Ligand-induced asymmetry between active sites of cytoplasmic malate dehydrogenase: a chemical modification study.
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.
Department of Biological Chemistry and Biomedical Sciences, Wright State University, Dayton, Ohio 45435.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine
D000596:Amino Acids; D007461:Iodoacetates; D013439:Sulfhydryl Reagents; D008291:Malate Dehydrogenase; D004229:Dithiothreitol; D019807:Iodoacetic Acid; D007460:Iodoacetamide