1fta
|
FRUCTOSE-1,6-BISPHOSPHATASE(D-FRUCTOSE-1,6-BISPHOSPHATE, 1-PHOSPHOHYDROLASE) (E.C.3.1.3.11) COMPLEXED WITH THE ALLOSTERIC INHIBITOR AMP
OverviewOverview
The molecular structure of human liver fructose-1,6-bisphosphatase, complexed with AMP was determined by x-ray diffraction using molecular, replacement, starting from the pig kidney enzyme AMP complex. Of the 34, amino acid residues which differ between these two sequences, only one, interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this, analysis, six sites in which side chains of amino acid residues are in, contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were, mutated by polymerase chain reaction. The wild-type and mutant forms were, expressed in Escherichia coli, purified, and their kinetic properties, determined. Circular dichroism spectra of the mutants were, indistinguishable from that of the wild-type enzyme. Kinetic analyses, revealed that all forms had similar turnover numbers, Km values for, fructose 2,6-bisphosphate, and inhibition constants for fructose, 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 -->, Phe and Met177 --> Ala mutants were similar to those of the wild-type, enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 -->, Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser, mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation, of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the, Tyr113 --> Phe mutant was undetectable even at millimolar AMP, concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of, the mutants to the same extent as for the wild-type enzyme, except in the, case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 -->, Ala mutant suggests that the side chain beyond C alpha is not needed for, AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts, with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds, to AMP consistent with strong side chain interactions in the wild-type, enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on, AMP inhibition observed in the Thr31 --> Ala mutant may be an important, clue relating to the mechanism of synergism of these two inhibitors.
DiseaseDisease
Known diseases associated with this structure: Fructose-bisphosphatase deficiency OMIM:[229700]
About this StructureAbout this Structure
1FTA is a Single protein structure of sequence from Homo sapiens with AMP as ligand. Active as Fructose-bisphosphatase, with EC number 3.1.3.11 Full crystallographic information is available from OCA.
ReferenceReference
The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure., Gidh-Jain M, Zhang Y, van Poelje PD, Liang JY, Huang S, Kim J, Elliott JT, Erion MD, Pilkis SJ, Raafat el-Maghrabi M, et al., J Biol Chem. 1994 Nov 4;269(44):27732-8. PMID:7961695
Page seeded by OCA on Mon Nov 12 16:57:19 2007