Directed Mutations in the Poorly Defined Region of Porcine Liver Fructose-1,6-bisphosphatase Significantly Affect Catalysis and the Mechanism of AMP Inhibition*

Feruz T. Kurbanov, Jun-yong Choe, Richard B. Honzatko, and Herbert J. FrommDagger

From the Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011

    ABSTRACT
Top
Abstract
Introduction
Results
Discussion
References

Asn64, Asp68, Lys71, Lys72, and Asp74 of porcine liver fructose-1,6-bisphosphatase (FBPase) are conserved residues and part of a loop for which no electron density has been observed in crystal structures. Yet mutations of the above dramatically affect catalytic rates and/or AMP inhibition. The Asp74 right-arrow Ala and Asp74 right-arrow Asn mutant enzymes exhibited 50,000- and 2,000-fold reductions, respectively, in kcat relative to wild-type FBPase. The pH optimum for the catalytic activity of the Asp74 right-arrow Glu, Asp68 right-arrow Glu, Asn64 right-arrow Gln, and Asn64 right-arrow Ala mutant enzymes shifted from pH 7.0 (wild-type enzyme) to pH 8.5, whereas the Lys71 right-arrow Ala mutant and Lys71,72 right-arrow Met double mutant had optimum activity at pH 7.5. Mg2+ cooperativity, Km for fructose 1,6-bisphosphate, and Ki for fructose 2,6-bisphosphate were comparable for the mutant and wild-type enzymes. Nevertheless, for the Asp74 right-arrow Glu, Asp68 right-arrow Glu, Asn64 right-arrow Gln, and Asn64 right-arrow Ala mutants, the binding affinity for Mg2+ decreased by 40-125-fold relative to the wild-type enzyme. In addition, the Asp74 right-arrow Glu and Asn64 right-arrow Ala mutants exhibited no AMP cooperativity, and the kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive to noncompetitive. The double mutation Lys71,72 right-arrow Met increased Ki for AMP by 175-fold and increased Mg2+ affinity by 2-fold relative to wild-type FBPase. The results reported here strongly suggest that loop 51-72 is important for catalytic activity and the mechanism of allosteric inhibition of FBPase by AMP.

    INTRODUCTION
Top
Abstract
Introduction
Results
Discussion
References

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase1), a key regulatory enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P2) to fructose 6-phosphate and Pi in the presence of divalent metal ions such as Mg2+, Mn2+, and Zn2+ (1, 2). The activation of the enzyme by Mg2+ exhibits sigmoidal kinetics with a Hill coefficient of 2 at neutral pH, but is hyperbolic at pH 9.6 (3, 4). FBPase is inhibited synergistically by AMP (as an allosteric effector) and fructose 2,6-bisphosphate (Fru-2,6-P2; as a substrate analog) (5-7). In contrast, AMP and Fru-2,6-P2 act as strong activators for phosphofructokinase, a major regulatory enzyme in the opposing glycolytic pathway.

In mammals, FBPase is a homotetramer with a subunit Mr of 37,000 (8). Each subunit of the tetramer (designated C1, C2, C3, and C4) has an allosteric AMP domain (residues 1-200) and a catalytic Fru-1,6-P2 domain (residues 201-335), with the AMP-binding site being ~28 Å away from the substrate-binding site (9-11). Structures of AMP complexes of the enzyme define the T-state, whereas structures in the presence substrate analogs and without AMP represent the R-state. The structural transition (R- to T-state) involves a 17° rotation of the C1-C2 dimer with respect to the C3-C4 dimer and a 1.9° rotation of the AMP domain relative to the Fru-1,6-P2 domain within each subunit (10). The R- to T-state transition results in conformational changes at interfaces between subunits C1 and C2 and subunits C1 and C4 (as well as interfaces related to these by the symmetry of the tetramer). Metal-binding sites (up to three total) are at or near the active site at the interface between the two domains of the enzyme (12, 13). Mg2+ and AMP are mutually exclusive in their binding to FBPase (14, 15). In fact, AMP inhibition is nonlinear and noncompetitive with respect to Fru-1,6-P2 and nonlinear and competitive with respect to Mg2+. Yet crystallographic studies reveal similar metal coordination in the absence and presence AMP (either one Mg2+ ion or two Zn2+ or two Mn2+ ions), although small perturbations in the active site are attributed to the 1.9° rotation of the AMP relative to the Fru-1,6-P2 domain due to AMP binding (13).

