Syncatalytic conformational changes in aspartate aminotransferase determined by hydrogen-deuterium exchange.
Catalysis-linked conformational transitions of aspartate aminotransferase (cytosolic isoenzyme from pig heart; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been probed by infrared spectrophotometric measurement of hydrogen-deuterium exchange. In the unliganded pyridoxal form of the enzyme at pH 6.0 and 20 degrees, 43% of the total 411 peptide hydrogens per subunit exchange within the first 10 min. An additional 9% exchange slowly in the following time period to 360 min. A quite similar exchange curve is obtained with the pyridoxamine form of the enzyme, indicating close correspondence in conformation of both unliganded forms of the enzyme. Formation of a nonproductive adsorption complex of the pyridoxal enzyme with 2-oxoglutarate or of the pyridoxamine enzyme with glutamate alters the exchange characteristics only slightly. In contrast, the formation of an equilibrium mixture of the covalent transamination intermediates, which occurs in the silultaneous presence of the amino acid and the keto acid substrate, results in a marked retardation of hydrogen exchange, reflecting a substantial tightening of the structure of the enzyme. The exchange reactions of at least 26 peptide hydrogens per subunit (6% of the total) are retarded by a factor of 6 on the average. The occurrence of such syncatalytic conformational changes reflects energetic coupling of the covalency changes at the active site with conformational changes of the macromolecular protein matrix that may contribute to optimizing the free energy profile of enzymic transamination.
D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D002384:Catalysis; D003903:Deuterium; D006859:Hydrogen; D007700:Kinetics; D008024:Ligands; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D011732:Pyridoxal Phosphate; D013552:Swine
D008024:Ligands; D011732:Pyridoxal Phosphate; D006859:Hydrogen; D003903:Deuterium; D001219:Aspartate Aminotransferases
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