Syncatalytic conformational changes in aspartate aminotransferase determined by hydrogen-deuterium exchange.

Abstract

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 200, 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 simultaneous 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.Conformational changes of enzymes concomitant to the binding of substrate or other specific ligands are amply documented by experiment. The changes are established as the basis for mechanisms of regulation and are also thought to contribute to both the specificity and efficiency of enzymic catalysis (1, 2). The occurrence of conformational adaptations of enzymes during the subsequent phase of catalysis, i.e., during the covalency changes, has been postulated on the basis of thermodynamic arguments (3,4). Syncatalytic adjustment of the conformation of an enzyme might optimize the free energy profile of the bond rearrangement steps. However, experimental evidence for such transient changes in conformation is scarce; their detection might be possible only under especially favorable circumstances.In aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) the occurrence of syncatalytic conformational changes has been indicated by differential chemical modifications. Both in the mitochondrial isoenzyme from chicken and pig (5) and in the cytosolic isoenzyme from pig (6), the reactivity of a nonessential cysteinyl residue toward sulfhydryl reagents is enhanced by one or two orders of magnitude, respectively, when the enzyme undergoes the bond rearrangement steps of transamination. In the present study, the conformational behavior of aspartate aminotransferaseThe costs of publication of this article were defrayed in part by the payment of page ch...

Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase

“…It has been observed in solution studies (Gehring & Christen, 1978; Pfister et al, 1978) as well as by X-ray crystallographic analyses (Kirsch et al, 1984; C. Almo and A. T. Danishefsky, unpublished results) that the enzyme undergoes a conformational change upon binding dicarboxylic acid substrates or inhibitors. This motion can be modeled, based on crystal structures of the open and closed forms, as a rigid-body rotation of the small domain with respect to the large domain, resulting in the closing of the enzyme around the substrate.…”

Catalytic activity of non-cross-linked microcrystals of aspartate aminotransferase in poly(ethylene glycol)

“…How do lattice forces decrease the catalytic efficiency of the enzyme? Lattice forces must either distort the geometry of the active site or impede ligand-induced or syncatalytic conformational adaptations that are integral features of the catalytic mechanism (Gehring & Christen, 1978;Pfister et al, 1978). A detailed kinetic analysis of crystalline AspAT in conjunction with mechanismbased chemical modifications has even indicated that its measured molar activity corresponds to the mean of two functionally non-equivalent active sites of the crystalline enzyme dimer.…”