Oxidation-reduction and activation properties of chloroplast fructose 1,6-bisphosphatase with mutated regulatory site.
The concentration of Mg(2+) required for optimal activity of chloroplast fructose 1,6-bisphosphatase (FBPase) decreases when a disulfide, located on a flexible loop containing three conserved cysteines, is reduced by the ferredoxin/thioredoxin system. Mutation of either one of two regulatory cysteines in this loop (Cys155 and Cys174 in spinach FBPase) produces an enzyme with a S(0.5) for Mg(2+) (0.6 mM) identical to that observed for the reduced WT enzyme and significantly lower than the S(0.5) of 12.2 mM of oxidized WT enzyme. E(m) for the regulatory disulfide in WT spinach FBPase is -305 mV at pH 7.0, with an E(m) vs pH dependence of -59 mV/pH unit, from pH 5.5 to 8.5. Aerobic storage of the C174S mutant produces a nonphysiological Cys155/Cys179 disulfide, rendering the enzyme partially dependent on activation by thioredoxin. Circular dichroism spectra and thiol titrations provide supporting evidence for the formation of nonphysiological disulfide bonds. Mutation of Cys179, the third conserved cysteine, produces FBPase that behaves very much like WT enzyme but which is more rapidly activated by thioredoxin f, perhaps because the E(m) of the regulatory disulfide in the mutant has been increased to -290 mV (isopotential with thioredoxin f). Structural changes in the regulatory loop lower S(0.5) for Mg(2+) to 3.2 mM for the oxidized C179S mutant. These results indicate that opening the regulatory disulfide bridge, either through reduction or mutation, produces structural changes that greatly decrease S(0.5) for Mg(2+) and that only two of the conserved cysteines play a physiological role in regulation of FBPase.
Balmer
Y
Y
Laboratoire de Biochimie Végétale, Université de Neuchâtel, CH-2007 Neuchâtel, Switzerland.
Stritt-Etter
A L
AL
Hirasawa
M
M
Jacquot
J P
JP
Keryer
E
E
Knaff
D B
DB
Schürmann
P
P
D020134:Catalytic Domain / Q000235:genetics; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000201:enzymology*; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004789:Enzyme Activation; D006597:Fructose-Bisphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics; D018724:Spinacia oleracea / Q000201:enzymology / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
D054479:Chloroplast Thioredoxins; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D006597:Fructose-Bisphosphatase; D008274:Magnesium; D003545:Cysteine