Site-directed mutagenesis of the residues involved at subunit interfaces of porcine liver FBPase revealed significant changes in the kinetic mechanism of AMP inhibition and cooperativity (16-19). However, a detailed mechanism of catalysis and allosteric regulation based on changes in interacting residues has been elusive. Perhaps published structures of FBPase do not reveal all of the essential structural elements for catalysis and regulation. Mutation of Arg49, a residue that precedes a disordered loop (residues 54-71), causes dramatic changes in FBPase kinetics (17). Comparison of the amino acid sequences of mammalian FBPases reveals Gly58, Gly61, Asn64, Asp68, Asp74, Lys71, and Lys72 as conserved residues within this disordered loop. Furthermore, loop 54-71 is proteolytically sensitive in all known FBPases (20). To determine whether loop 54-71 plays a major role in FBPase kinetics, mutant enzymes Asn64 right-arrow Gln, Asn64 right-arrow Ala, Asp68 right-arrow Glu, Lys71 right-arrow Ala, Lys71,72 right-arrow Met (double mutation), Asp74 right-arrow Ala, Asp74 right-arrow Asn, and Asp74 right-arrow Glu were expressed in Escherichia coli, purified to homogeneity, and evaluated by initial velocity kinetics. The above mutations have profound effects on both catalysis and regulation of FBPase.

    EXPERIMENTAL PROCEDURE

Materials-- Fru-1,6-P2, Fru-2,6-P2, NADP, AMP, ampicillin, tetracycline, and isopropyl-beta -D-thiogalactopyranoside were purchased from Sigma. DNA-modifying and restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were either from New England Biolabs Inc. or Promega. Glucose-6-phosphate dehydrogenase and phosphoglucose isomerase were supplied by Boehringer Mannheim. Tryptone, yeast extract, and agar were from Difco. Other chemicals were of reagent grade or the equivalent.

E. coli strains BMH 71-18 mutS (thi, supE, Delta (lac-proAB), [mutS::Tn10] [F'proAB, lacIqZDelta M15]) and XL-1Blue (recA1, endA1, gyrA96, thi-1, hsdR-17, supE44, thi-1, relA1, lac [F' proAB, LacIqZDelta M15, Tn10(Tetr)] came from CLONTECH and Stratagene, respectively. FBPase-deficient E. coli strain DF 657 (tonA22, ompF627(T2r), relA1, pit-10, spoT1, Delta (fbp)287) was from the Genetic Stock Center at Yale University.

Mutagenesis of FBPase-- Mutations were accomplished by the introduction of specific base changes into a double-stranded DNA plasmid. Eight mutagenic primers (5'-TGGCTCCACGCAGGTGACAGG-3', 5'-TGGCTCCACGGCCGTGACAGGT-3', 5'-GTGACAGGTGAGCAAGTGAAG-3', 5'-GTGATCAAGTGGCGAAGTTGGATG-3', 5'-GTGATCAAGTGATGATGTTGGATGT-3', 5'-GAAGTTGGCGGTCCTCTCCA3', 5'-GAAGAAGTTGGAAGTCCTCTC-3', and 5'-GAAGTTGGAGGTCCTCTCCAA-3') were used to mutate Asn64 right-arrow Gln, Asn64 right-arrow Ala, Asp68 right-arrow Glu, Lys71 right-arrow Ala, Lys71,72 right-arrow Met, Asp74 right-arrow Ala, Asp74 right-arrow Asn, and Asp74 right-arrow Glu. A selective primer (5'-CAGCCTCGCGTCGCGAACGCCAG-3') was used to change the unique XhoI site to the original NruI site on the pET-11a vector. The double-stranded FBPase expression plasmid (pET-FBP) and mutagenic and selective primers were denatured, annealed, and polymerized using the TransformerTM site-Directed mutagenesis kit (21). Mutations were confirmed by XhoI/NruI digestion and by fluorescent dideoxy chain termination sequencing at the Nucleic Acid Facility at Iowa State University. The plasmids with mutations were transformed into E. coli strain DF 657. The plasmid DNA was isolated from the overnight culture, and sequence analysis was performed on the entire FBPase gene.

