ISSN:
1573-4943
Schlagwort(e):
crystals of aspartate aminotransferase
;
equilibrium kinetics in enzyme crystals
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Chemie und Pharmazie
Notizen:
Abstract Orthorhombic single crystals of cytoplasmic aspartate aminotransferase were examined alone or in the presence of substrates or inhibitors to quantitatively compare the interaction of ligands with the active-site chromophore between soluble and crystalline enzyme. As in enzyme solutions, equilibrium kinetic measurements can be made between substrates and single crystals of cytoplasmic aspartate aminotransferase. The absorption spectra of ligand-free enzyme forms and of enzyme-substrate or-inhibitor complexes are as distinctive as when the enzyme is in solution. The dissociation constants for glutamate with the pyridoxal form of the enzyme are identical to those in solution. The substrate analog erythro-β-hydroxyaspartate also binds with equal affinity to the active site in enzyme crystals as in solution; and the affinity of α-ketoglutarate to bind in nonproductive complexes with the pyridoxal form of the enzyme is also unimpaired in the crystal (K d =2 mM). In contrast to the affinity constants, the stoichiometry of the interactions does not appear to correlate to those in solution. In the presence of an amino acid plus keto acid substrates pair, the absorbance values of the enzyme-substrate complex(es) could be interpreted as for occupany of only half the available sites in the crystals. Yet an amino acid, cysteine sulfinate, and α-keto acids such as β, β-difluorooxalacetate convert all active sites in the crystal to the pyridoxamine or pyridoxal form when added to the pyridoxal or pyridoxamine forms, respectively. This ability to completely undergo substrate-induced half-transamination and the apparently conflicting results in trapping half the sites in enzyme-substrate complexes are incorporated into a proposed reciprocating mechanism applicable only to the crystalline state of the enzyme and dictated by crystal packing forces rather than an intrinsic property of the enzyme. Active-site bound pyridoxal phosphate continues to behave as a pH indicator; nevertheless, the pK value of the single crystals is a pH unit (pK=7.15) higher than that in solution. This variation is interpreted as indication of a difference in the environment of the chromophore between the crystal and solution states. While the environmental difference does not significantly alter the affinity for substrates, it could account for the reduced rates in transformation of the enzyme-substrate complexes in half-transamination reactions in the crystalline state.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/BF01024997
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