Crystallization-induced modification of cytoplasmic malate dehydrogenase structure and function.
In an effort to assess the effects of phase-state changes on protein conformations, we have compared several properties of cytoplasmic malate dehydrogenase in the crystalline and solution states. Two crystalline forms of the enzyme have been examined: one crystallized in the presence of nicotinamide adenine dinucleotide and the other in its absence. Though both forms catalyze cytoplasmic malate dehydrogenase's normal substrate conversions, they have specific activities 150-3000-fold less than the solution-state enzyme. These dramatic activity decreases cannot be accounted for by diffusion constraints imposed by the crystal lattice nor do they result from the manipulations necessary to crystallize or cross-link the enzyme. Further, crystal- and solution-state enzymes have different pH dependences of their enzymatic activities, have different sensitivities toward inactivation by the covalent inhibitor iodoacetate, and respond differently to nicotinamide adenine dinucleotide protection against this inactivation. Finally, crystals of the enzyme grown in the presence and absence of cofactor are also distinguishable from one another by using the same criteria. Taken together, these results suggest that crystallization perturbs the dynamics and, perhaps, the average conformation of cytoplasmic malate dehydrogenase.
D000818:Animals; D003432:Cross-Linking Reagents; D003460:Crystallization; D003593:Cytoplasm / Q000201:enzymology; D004058:Diffusion; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D012996:Solutions; D013329:Structure-Activity Relationship; D013552:Swine
D003432:Cross-Linking Reagents; D007461:Iodoacetates; D012996:Solutions; D008291:Malate Dehydrogenase; D019807:Iodoacetic Acid