In vivo and in vitro phosphorylation of rat liver fructose-1,6-bisphosphatase.

Riou, J. P.; Claus, Thomas H.; Flockhart, David A.; Corbin, Jackie D.; Pilkis, S J

doi:10.1073/pnas.74.10.4615

Cited by 96 publications

(41 citation statements)

References 25 publications

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“…It is noteworthy that Kemp et al (19,32) demonstrated that a basic amino acid residue near the serine is an important determinant of substrate specificity for the cAMP kinase. Substitutions at the first of the t It has been shown that the C subunit catalyzes the phosphorylation of rat liver fructose-1,6-diphosphatase (44). We have observed that the cGMP kinase also catalyzes the phosphorylation of this protein in a manner similar to that for the substrates shown in Fig.…”

Section: Resultsmentioning

confidence: 60%

Adenosine 3′:5′-cyclic monophosphate- and guanosine 3′:5′-cyclic monophosphate-dependent protein kinases: Possible homologous proteins

Lincoln

Corbin

1977

Proc. Natl. Acad. Sci. U.S.A.

148

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The properties of purified mammalian adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP)dependent protein kinases were compared. Several physical characteristics of the two enzymes were similar, including size, shap, affinity for cyclic nucleotide binding, and Km for ATP. In addition, the amino acid compositions of the two proteins indicated a close composition homology (70-90% Adenosine 3':5'-cyclic monophosphate (cAMP)-and guanosine 3':5'-cyclic monophosphate (cGMP)-dependent protein kinases (cAMP kinase and cGMP kinase, respectively) have been described in several vertebrate and invertebrate tissues (1-8). Both enzymes have been purified to homogeneity and the subunit compositions characterized (9-14). While the distribution of the cAMP kinase is ubiquitous in mammalian tissues, there does not appear to be a similar distribution of the cGMP kinase since only lung, cerebellum, heart, and small intestine have appreciable amounts of specific cGMP binding activity (8). On the other hand, cGMP kinase is more widely distributed in the lower species of animals especially in the arthropods where the enzyme was first described (3, 4). Abundant evidence has been provided that indicates that cyclic nucleotide-dependent protein kinases are the major receptors for cyclic nucleotides in eukaryotic cells.Several reports have appeared comparing the properties of cAMP and cGMP kinases from several sources (6-9, 15). In general, these studies emphasized the differences between the two enzymes. One study by Hashimoto et al. (16), however, disclosed similarities in substrate specificity between the two cyclic nucleotide-dependent protein kinases. Since relatively crude enzyme preparations were used, few details concerning substrate specificity and physical nature of the enzymes could be obtained.In this paper, we present data showing that purified mammalian cAMP and cGMP kinases have several similarities in physical properties and substrate specificity. Furthermore, we propose, on the basis of several lines of indirect evidence, that the two enzymes are homologous proteins.MATERIALS AND METHODS Purification of Enzymes. Homogeneous bovine liver catalytic subunit of cAMP kinase (C subunit) was prepared by the method of Sugden et al. (17); homogeneous bovine lung cGMP kinase was purified as described (13,14). The regulatory subunit of cAMP kinase (R subunit) from bovine heart was prepared by a method modified from that of Dills et al. (12)

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Section: Resultsmentioning

confidence: 60%

Adenosine 3′:5′-cyclic monophosphate- and guanosine 3′:5′-cyclic monophosphate-dependent protein kinases: Possible homologous proteins

Lincoln

Corbin

1977

Proc. Natl. Acad. Sci. U.S.A.

148

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“…Determination of Fru-1,6-P 2 ase Activity and Data Analysis-A spectrophotometric, coupled-enzyme assay was employed to measure Fru-1,6-P 2 ase activity (18). Standard conditions (2 mM magnesium chloride) were used to determine specific activity.…”

Section: Methodsmentioning

confidence: 99%

Importance of the Dimer-Dimer Interface for Allosteric Signal Transduction and AMP Cooperativity of Pig Kidney Fructose-1,6-Bisphosphatase

Giroux

Kantrowitz

1997

Journal of Biological Chemistry

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The role of the 190's loop of fructose-1,6-bisphosphatase (Fru-1,6-P 2 ase) in the allosteric regulation of Fru-1,6-P 2 ase has been investigated through kinetic studies on three mutant enzymes, Glu-192 3 Ala, Glu-192 3 Gln, and Asp-187 3 Ala. AMP is an allosteric inhibitor, which binds to the regulatory sites and induces the R-to T-state transition; for wild-type Fru-1,6-P 2 ase AMP inhibition is cooperative with a Hill coefficient of 2.0. The replacement of Asp-187, which forms an interaction across the C1:C2 monomer-monomer interface, with alanine did not change the catalytic efficiency, and it had no effect on the cooperativity of AMP inhibition; however, the apparent dissociation constant for AMP increased more than 4-fold as compared to the value for the wild-type enzyme. The replacement of Glu-192, which forms interactions across the C1:C4 dimer-dimer interface, with Ala and Gln lowered k cat from 21 s ؊1 for wild-type enzyme to 15 s ؊1 and 13 s ؊1 , respectively, for the mutant enzymes, while their respective K m values were not changed. However, these replacements did have dramatic effects on AMP inhibition; first, cooperative AMP inhibition was lost; second, the AMP inhibition was biphasic, which can be interpreted as due to AMP binding to two classes of binding sites. The high affinity class of sites corresponds to the regulatory sites, while the low affinity class of sites may be the active sites. The results reported here, combined with the structural and kinetic results from the Lys-42 3 Ala enzyme, strongly suggest that the C1:C4 dimer-dimer interface, rather than the C1:C2 monomer-monomer interface, is critical for the propagation of the allosteric signal between the AMP sites on different subunits; in addition, cooperative AMP inhibition is essential for the enzyme to be fully inhibited by the binding of AMP to the allosteric site.Fructose-1,6-bisphosphatase (Fru-1,6-P 2 ase, EC 3.1.3.11), 1 a key regulatory enzyme in gluconeogenesis (1), catalyzes the hydrolysis of the ␣ anomer of fructose 1,6-bisphosphate (Fru-1,6-P 2 ) to ␣-D-fructose 6-phosphate and inorganic phosphate in the presence of divalent metal ions. Pig kidney Fru-1,6-P 2 ase is an allosteric enzyme composed of four identical subunits, each with a molecular weight of 34,000 (2). Although the enzyme is normally in the active R state, upon the binding of AMP it is transformed into the completely inactive T state. X-ray structural data for both the R-and T-states are available for pig kidney Fru-1,6-P 2 ase. The AMP complexes (3) both in the absence and the presence of fructose 6-phosphate are identified as T-state structures, while those complexes with bound fructose 6-phosphate (4) or fructose 2,6-bisphosphate (Fru-2,6-P 2 ) (5) are identified as R-state structures. The tertiary structure of each monomer is composed of two folding domains (Fig. 1), the AMP domain with the AMP binding site and the FBP domain containing the active site.The activity of pig kidney Fru-1,6-P 2 ase is negatively modulated by the allosteric inhibitor AMP an...