Expression and Purification of Wild-type and Mutant FBPases-- The expression and purification of wild-type and mutant forms of FBPase were carried out as described previously (22) with slight modification. After centrifugation to remove cell debris, the supernatant was subjected to heat treatment, 30-70% ammonium sulfate precipitation, Sephadex G-200 column chromatography, and CM-Sepharose column chromatography. Wild-type and mutant FBPases eluted as a single peak from the CM-Sepharose column by a NaCl gradient from 20 to 400 mM in 10 mM malonate buffer, pH 6.0. Protein purity was evaluated by 12% SDS-polyacrylamide gel electrophoresis according to Laemmli (23). Protein concentrations were determined as described by Bradford (24) using bovine serum albumin as the standard.

Kinetic Studies-- Specific activity during purification was determined by the phosphoglucoisomerase and glucose-6-phosphate dehydrogenase coupled spectrometric assay at either pH 7.5 or 9.6 (1). All other kinetic experiments were done at either pH 7.5 or 8.5 and 25 °C using a coupled spectrofluorometric assay (15). Initial rate data were analyzed using a computer program written in MINITAB language with an alpha  value of 2.0 (25). Cooperativity was evaluated using either the ENZFITTER program (26) or the MINITAB program.

Circular Dichroism Spectrometry-- CD studies on the wild-type and mutant FBPases were carried out in a Jasco J710 CD spectrometer in a 1-mm cell at room temperature using a 0.2 mg/ml concentration of the enzyme. Spectra were collected from 200 to 260 nm in increments of 1.3 nm, and each spectrum was blank-corrected and smoothed using the software package provided with the instrument.

    RESULTS
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Abstract
Introduction
Results
Discussion
References

Purification of Wild-type and Mutant Forms of FBPase-- Purification of mutant and wild-type enzymes followed previous protocols (22). Mutant enzymes exhibited elution patterns similar to wild-type FBPase, except for Lys71 right-arrow Ala and Lys71,72 right-arrow Met, which eluted from the CM-Sepharose column at 100 mM NaCl. On the basis of SDS-polyacrylamide gel electrophoresis, all enzymes exhibit identical mobilities (Mr ~ 37, 500) with no evidence of proteolysis (27) and purity greater than 95% (data not shown). Activity ratios (pH 7.5:9.6) for the wild-type, Lys71 right-arrow Ala, and double mutant Lys71,72 right-arrow Met enzymes confirmed the absence of proteolysis.

Secondary Structure Analysis-- The CD spectra of wild-type and mutant FBPases are essentially superimposable from 200 to 260 nm (data not shown), indicating the absence of global structural alteration changes caused by the mutations.

Catalytic Rates of FBPase Mutants-- Initial rate studies were done at saturating concentrations of Fru-1,6-P2 or Mg2+ that do not cause substrate inhibition. Kinetic parameters are in Table I. The kcat values for the Asp74 right-arrow Ala and Asp74 right-arrow Asn mutants decreased 50,000 and 2,000 times, respectively, whereas a shift in the pH of optimum activity from pH 7.5 to 8.5 was exhibited by the Asp74 right-arrow Glu mutant with only a 20-fold reduction in kcat. A similar alteration in the pH of optimum activity occurred with mutants Asn64 right-arrow Gln, Asn64 right-arrow Ala, and Asp68 right-arrow Glu. On the other hand, replacements Lys71 right-arrow Ala and Lys71,72 right-arrow Met did not alter the pH of optimum activity. In Table I, the kinetic parameters for the Asp74 right-arrow Glu, Asn64 right-arrow Gln, Asn64 right-arrow Ala, and wild-type FBPases were measured at pH 8.5, and for mutants Asp68 right-arrow Glu, Lys71 right-arrow Ala, and Lys71,72 right-arrow Met at pH 7.5. A slight change in Fru-1,6-P2 affinity was observed in all mutants relative to wild-type FBPase.

                              
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Table I
Kinetic parameters for wild-type and mutant forms of fructose-1,6-bisphosphatase

