5 research outputs found
Enhancement of the reductive activation of chloroplast fructoseâ1,6âbisphosphatase by modulators and protein perturbants
To characterize the mechanism of chloroplast fructoseâ1,6âbisphosphatase activation, we have examined kinetic and structural changes elicited by protein perturbants and reductants. At variance with its wellâknown capacity for enzyme inactivation, 150 mM sodium trichloroacetate yielded an activatable chloroplast fructoseâ1,6âbisphosphatase in the presence of 1.0 mM fructose 1,6âbisphosphate and 0.1 mM Ca2+. Other sugar bisphosphates did not replace fructose 1,6âbisphosphate whereas Mg2+ and Mn2+ were functional in place of Ca2+. Variations of the emission fluorescence of intrinsic fluorophores and a noncovalently bound extrinsic probe [2â(Pâtoluidinyl)naphthaleneâ6âsulfonate] indicated the presence of conformations different from the native form. A similar conclusion was drawn from the analysis of absorption spectra by means of fourthâderivative spectrophotometry. The effect of these conformational changes on the reductive process was studied by subsequently incubating the enzyme with dithiothreitol. The reaction of chloroplast fructoseâ1,6âbisphosphatase with dithiothreitol was accelerated 13âfold by the chaotropic anion: secondâorder rate constants were 48.1 Mâ1· minâ1 and 3.7 Mâ1· minâ1 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 fructoseâ1,6âbisphosphatase 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 lightâdependent activation mediated by the ferredoxinâthioredoxin 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. NÎ3 and NÎ7 did not change the kinetic parameters with respect to the wild-type. NÎ11 and NÎ15 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. GĂłmez 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 NÎ15 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 CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Santa Fe. Instituto de AgrobiotecnologĂa del Litoral. Universidad Nacional del Litoral. Instituto de AgrobiotecnologĂa del Litoral; ArgentinaFil: Preiss, J.. Michigan State University; Estados Unido