The N-terminal segment of recombinant porcine fructose-1,6-bisphosphatase participates in the allosteric regulation of catalysis.
Residues 1--10 of porcine fructose-1,6-bisphosphatase (FBPase) are poorly ordered or are in different conformations, sensitive to the state of ligation of the enzyme. Deletion of the first 10 residues of FBPase reduces k(cat) by 30-fold and Mg(2+) affinity by 20-fold and eliminates cooperativity in Mg(2+) activation. Although a fluorescent analogue of AMP binds with high affinity to the truncated enzyme, AMP itself potently inhibits only 50% of the enzyme activity. Additional inhibition occurs only when the concentration of AMP exceeds 10 mm. Deletion of the first seven residues reduces k(cat) and Mg(2+) affinity significantly but has no effect on AMP inhibition. The mutation of Asp(9) to alanine reproduces the weakened affinity for Mg(2+) observed in the deletion mutants, and the mutation of Ile(10) to aspartate reproduces the AMP inhibition of the 10-residue deletion mutant. Changes in the relative stability of the known conformational states for loop 52--72, in response to changes in the quaternary structure of FBPase, can account for the phenomena above. Some aspects of the proposed model may be relevant to all forms of FBPase, including the thioredoxin-regulated FBPase from the chloroplast.
Nelson
S W
SW
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Kurbanov
F T
FT
Honzatko
R B
RB
Fromm
H J
HJ
D000494:Allosteric Regulation; D000818:Animals; D002384:Catalysis; D005453:Fluorescence; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D005635:Fructosediphosphates / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013552:Swine
D005635:Fructosediphosphates; D011994:Recombinant Proteins; C027652:fructose 2,6-diphosphate; D006597:Fructose-Bisphosphatase; D008274:Magnesium