Mg2+ Activation-- The activity of wild-type FBPase as a function of Mg2+ concentration is sigmoidal at neutral pH with a Hill coefficient of ~2, but is hyperbolic at pH 9.6 (3, 4). The Hill coefficient for Mg2+ and the Ka for Mg2+ of the wild-type and mutant forms of FBPase were determined here at a saturating Fru-1,6-P2 concentration (30 µM) at pH 7.5 or 8.5. Hill coefficients at pH 8.5 dropped from 2.2 for the wild-type enzyme to ~1.4 for the Asp74 right-arrow Glu, Asn64 right-arrow Gln, and Asn64 right-arrow Ala mutants, and at pH 7.5 from 2.0 to 1.4 for the Asp68 right-arrow Glu mutant. Increases of 126-, 51-, 47-, and 39-fold in Ka values for Mg2+ were found for the Asp74 right-arrow Glu, Asp68 right-arrow Glu, Asn64 right-arrow Gln, and Asn64 right-arrow Ala mutants, respectively, relative to the wild-type enzyme. The Lys71 right-arrow Ala and Lys71,72 right-arrow Met mutant enzymes as well as wild-type FBPase showed sigmoidal kinetics for Mg2+ at pH 7.5. On the other hand, the affinity for Mg2+ at pH 7.5 increased 2-fold for the Lys71 right-arrow Ala and Lys71,72 right-arrow Met mutant enzymes relative to wild-type FBPase.

Fru-2,6-P2 Inhibition-- Fru-2,6-P2 is a competitive inhibitor of Fru-1,6-P2 and competes with the substrate for the active site of FBPase (7, 29). Ki values for Fru-2,6-P2 decreased by <4-fold for all mutant enzymes compared with the wild-type enzyme. On the other hand, the Lys71,72 right-arrow Met mutant exhibited a 7-fold increase in Ki for Fru-2,6-P2.

Kinetics of AMP Inhibition-- AMP is an allosteric regulator of FBPase (30). The action of AMP inhibition is nonlinear and noncompetitive with respect to Fru-1,6-P2 (31), but nonlinear and competitive relative to Mg2+ for wild-type FBPase at either neutral or alkaline pH (15). AMP binding to FBPase is cooperative with a Hill coefficient of ~2 (4). The wild-type and Lys71 right-arrow Ala, Lys71,72 right-arrow Met, Asp68 right-arrow Glu, and Asn64 right-arrow Gln FBPases exhibited competitive inhibition patterns for AMP relative to Mg2+, whereas the Asp74 right-arrow Glu and Asn64 right-arrow Ala mutants showed noncompetitive inhibition. Fig. 1 shows double-reciprocal plots of 1/velocity versus 1/[Mg2+]2 at various fixed concentrations of AMP for the Lys71,72 right-arrow Met mutant of FBPase. The data of Fig. 1 are consistent with a steady-state random mechanism represented by Equation 1,
<FR><NU>1</NU><DE>v</DE></FR>=<FR><NU>1</NU><DE>V<SUB>M</SUB></DE></FR><FENCE>1+<FR><NU>K<SUB>a</SUB></NU><DE>A<SUP>2</SUP></DE></FR><FENCE>1+<FR><NU>I</NU><DE>K<SUB>i</SUB></DE></FR>+<FR><NU>I<SUP>n</SUP></NU><DE>K<SUB>ii</SUB></DE></FR>+<FR><NU>I</NU><DE>K<SUB>iii</SUB></DE></FR>+<FR><NU>I<SUP>n</SUP></NU><DE>K<SUB>iv</SUB></DE></FR></FENCE></FENCE> (Eq. 1)
<FENCE>+<FR><NU>K<SUB>b</SUB></NU><DE>B</DE></FR>+<FR><NU>K<SUB>ia</SUB></NU><DE>A<SUP>2</SUP></DE></FR> <FR><NU>K<SUB>b</SUB></NU><DE>B</DE></FR><FENCE>1+<FR><NU>I</NU><DE>K<SUB>I</SUB></DE></FR>+<FR><NU>I<SUP>n</SUP></NU><DE>K<SUB>ii</SUB></DE></FR></FENCE></FENCE>
where v, Vm, A, B, I, Ka, Kb, Kia, Ki, Kii, Kiii, and Kiv represent initial velocity; maximal velocity; the concentrations of Mg2+, Fru-1,6-P2, and AMP; the Michaelis constants for Mg2+ and Fru-1,6-P2; the dissociation constant for Mg2+; and the dissociation constants for AMP from the enzyme·AMP, enzyme·AMP·AMP, enzyme·Fru-1,6-P2·AMP, and enzyme·Fru-1,6-P2·AMP·AMP complexes, respectively. n represents the Hill coefficient for AMP. When n = 2, the binding of AMP to FBPase is cooperative; on the other hand, there is no cooperativity when n = 1. In the case of the wild-type, Lys71 right-arrow Ala, Asp68 right-arrow Glu, and Asn64 right-arrow Gln FBPases, the kinetic data (similar to those shown in Fig. 1) fit best to Equation 1 when n = 2. The "goodness of fit" was 4% when n = 2 as opposed to 11% when n = 1. 


