Conformational changes in aspartate aminotransferase. Effect of active site ligands on peptide hydrogen-deuterium exchange.
The conformational responses of aspartate aminotransferase (cytosolic isoenzyme from pig) to the binding of the coenzyme and competitive inhibitors and to the bond rearrangement steps during the transamination reaction were probed by the method of peptide hydrogen deuterium exchange. Binding of the coenzyme to the apoenzyme results in a marked retardation of hydrogen exchange; binding of the competitive inhibitor maleate to the pyridoxal enzyme induces a retardation of exchange somewhat exceeding that observed in the presence of the transaminating substrate pair glutamate and 2-oxoglutarate (Pfister, K., Kägi, J.H.R., and Christen, P. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 145-148). On formation of the complex of apoenzyme with N-(5'-phosphopyridoxyl)-L-glutamate or-L-aspartate, analogs of the covalent coenzyme substrate intermediates, a similar exchange retardation occurs. The extent of the exchange retardation in these different functional states of the enzyme correlates with previous results of differential chemical and proteolytic modifications. Apparently, the diverse methods register shifts in one and the same conformational equilibrium. Moreover, the conditions under which peptide hydrogen exchange indicates a pronounced tightening of the protein matrix correspond with those inducing crystallization of the enzyme in the "closed" form. Thus, the transition between the "open" and "closed" form of the enzyme, i.e. the bulk movement of the small domain, as observed and defined by x-ray crystallography (Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525) is the major structural correlate of the conformational changes undergone by the enzyme in solution.
D000818:Animals; D001219:Aspartate Aminotransferases; D001665:Binding Sites; D003903:Deuterium; D006859:Hydrogen; D008024:Ligands; D008298:Maleates; D010455:Peptides; D011487:Protein Conformation; D013552:Swine; D014961:X-Ray Diffraction
D008024:Ligands; D008298:Maleates; D010455:Peptides; D006859:Hydrogen; C030272:maleic acid; D003903:Deuterium; D001219:Aspartate Aminotransferases