently to the enzyme's active site (Zimmerle et al, 1987). Representative results are shown in Figure 2.…”

“…Finally, previous studies from this laboratory have shown that under standard assay conditions, all active sites of the sMDH preparation are identical by the criterion of reactivity toward the active-site-specific reagent, iodoacetic acid. This reagent specifically modifies Met 98 of the enzyme's active site in a reaction whose rate is sensitive to changes in the active-site environment (Zimmerle & Alter, 1983;Zimmerle et al, 1987).…”

“…Partially inactivated sMDH was prepared by reacting purified sMDH (1 mg/mL) with 250 mM iodoacetic acid in 50 mM HEPES buffer, pH 8.0, 37 "C, and quenching with 0.3 M dithiotheitol buffered in 50 mM PIPES, pH 6.0 (Zimmerle et al, 1987), after various incubation times. These enzyme preparations were exhaustively dialyzed versus the appropriate buffer prior to kinetic or equilibrium-binding analyses.…”

Mechanism of complexing of chlorin e 6 and tetra(p-carboxyphenyl)porphyrin with malate dehydrogenase

“…It should also be noted that, in the absence of ligands, the subunits forming the MDG molecule are chemically identical and are arranged symmetrically [22,23]. However, the bonding of a ligand to one of the subunits of the enzyme can cause an asymmetry in the arrangement of the functional groups belonging to the neighboring subunits [33] and influence the access to the unoccupied sites of bonding.…”