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Fig. 1.   Plot of reciprocal of initial velocity in arbitrary fluorescent units versus reciprocal of [Mg2+]2 for Lys71,72 right-arrow Met FBPase. The concentrations of AMP used were 0 (bullet ), 6.6 (open circle ), 13.3 (black-square), and 26.6 (square ) µM. The coupled fluorescence assay was used at 25 °C in 50 mM Tris buffer, pH 7.5, containing 0.1 M KCl and 20 µM Fru-1,6-P2. The lines are theoretical based upon Equation 1 when n = 2, and the points were experimentally determined.

Fig. 2 illustrates double-reciprocal plots of 1/velocity versus 1/[Mg2+]2 at various fixed concentrations of AMP for the Asp74 right-arrow Glu mutant of FBPase. The family of lines intersect to the left of the 1/velocity axis in Fig. 2. A similar result was found with the Asn64 right-arrow Ala mutant (data not shown). The data for both mutants are indicative of noncompetitive inhibition by AMP relative to Mg2+ and are consistent with Equation 2,
<FR><NU>1</NU><DE>v</DE></FR>=<FR><NU>1</NU><DE>V<SUB>M</SUB></DE></FR><FENCE>1+<FR><NU>I<SUP>n</SUP></NU><DE>K<SUB>ii</SUB></DE></FR>+<FR><NU>K<SUB>a</SUB></NU><DE>A<SUP>2</SUP></DE></FR><FENCE>1+<FR><NU>I<SUP>n</SUP></NU><DE>K<SUB>i</SUB></DE></FR></FENCE></FENCE> (Eq. 2)
where v, VM, A, I, Ka, Ki, and Kii are defined as above, and n represents the Hill coefficient for AMP. The data for Asp74 right-arrow Glu and Asn64 right-arrow Ala all fit best to Equation 2 when n = 1. The goodness of fit is 5 and 6% when n = 1 for Asp74 right-arrow Glu and Asn64 right-arrow Ala, respectively, and >18% when n = 2. 


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Fig. 2.   Plot of reciprocal of initial velocity in arbitrary fluorescent units versus reciprocal of [Mg2+]2 for Asp74 right-arrow Glu FBPase. The concentrations of AMP used were 0 (bullet ), 2 (open circle ), 4 (black-square), and 10 (square ) µM. The coupled fluorescence assay was used at 25 °C in 50 mM Tris buffer, pH 8.5, containing 0.1 M KCl and 20 µM Fru-1,6-P2. The lines are theoretical based upon Equation 2 when n = 1, and the points were experimentally determined.

The double-reciprocal plots of 1/velocity versus 1/[Fru-1,6-P2] at various fixed concentrations of AMP for the wild-type, Lys71 right-arrow Ala, Lys71,72 right-arrow Met, and Asp68 right-arrow Glu enzymes gave a family of lines intersecting to the left of the 1/velocity axis (data not shown). Consequently, the mechanism of AMP inhibition for the Lys71 right-arrow Ala, Lys71,72 right-arrow Met, Asp68 right-arrow Glu, and wild-type FBPases is noncompetitive. The Ki values for the Lys71,72 right-arrow Met and Lys71 right-arrow Ala mutants increased 175- and 10-fold relative to the wild-type enzyme, respectively. AMP inhibition relative to Fru-1,6-P2 for Asp74 right-arrow Glu, Asn64 right-arrow Gln, and Asn64 right-arrow Ala gave a family of parallel lines (Fig. 3), indicating uncompetitive inhibition. Equation 3 with n = 1 best accounts for the data,
<FR><NU>1</NU><DE>v</DE></FR>=<FR><NU>1</NU><DE>V<SUB>M</SUB></DE></FR><FENCE>1+<FR><NU>I<SUP>n</SUP></NU><DE>K<SUB>i</SUB></DE></FR>+<FR><NU>K<SUB>a</SUB></NU><DE>A</DE></FR></FENCE> (Eq. 3)
where v, VM, A, I, n, Ka, and Ki represent initial velocity, maximal velocity, the concentration of Fru-1,6-P2, the concentration of AMP, the Hill coefficient, the Michaelis constants for Fru-1,6-P2, and the dissociation constant for AMP from the enzyme·AMP complex, respectively. The goodness of fit was <6%. Thus, cooperativity for AMP inhibition is lost in addition to the change of the kinetic mechanism.


