©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glycine 122 Is Essential for Cooperativity and Binding of Mg to Porcine Fructose-1,6-bisphosphatase (*)

(Received for publication, June 10, 1994; and in revised form, October 21, 1994)

Rulin Zhang (1) Lirong Chen (1) Vincent Villeret (2) Herbert J. Fromm (1)(§)

From the  (1)Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011 and (2)Gibbs Chemical Laboratory, Harvard University, Cambridge, Massachusetts 02138

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Site-directed mutagenesis of an amino acid residue in the substrate binding site of porcine fructose-1,6-bisphosphatase was carried out based on the crystal structure of the enzyme (Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W. N.(1993) Biochemistry 32, 1844-1857). A mutant enzyme form of fructose-1,6-bisphosphatase, G122A, was purified and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectrometry (CD), and initial rate kinetics. There were no discernible differences between the secondary structures of the wild-type and the mutant enzyme on the basis of CD data. Altering Gly-122 to alanine caused a significant decrease in the enzyme's activity and affinity for Mg. The k for this mutant enzyme was only about 5% of that of wild-type fructose-1,6-bisphosphatase, and the K for Mg was about 20-fold higher than that of the wild-type enzyme. The K for AMP was increased 77-fold in the case of the mutant enzyme; however, the Hill coefficient was unaltered. Most importantly, it was observed that replacement of Gly-122 with alanine caused the total loss of cooperativity for Mg. It is concluded that Gly-122 is essential for Mg cooperativity and important for binding of Mg and AMP as well as for enzyme activity.


INTRODUCTION

It is well known that fructose-1,6-bisphosphatase (FBPase) (^1)is a crucial step in the regulation of gluconeogenesis(1, 2, 3) . The enzyme catalyzes the hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P(2)) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate and exhibits a rapid equilibrium random Bi Bi kinetic mechanism(4) . The reaction is competitively inhibited by fructose 2,6-bisphosphate (Fru-2,6-P(2)) and noncompetitively inhibited by AMP(4) . These two compounds are also involved in the activation of phosphofructokinase(5) . Therefore, it is thought that Fru-2,6-P(2) plays crucial roles in the regulation of glycolysis and gluconeogenesis. AMP is also an important element in the regulation of FBPase because it acts synergistically with Fru-2,6-P(2) to inhibit FBPase(5) . The role of AMP is thought to prevent divalent metal binding to FBPase, and divalent cations such as Mg and Mn are absolutely required for FBPase activity(4, 6) . One of the functions of Fru-2,6-P(2) is to keep AMP on the enzyme (make it stickier) (7) , thus enhancing the action of AMP.

FBPase has long been recognized to be a metal-requiring enzyme(8) . Univalent cations can activate the enzyme(9) , but divalent cations are absolutely required for enzyme activity(8) . The crystallographic structure, reported by the Lipscomb group(10) , suggests that two divalent cations are bound at the metal-binding site. Benkovic et al.(11, 12) suggested that catalysis requires the sequential addition of metal and substrate in the order, structural metal, substrate, and catalytic metal to form a catalytically competent quaternary complex of enzyme-M(1)-M(2)-Fru-1,6-P(2). Nimmo and Tipton (13, 14) showed that pH could affect the divalent metal kinetics of bovine liver FBPase, i.e. the plots of velocity versus Mg are sigmoidal at neutral pH, but they are hyperbolic at pH 9.6. Furthermore, cooperativity with respect to metal ion binding and its activation of the mammalian FBPase reaction has long been recognized(11, 13, 14, 15) . Recently, Chen et al.(16) and El-Maghrabi et al.(17) carried out site-directed mutagenesis experiments in the metal binding sites of mammalian FBPase. Their investigations support the suggestion that there are two metal binding sites associated with FBPase.

FBPase from mammalian liver and kidney, two highly gluconeogenic tissues, is a homotetramer(18) . Alteration of the amino acid residues in the metal binding site can affect not only the cooperativity and ligand affinity of metal ions but also the enzyme's affinity for AMP (16) . These observations suggest that metal binding sites and AMP binding sites may somehow communicate with each other.

X-ray diffraction results of FBPase have pinpointed a number of amino acid residues that are associated with substrate binding(10) . These findings (10) suggest that the main chain NH group of the Gly-122 residue forms a bifurcated hydrogen bond to the ester oxygen and a 1-phosphoryl oxygen of the substrate. Thus, this residue is postulated to have the function of fixing the 1-phosphoryl group in the required position for metal binding and catalysis.

To gain some insight into the residues involved in substrate binding and catalysis, we have prepared a mutant of FBPase at position 122 by site-directed mutagenesis and have studied its properties. In this study, we report that replacement of Gly-122 with alanine causes the total loss of cooperativity of Mg and very significant decreases in enzyme activity, AMP binding, and affinity for metal ions.


