Study of subunit interactions in immobilized D-glyceraldehyde-3-phosphate dehydrogenase

Abstract

Under conditions which cause dissociation of soluble tetrameric glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) into inactive dimers, immobilized apoenzymes from yeast and rat skeletal muscle coupled to CnBr-activated Sepharose via one subunit retain 50% of matrix-bound protein with unaltered specific activity. The solubilized dissociated species are inactive. Two molecules of NAD+ (NADH) firmly bound to the immobilized rat muscle tetramer can prevent the dissociation. Immobilized dimer was demonstrated to bind one molecule of coenzyme with high affinity. Using various combinations of immobilized and soluble rat muscle and yeast dimers, we succeeded in reconstituting tetramers, containing one molecule of NAD+ bound either to a matrix-linked or to a non-covalently bound dimer. In the latter case, the dissociation of the tetramer was completely prevented. This suggests that the binding of a single coenzyme molecule is sufficient to stabilize the interdimeric contacts provided the neighbouring dimer is stabilized independently. Such stabilization is produced by the covalent binding of one of the subunits comprising the dimer to the matrix. The structure of the dimer as a whole becomes resistant to the action of the dissociating agent. The effect appears to be cooperative and similar to that of NAD+ or NADH. The dissociation of the immobilized tetramer is, most likely, the result of conformational changes, affecting the structure of the non-covalently bound dimer. Any factor, capable of preventing these changes, would stabilize the interdimeric contacts. The latter conclusion is substantiated by the effect of specific antibodies, which prevent the dissociation of the immobilized tetramer by forming a complex with the dimer, non-covalently bound to the matrix. The evidence obtained in the present investigation supports the conclusion that the isolated dimer of glyceraldehyde-3-phosphate dehydrogenase represents a relatively independent structural and functional 'unit' of the enzyme. It can be stabilized in a catalytically active form by interactions other than those involved in inter-dimeric contacts in the tetramer. The kinetics of the association of immobilized and soluble dimers have been studied. Association rate constants were determined for homologous (yeast-yeast, rat-rat) and heterologous (yeast-rat, yeast-rabbit) dimer combinations. The binding of one molecule of specific antibody to the immobilized dimer was shown to increase the rate constant of association.