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Fig. 3.   Plot of reciprocal of initial velocity in arbitrary fluorescent units versus reciprocal of Fru-1,6-P2 for Asn64 right-arrowAla FBPase. The concentrations of AMP used were 0 (bullet ), 3.3 (open circle ), 6.6 (black-square), and 10 (square ) µM. The coupled fluorescence assay was used at 25 °C in 50 mM Tris buffer, pH 8.5, containing 0.1 M KCl and 5 mM Mg2+. The lines are theoretical based upon Equation 3 when n = 1, and the points were experimentally determined.

    DISCUSSION
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Abstract
Introduction
Results
Discussion
References

Amino acid sequences of all known mammalian FBPases are well conserved, particularly in segment 52-72, where 10 amino acids are highly conserved, suggesting an important functional or structural role. In all published crystal structures of FBPase (9-13), however, little or no electron density is present for residues 52-72, which is indicative of conformational disorder or proteolytic damage. The latter possibility is unlikely as microsequencing detected no proteolysis (10). Reduced kcat values for the Asp74 right-arrow Ala, Asp74 right-arrowAsn, and Asp74 right-arrow Glu FBPases by 50,000-, 2,000-, and 20-fold, respectively, along with the shift in the pH of optimum activity from neutral to alkaline for the latter mutant, demonstrate the importance of position 74 in catalysis. Given the sensitive nature of position 74, conformational changes in the adjacent segment 52-72 could leverage significant alterations in the catalytic properties of FBPase. Mutations Asn64 right-arrow Ala, Asn64 right-arrow Gln, and Asp68 right-arrow Glu, for instance, change the pH of optimum activity from neutral to alkaline and decrease kcat values from 3- to 4-fold relative to that of the wild-type enzyme, and the double mutation Lys71,72 right-arrow Met elevates the Ki for AMP nearly 175-fold. The activity profiles of the Asp74 right-arrow Glu, Asn64 right-arrow Ala, Asn64 right-arrow Gln, and Asp68 right-arrow Glu mutants are very similar to those obtained with the proteolyzed enzyme in which residues 1-64 and 1-25 are missing (32-34).

The analysis above is strengthened by the crystal structure2 (2.3-Å resolution, R-factor = 0.165, R-free = 0.24) of recombinant wild-type porcine liver FBPase with the products Fru-6-P, Pi, and Zn2+, which reveals strong electron density for loop 52-72 and a significant network of hydrogen bonds involving Asp68 , Asn64, and other residues of the active site. In the proposed catalytic mechanism of hydrolysis of Fru-1,6-P2 (12, 13), a water molecule, bound to a metal cation, is activated for a nucleophilic attack of the 1-phosphate. The side chains of Asp74 and Glu98, relative to the attacking water and to the metal ion at the "catalytic" site (called metal site 2 in crystal structures of FBPase) (Fig. 4), are in position to act as general base catalysts. The distance between OD2 of Asp74 and OE2 of Glu98 and the cation at site 2 is ~3.3 Å, but neither side chain is in the inner coordination sphere of that cation. Replacement of Glu98 by glutamine resulted in a 1,600-fold reduction in kcat relative to wild-type FBPase (35). The Asp74 right-arrow Asn mutation caused a decrease in catalytic rate of the same order of magnitude, whereas the Asp74 right-arrow Ala mutation resulted in almost the total loss of catalytic activity. Thus, Asp74 and Glu98 may act in concert to remove a proton from the attacking water molecule.


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Fig. 4.   Stereoview of the location of Asp74 and Glu98 relative to the metal ion at site 2, the 1-phosphate group, and the attacking water molecule. Wat, water; F6P, fructose 6-phosphate.

Asn64 and Asp68 are also in the active site (Fig. 5), with the former hydrogen bonding with Glu97 and Glu98 and the latter with Arg276. Evidently, Asn64 maintains Glu97 and Glu98 in proper orientations for metal binding and the activation of the attacking water molecule. The above is consistent with the reduction in the Hill coefficient for Mg2+ and the 50-fold decrease in affinity for Mg2+, exhibited by the Asn64 right-arrow Ala and Asn64 right-arrow Gln mutants relative to wild-type FBPase. Mutation of Arg276 to methionine reduces activity by 2,000-fold, abolishes Mg2+ cooperativity, and alters the kinetic mechanism of FBPase (36). Evidently, the Asp68-Arg276 hydrogen bond is a determinant for many of the kinetic properties of FBPase; its loss could destabilize loop 52-72 and impair the catalytic function of Asp74.


