Enhancement of the reductive activation of chloroplast fructose‐1,6‐bisphosphatase by modulators and protein perturbants

To characterize the mechanism of chloroplast fructose‐1,6‐bisphosphatase activation, we have examined kinetic and structural changes elicited by protein perturbants and reductants. At variance with its well‐known capacity for enzyme inactivation, 150 mM sodium trichloroacetate yielded an activatable chloroplast fructose‐1,6‐bisphosphatase in the presence of 1.0 mM fructose 1,6‐bisphosphate and 0.1 mM Ca2+. Other sugar bisphosphates did not replace fructose 1,6‐bisphosphate whereas Mg2+ and Mn2+ were functional in place of Ca2+. Variations of the emission fluorescence of intrinsic fluorophores and a noncovalently bound extrinsic probe [2‐(P‐toluidinyl)naphthalene‐6‐sulfonate] indicated the presence of conformations different from the native form. A similar conclusion was drawn from the analysis of absorption spectra by means of fourth‐derivative spectrophotometry. The effect of these conformational changes on the reductive process was studied by subsequently incubating the enzyme with dithiothreitol. The reaction of chloroplast fructose‐1,6‐bisphosphatase with dithiothreitol was accelerated 13‐fold by the chaotropic anion: second‐order rate constants were 48.1 M−1· min−1 and 3.7 M−1· min−1 in the presence and in the absence of trichloroacetate, respectively. Thus, the enhancement of the reductive activation by compounds devoid of redox activity illustrated that the modification of intramolecular noncovalent interactions of chloroplast fructose‐1,6‐bisphosphatase plays an essential role in the conversion of enzyme disulfide bonds to sulfhydryl groups. In consequence, a conformational change would operate concertedly with the reduction of disulfide bridges in the light‐dependent activation mediated by the ferredoxin–thioredoxin system. Copyright © 1994, Wiley Blackwell. All rights reservedFil:Ballicora, M.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Wolosiuk, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

ADPglucose pyrophosphorylase's N-terminus: Structural role in allosteric regulation

We studied the functional role of the Escherichia coli ADPglucose pyrophosphorylase's N-terminus in allosteric regulation, and the particular effects caused by its length. Small truncated mutants were designed, and those lacking up to 15-residues were active and highly purified for further kinetic analyses. NΔ3 and NΔ7 did not change the kinetic parameters with respect to the wild-type. NΔ11 and NΔ15 enzymes were insensitive to allosteric regulation and highly active in the absence of the activator. Co-expression of two polypeptides corresponding to the N- and C-termini generated an enzyme with activation properties lower than those of the wild-type [C.M. Bejar, M.A. Ballicora, D.F. Gómez Casati, A.A. Iglesias, J. Preiss, The ADPglucose pyrophosphorylase from Escherichia coli comprises two tightly bound distinct domains, FEBS Lett. 573 (2004) 99-104]. Here, we characterized a NΔ15 co-expression mutant, in which the allosteric regulation was restored to wild-type levels. Unusual allosteric effects caused by either an N-terminal truncation or co-expression of individual domains may respond to structural changes favoring an up-regulated or a down-regulated conformation rather than specific activator or inhibitor sites' disruption.Fil: Bejar, C.M.. Michigan State University; Estados UnidosFil: Ballicora, M.A.. Loyola University Of Chicago; . Michigan State University; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Preiss, J.. Michigan State University; Estados Unido