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“…A spectrophotometric coupled enzyme assay was employed to measure Fru-l,6-P2ase activity (Riou et al, 1977). Standard conditions (2 mM magnesium chloride, 17.5 p M F~-l , 6 -P 2 ) were used to determine specific activity.…”

Section: Determination Of Fru-l4-p2ase Activity and Data Analysismentioning

confidence: 99%

Evidence for an active T‐state pig kidney fructose 1,6‐bisphosphatase: Interface residue Lys‐42 is important for allosteric inhibition and AMP cooperativity

Stec

Giroux

et al. 1996

Protein Science

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During the R 3 T transition in the tetrameric pig kidney fructose-1,6-bisphosphatase (Fru-1 ,6-P2ase, EC 3.1.3.1 1) a major change in the quaternary structure of the enzyme occurs that is induced by the binding of the allosteric inhibitor AMP (Ke HM, Liang JY, Zhang Y, Lipscomb WN, 1991, Biochemistry 30:4412-4420). The change in quaternary structure involving the rotation of the upper dimer by 17" relative to the lower dimer is coupled to a series of structural changes on the secondary and tertiary levels. The structural data indicate that Lys-42 is involved in a complex set of intersubunit interactions across the dimerdimer interface with residues of the 190's loop, a loop located at the pivot of the allosteric rotation. In order to test the function of Lys-42, we have replaced it with alanine using site-specific mutagenesis. The kc,, and K , values for Lys-42 +Ala Fru-1 ,6-P2ase were 1 1 s" and 3.3 FM, respectively, resulting in a mutant enzyme that was slightly less efficient catalytically than the normal pig kidney enzyme. Although the Lys-42 -+ Ala Fru-1,6-P2ase was similar kinetically in terms of K , and k,,,, the response to inhibition by AMP was significantly different than that of the normal pig kidney enzyme. Not only was AMP inhibition no longer cooperative, but also it occurred in two stages, corresponding to high-and low-affinity binding sites. Saturation of the high-affinity sites only reduced the activity by 30%, compared to 100% for the wild-type enzyme. In order to determine in what structural state the enzyme was after saturation of the highaffinity sites, the Lys-42 -+ Ala enzyme was crystallized in the presence of Mn2+, fructose-6-phosphate (Fru-6-P), and 100 /LM AMP and the data collected to 2.3 8, resolution. The X-ray structure showed the T state with AMP binding with full occupancy to the four regulatory sites and the inhibitor Fru-6-P bound at the active sites. The results reported here suggest that, in the normal pig kidney enzyme, the interactions between Lys-42 and residues of the 190's loop, are important for propagation of AMP cooperativity to the adjacent subunit across the dimerdimer interface as opposed to the monomer-monomer interface, and suggest that AMP cooperativity is necessary for full allosteric inhibition by AMP.Keywords: allostery; metalloenzymes; protein structure-function; site-specific mutagenesis; X-ray diffraction Fructose 1,6-bisphosphatase (EC.3.1.3.11) catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate, a key control step in the gluconeogenesis Abbreviarionsr Fru-1 ,6-P2ase, fructose-1,6-bisphosphatase; Fru-1,6-P2, fructose 1.6-bisphosphate; Fru-2,6-P~, fructose 2,6-bisphosphate; Fru-6-P, fructose 6-phosphate; Lys-42 + Ala, the mutant pig kidney fructose-1.6-bisphosphatase with alanine in place of lysine at position 42; RMSD, RMS displacement.

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In vivo and in vitro phosphorylation of rat liver fructose-1,6-bisphosphatase.

Cited by 96 publications

References 25 publications

Adenosine 3′:5′-cyclic monophosphate- and guanosine 3′:5′-cyclic monophosphate-dependent protein kinases: Possible homologous proteins

Adenosine 3′:5′-cyclic monophosphate- and guanosine 3′:5′-cyclic monophosphate-dependent protein kinases: Possible homologous proteins

Importance of the Dimer-Dimer Interface for Allosteric Signal Transduction and AMP Cooperativity of Pig Kidney Fructose-1,6-Bisphosphatase

Evidence for an active T‐state pig kidney fructose 1,6‐bisphosphatase: Interface residue Lys‐42 is important for allosteric inhibition and AMP cooperativity

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