Effects of mutations of the cysteines in the regulatory loop on structure and activity of chloroplast fructose-1,6-bisphosphatase (FBPase)

Abstract

Chloroplast FBPase is a redox-regulated enzyme of the Calvin cycle activated by thioredoxin f via reduction of a disulfide bridge located in a loop, absent from the cytosolic FBPases, consisting of an insertion of about twenty amino acids. This loop contains three strictly conserved cysteines, two forming the regulatory disulfide (Cys155, Cys174 in spinach FBPase), and a third of yet unclear function (Cys179). To gain more information on structural and functional roles of these cysteines in spinach FBPase we replaced them individually or in pairs. Mutation of either one or both Cys of the regulatory disulfide (Cys155, Cys174) yields an enzyme comparable to reduced WT, with identical affinity for Mg2+ of 0.6 mM. However, during aerobic storage of the Cys174Ser mutant an artefactual disulfide bond between Cys155 and Cys179 is formed rendering the enzyme partially dependent on thioredoxin activation. Circular dichroism spectra and SH-titrations of WT and mutant proteins support the appearance of an artifactual disulfide bridge. Mutation of the third conserved cysteine leads to an enzyme behaving essentially like WT FBPase, but which is much more rapidly activated, due partially to a redox potential of the regulatory disulfide that is 15 mV more positive than that of WT enzyme, (Em = -305 mV at pH 7). The affinity for Mg2+ is slightly decreased to 1.2 mM. Our results confirm that opening of the regulatory disulfide bridge by reduction or mutation greatly decreases the affinity for Mg2+ due to structural changes originating in the regulatory loop.