Interaction of a Self-Associating Protein with Its Cofactor

Abstract

Solutions of the β2 component of tryptophan synthetase which are paucidisperse in the ultracentrifuge can be rendered monodisperse by affixing the cofactor, pyridoxal 5′-phosphate (pyridoxal-P), to the enzyme by chemical reduction with sodium borohydride. Unlike the apoenzyme, the distribution of the reduced form in the centrifuge is unaffected by Tris even above pH 9. Investigation of the effect of pyridoxal 5′-phosphate on the isopotential apparent specific volume of the β2 protein (φ′) showed that the cofactor is altering the molecular weight distribution of the association. Furthermore, study of the φ′ parameter under conditions favoring subunit dissociation shows that a volume change does not accompany the self-association process. Molecular weight determinations in the presence of high concentrations of mercaptoethanol lead to a reduction in the Z average molecular weight. Comparison of holoenzyme and apoenzyme preparations shows that the apoenzyme contains substantially more of a heavy component. These results together with the finding that both pyridoxal-P removal and denaturation lead to the unmasking of a second and third reactive sulfhydryl suggest the presence of a higher molecular weight aggregate resulting from intermolecular disulfide formation. Previous results may therefore be interpreted in terms of a simple monomer-dimer equilibrium which is more in keeping with the proposed a2β2 form of the native complex.