Kinetics of the conformational transition of the spinach chloroplast fructose-1,6-bisphosphatase induced by fructose 2,6-bisphosphate.
The activation of oxidized chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate and magnesium previously described at pH 7.5 [Soulié et al. (1988) Eur. J. Biochem. 176, 111-117] has now been studied at pH 8, the pH which prevails under light conditions in the chloroplast stroma. The process obeys a hysteretic mechanism but the rate of activation is considerably increased with half-times down to 50 s and the apparent dissociation constant of fructose 2,6-bisphosphate from the enzyme is lowered from 1 mM at pH 7.5 to 3.3 microM at pH 8. The process is strictly metal-dependent with a half-saturation concentration of 2.54 mM for magnesium. The conformational transition postulated in our hysteretic model has been investigated through both the spectrophometric and chemical modification approaches. The activation of the enzyme by fructose 2,6-bisphosphate in the presence of magnesium results in a slow modification of the ultraviolet absorption spectrum of the enzyme with an overall increase of 3% at 290 nm. The same treatment leads to the protection of two free sulfhydryls and an increased reactivity of one sulfhydryl group/enzyme monomer to modification by 5,5'-dithiobis(2-nitrobenzoic acid). The titration of the exposed cysteinyl residue prevents the relaxation of enzyme species induced by fructose 2,6-bisphosphate to the native form. The activation of chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate is discussed both with respect to the understanding of the overall regulation properties of the enzyme and to a possible physiological significance of this process.
Soulié
J M
JM
Centre de Biochimie et de Biologie Moléculaire, C.N.R.S., Marseille, France.
Rivière
M
M
Baldet
P
P
Ricard
J
J
D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D005635:Fructosediphosphates; C027652:fructose 2,6-diphosphate; D006597:Fructose-Bisphosphatase