pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea).
The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 x 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 x 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.
Cadet
F
F
Centre de Biotechnologie Agro-Industrielle, Institut National Agronomique Paris-Grignon, Thiverval-Grignon, France.
Meunier
J C
JC
D002736:Chloroplasts / Q000201:enzymology*; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D005635:Fructosediphosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D010084:Oxidation-Reduction; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*
D005635:Fructosediphosphates; D046911:Macromolecular Substances; C027652:fructose 2,6-diphosphate; D006597:Fructose-Bisphosphatase; D010744:Phosphoric Monoester Hydrolases; C022627:sedoheptulose-bisphosphatase; D008274:Magnesium; C029063:fructose-1,6-diphosphate