EXPERIMENTAL PROCEDURES

Materials

NADP, fructose 1,6-bisphosphate (Fru-1,6-P(2)), fructose 2,6-bisphosphate (Fru-2,6-P(2)), AMP, Hepes, and Tris were purchased from Sigma. Glucose-6-phosphate dehydrogenase and phosphoglucoisomerase were from Boehringer Mannheim. Distilled deionized water was used in all experiments. All other reagents were of the highest purity available commercially. Recombinant and mutant forms of porcine liver FBPase were prepared and purified as described elsewhere (19) with slight modifications. Mutant forms of the enzyme were obtained in yields comparable to the wild-type enzyme. Porcine liver and kidney FBPase are identical in their primary sequences(19) . There was no measurable endogenous expression of wild-type FBPase in the absence of added isopropyl-beta-D-thiogalactopyranoside.

Mutant of Fructose-1,6-bisphosphatase

A mutant of recombinant porcine liver FBPase, G122A, was obtained by site-directed mutagenesis. A mutagenic oligonucleotide primer, 5`-CCC-CTC-GAT-GCA-TCG-TCG-AAC-3`, was synthesized by the beta-cyanoethylphosphoramidite method at the Nucleic Acid Facility at Iowa State University. The codon GCA was used to mutate G122A. BamHI/SphI fragments encoding FBPase from pEt-11a were ligated into a previously digested PUC118 plasmid. The mutagenesis was done by using single-stranded DNA from recombinant pUC118 plasmid as the template and synthesized oligonucleotide as primer. The oligonucleotide-directed in vitro mutagenesis procedure was performed as described by Nakamaye and Eckstein(20) . Mutagenesis was verified by dideoxy chain termination sequencing(21) . The BamHI/XbaI fragments encoding the mutations were ligated back into previously digested pEt-11a expression vector. pEt-11a was used to transform Escherichia coli strain BL21 (DE3).

Protein Assay

Total protein was measured by the Bio-Rad method with bovine serum albumin (from Sigma) as a standard.

Circular Dichoism Spectrometry

CD studies on the wild-type and mutant form of FBPase were carried out in 50 mM Tris-HCl buffer (pH7.5) at room temperature in an AVIV CD spectrometer model 62DS kindly supplied by Dr. Earl Stellwagen at the University of Iowa. Samples were placed in a 1-mm cuvette, and data points were collected from 200 to 260 nm in 0.5-nm increments. Each spectrum was calibrated to remove the background of the buffer and smoothed by using a program in the computer of the spectrometer.

Kinetic Studies

FBPase activity during purification and the specific activity of pure enzyme were measured by using the phosphoglucoisomerase/glucose-6-phosphate dehydrogenase coupled spectrophotometric assay(22) . All other kinetic experiments were done using a fluorometric assay (4) at pH 7.5 (50 mM Hepes buffer) and 24 °C. The initial rate data were analyzed for kinetic mechanisms by using a Minitab language program with an alpha value of 2.0 (4) . Cooperativity was evaluated by using both the ENZFITTER program (23) and the Minitab program.


RESULTS

Enzyme Quality

The purity of wild-type recombinant FBPase and the G122A mutant of porcine FBPase was evaluated by SDS-PAGE. The results are shown in Fig. 1. It was found that the proteins are greater than 95% pure using the criterion of electrophoresis. In addition, the single band exhibiting a molecular mass of approximately 37 kDa in each lane indicates that the proteins had not undergone discernible degradation.


Figure 1: SDS-PAGE analysis of purified wild-type and mutant G122A porcine FBPase. All samples were run on a 12% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250. Lanes 1 and 4, protein standard; lane 2, G122A; lane 3, wild-type. Molecular mass of protein standards: A, 66 kDa; B, 45 kDa; C, 36 kDa; D, 29 kDa; E, 24 kDa; F, 20 kDa; G, 14 kDa.



Secondary Structure Analysis

The secondary structures of recombinant wild-type and the G122A mutant of FBPase were analyzed by CD spectrometry. The purpose of this study was to determine whether localized or global structural alterations were induced in the mutant. The CD spectral data showed that the spectrum of the mutant was essentially superimposable on that of the wild-type enzyme (data not shown). These results suggest that no major conformational changes occurred in FBPase when Gly-122 was mutated to alanine by using CD as a criterion of secondary protein structure.

Initial Rate Studies

Table 1shows the kinetic parameters for wild-type and the G122A mutant of FBPase. The data were obtained by measuring the initial rate at saturating Mg or Fru-1,6-P(2) concentrations. From Table 1, it can be seen that replacement of Gly-122 with alanine caused a significant decrease in enzyme activity. The k of the G122A mutant of FBPase was only about 5% of that of the wild-type enzyme, whereas the K(m) for Fru-1,6-P(2) of this mutant form of the enzyme did not exhibit a large alteration, i.e. only a 3-fold increase compared with that of the wild-type enzyme.



MgIon Activation

The Hill coefficient and K(a) for Mg of the wild-type and mutant form of FBPase were determined at their saturating substrate concentrations; i.e. wild-type enzyme at 12 µM and G122A at 60 µM. As expected, the Hill coefficient of wild-type FBPase for Mg was about 2.0, and Mg activation of FBPase was sigmoidal (data not shown). These results indicate that Mg activation of FBPase exhibits cooperativity. This is consistent with previous reports (13, 14, 15) . It was observed, however, that altering Gly-122 to alanine caused the Hill coefficient for Mg to decrease to about 1, i.e. the Mg activation of FBPase was hyperbolic rather than sigmoidal (data not shown). These results indicate that replacement of Gly-122 with alanine results in a total loss of Mg cooperativity. Furthermore, this mutant form of FBPase exhibited a significant decrease in its affinity for Mg, i.e. the apparent K(a) for Mg of this mutant form of the enzyme is about 20-fold greater than that of the wild-type enzyme.

