Stabilization of subunit enzyme by intersubunit crosslinking with bifunctional reagents: studies with glyceraldehyde-3-phosphate dehydrogenase

Abstract

Enzyme subunits, the first step of the conformation deactivation of which is the reversible dissociation into catalytically inactive subunits, can be stabilized against dissociative deactivation by intramolecular intersubunit crosslinking. Maximal stabilization can be achieved by using bifunctional crosslinking reagents whose molecular length corresponds to the distance between the protein functional groups to be crosslinked on different subunits. Glyceraldehyde-3-phosphate dehydrogenase (E.G.1.2.1.12), consisting of four identical subunits, can be stabilized against deactivation by using different diacids of HOOC-(CH 2) n -COOH type which interact with protein amino groups after preliminary activation with water-soluble carbodiimide. Maximal stabilization was observed in the case of succinic acid. The presence of intersubunit crosslinkages was proved by the data from SDS-polyacrylamide gel electrophoresis under dissociative conditions, following the intensity of the bands corresponding to enzyme oligomers. The disappearance of the dissociation step in the two-step process of enzyme inactivation after subunit crosslinking was also revealed by kinetic experiments. New methods of ESR spectroscopy (free radical recombination) were used to study the mobility of native and crosslinked protein. It was shown that the decrease in intramolecular protein mobility correlates directly with the increase in its stability against dissocation.