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Fig. 5.   Stereoview of the active-site area around Asn64 and Asp68. F6P, fructose 6-phosphate.

AMP induces a modest structural change in the helix (H2) immediately preceding loop 52-72, and in all AMP-bound complexes of FBPase, loop 52-72 is disordered (13, 28). The new crystal structure has an ordered loop in the absence of AMP. Hence, AMP could inhibit FBPase by the disruption of interactions between loop 52-72 and the active site. As Asn64 and Asp68 probably stabilize metal binding to the active site, the observed competition between AMP and Mg2+ in kinetics and NMR investigations may stem from the displacement of loop 52-72 from the active site due to the AMP-induced perturbation of helix H2.

The 175- and 10-fold increases in Ki of AMP inhibition with respect to Fru-1,6-P2 for the Lys71,72 right-arrow Met and Lys71 right-arrow Ala enzymes, respectively, could originate from the destabilization of loop 52-72 in its disordered, AMP-induced conformation. AMP may be less effective in the displacement of loop 52-72 of the double mutant (in which positions 71 and 72 are methionine instead of lysine) because of the unfavorable thermodynamics of exposing two methionyl side chains to the solvent. Indeed, the double mutant apparently exhibits preference toward the active R-state, relative to the less active or inactive T-state. Its kcat/Km increases 3-fold, and its Ka for Mg2+ decreases 2-fold, whereas its Ki for AMP and Fru-2,6-P2 increases 175- and 7-fold, respectively. In fact, the double mutant may represent the kinetic properties of FBPase locked into the R-state.

Mutations that bring about a change in the kinetic mechanism of FBPase from competitive to noncompetitive (AMP inhibition relative to Mg2+) are possible in the context of a steady-state random mechanism (16, 17) and now can be understood in terms of a conformational mechanism. To a first approximation, loop 52-72 is the instrument by which AMP exerts its allosteric effect on the active site. In the wild-type system with a fully functional loop, AMP and Mg2+ (presumably at site 2) are probably antagonists with respect to the conformation that they stabilize for loop 52-72. Hence, in wild-type FBPase, loop 52-72 strongly couples the AMP- and Mg2+-binding sites. In some mutants of loop 52-72, however, the strong coupling of the AMP and the cation site is diminished; AMP binds and perturbs the metal at site 2, thereby causing inhibition, but is no longer completely effective in displacing the cation from a catalytically productive association with the active site.

A rational basis for an uncompetitive mechanism is more challenging, however. Four separate mutations (Asp74 right-arrow Glu, Asn64 right-arrow Ala, Asn64 right-arrow Gln, and Arg49right-arrow Cys) change AMP inhibition with respect to Fru-1,6-P2 from noncompetitive to uncompetitive. The uncompetitive mechanism implies that AMP binds as an inhibitor only when Fru-1,6-P2 is productively bound at the active site. Hence, these mutations may stabilize a conformation of loop 52-72 that does not favor the binding of AMP (as an inhibitor), but permits the association of Fru-1,6-P2 with the active site. Productively bound Fru-1,6-P2 could then induce a conformational change in loop 52-72 that re-establishes the coupling between the AMP and the substrate-binding site. A more detailed explanation of the above phenomenon, however, must await crystallographic and ligand binding studies of mutant FBPases.

    FOOTNOTES

* This work was supported by Research Grant NS 10546 from the National Institutes of Health and Grant MCB-9603595 from the National Science Foundation. This is Journal Paper J-17874 of the Iowa Agriculture and Home Economics Experiment Station (Ames, IA), Project 3191.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, 1210 Molecular Biology Bldg., Iowa State University, Ames, IA 50011. Tel.: 515-294-6116; Fax: 515-294-0453; E-mail: hjfromm{at}iastate.edu.

1 The abbreviations used are: FBPase, fructose-1,6-bisphosphatase; Fru-1,6-P2, fructose 1,6-bisphosphate; Fru-2,6-P2, fructose 2,6-bisphosphate.

2 J.-Y. Choe, H. J. Fromm, and R. B. Honzatko, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Results
Discussion
References

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