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Equilibrium protection studies of the interaction of bovine glutamate dehydrogenase with purine nucleotide effectors
Author(s) -
Chen Soo-Se,
Engel Paul C.
Publication year - 1975
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(75)80259-6
Subject(s) - library science , citation , chemistry , stereochemistry , philosophy , computer science
Bovine liver glutamate dehydrogenase (GDH) is an allosteric enzyme [l-4]. Although its heterotropic effecters, ADP and GTP, are structurally related to the coenzymes and homotropic effecters, NAD(P)+ and NAD(P)H, there is now abundant evidence that the enzyme possesses separate, regulatory nucleotide sites in addition to the catalytic sites [2,5-lo]. It is still not entirely clear, however, to what extent these sites overlap. In recent studies [ II1.51 of reversible enzyme inactivation by pyridoxal 5’-phospate (PLP) we have developed a technique which we term ‘equilibrium protection’. Even at saturating concentrations, PLP causes only partial inactivation of GDH and other dehydrogenases [12-l 61, because, although in each case the covalently-modified enzyme (Schiff base) is completely inactive, it exists in equilibrium with a readily dissociable, non-covalent enzyme-PLP complex. The residual activity at equilibrium may be measured very precisely with small amounts of enzyme, and perturbation of the equilibrium thus provides a sensitive means of monitoring ligand binding and consequent conformational changes. Success in detecting a conformational change in GDH following the binding of the substrate, 2-0x0grutarate, or the substrate analogue, glutarate [ 1 I], prompted US to try the equilibrium protection method in studying the interactions of ADP and GTP with the enzyme.