Effects of pH and fructose 2,6-bisphosphate on oxidized and reduced spinach chloroplastic fructose-1,6-bisphosphatase.
This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-1,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange. The pK values relative to each redox form for the same substrate (either fructose 1,6-bisphosphate or sedoheptulose 1,7-bisphosphate) are identical, suggesting the same site for both substrates on the active molecule. The finding that the analog (fructose 2,6-bisphosphate) behaves like a competitive inhibitor for both substrates also favours this hypothesis. The inhibitory effect of this sugar is more important when the enzyme is reduced than when it is oxidized. The shift in the optimum pH observed when [Mg2+] was raised is interpreted as a conformational change of oxidized enzyme demonstrated by a change in fluorescence. The reduced and oxidized forms have the same theoretical rates relative to both substrates, but the reduced form has an observed Vmax which is 60% of the theoretical Vmax while that of the oxidized form is only 37% of the theoretical Vmax. The reduced enzyme appears more efficient than the oxidized one in catalysis.
D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000201:enzymology*; D006597:Fructose-Bisphosphatase / Q000302:isolation & purification / Q000378:metabolism*; D005635:Fructosediphosphates / Q000494:pharmacology*; D006598:Hexosediphosphates / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity; D013879:Thioredoxins
D054479:Chloroplast Thioredoxins; D005635:Fructosediphosphates; D006598:Hexosediphosphates; D010940:Plant Proteins; D013879:Thioredoxins; C027652:fructose 2,6-diphosphate; D006597:Fructose-Bisphosphatase