Kinetics of Fru-2,6-P(2) Inhibition

It is well known that Fru-2,6-P(2), like the substrate Fru-1,6-P(2), binds at the active site of FBPase(24, 25, 26) . Since one is a substrate and the other a competitive inhibitor, FBPase must have the ability to distinguish between these two molecules. The K(i) for Fru-2,6-P(2) and the K(m) for Fru-1,6-P(2) of the G122A mutant did increase slightly (about 3-fold) relative to wild-type FBPase. These results indicate that the Gly-122 residue is not important in permitting FBPase to discriminate between the substrate and inhibitor. In other words, it is not directly involved in Fru-2,6-P(2) inhibition of FBPase.

The effects of Fru-2,6-P(2) inhibition on Mg with the G122A mutant form of FBPase were also studied. The K(i) increased with the increase of Mg concentration as described previously(24) . At 5 mM Mg, the K(i) for Fru-2,6-P(2) is about 22 µM.

Inhibition by AMP

AMP is known to be an allosteric inhibitor of FBPase(4, 27) , and the role of AMP is to remove divalent metal ions from FBPase(7) . When the Gly-122 residue was converted to alanine, the K(i) for AMP of this mutant increased about 77-fold relative to that of the wild-type enzyme (Table 1). This is a reasonable finding in light of the fact that, with this mutant, the K(a) for Mg also increased about 20-fold compared with that of the wild-type enzyme. It is believed that the AMP and metal binding sites can somehow communicate with each other, presumably though conformational changes(4, 7, 26) .

The binding of AMP to wild-type FBPase is known to exhibit cooperativity(27) . The kinetic data for AMP inhibition with the two mutant forms of FBPase gave excellent fits to a cooperative model (28) in which the Hill coefficient is 2.0 (data not shown). Thus, the Gly-122 residue is not directly involved in the cooperativity of AMP binding to FBPase.

Kinetic Studies in the Absence of Inhibitors

To confirm the finding that altering Gly-122 to alanine results in the total loss of cooperativity for Mg with FBPase, we studied the kinetics of the FBPase reaction in the absence of inhibitors. Fig. 2shows the double-reciprocal plot of initial velocity against the concentration of Mg at different fixed levels of Fru-1,6-P(2). When Fru-1,6-P(2) concentration was varied at different fixed concentrations of Mg, a family of lines intersecting to the left of the 1/velocity axis was obtained for double-reciprocal plots (data not shown). The data in Fig. 2gave excellent fits to where n = 1 but fit poorly when n = 2. The form of is:


Figure 2: Plot of reciprocal of initial velocity in arbitrary fluorescent units against reciprocal of Mg concentration for G122A FBPase. The concentrations of Fru-1,6-P(2) are 25 µM (times), 15 µM (box), 10 µM (), 6 µM (+), and 3 µM (). The lines are theoretical based on when n = 1, and the points are experimentally determined.



where V, V(m), A, B, K(a), K(b), and K represent the initial velocity, maximum velocity, the concentration of free Mg, the concentration of free Fru-1,6-P(2), the Michaelis constant for Mg, the Michaelis constant for Fru-1,6-P(2), and the dissociation constant for Mg, respectively; n represents the Hill coefficient for Mg with FBPase. When n = 1, there is no cooperativity; when n = 2, the binding of Mg to FBPase is cooperative, with a Hill coefficient of 2. is the fundamental initial rate equation for the sequential kinetic mechanism shown in . (^2)

On the other hand, the kinetic data in the absence of inhibitors with wild-type FBPase (shown in Fig. 3) gave excellent fits to when n = 2 and did not fit well to when n = 1. Note that the only difference in the rate equations between the G122A mutant enzyme and the wild-type enzyme is the term n. The data shown in Fig. 3are consistent with previous reports that show that binding of divalent metal ions to FBPase exhibits cooperativity(11, 13, 14, 15) , whereas the findings illustrated in Fig. 2exclude the possibility of cooperativity for Mg with the G122A mutant enzyme. Had Mg ions bound cooperatively to FBPase, the data would have fit to much better when n = 2 than when n = 1. On the basis of these results and the Hill coefficient obtained for G122A, it is reasonable to conclude that the Gly-122 residue is required for the cooperativity observed for Mg with FBPase.


Figure 3: Plot of reciprocal of initial velocity in arbitrary fluorescent units versus reciprocal of [Mg]^2 for wild-type FBPase. The concentrations of Fru-1,6-P(2) are 9.0 µM (times), 5.0 µM (box), 3.0 µM (), 2.0 µM (+), and 1.2 µM (), The lines are theoretical based on when n = 2, and the points are experimentally determined.




DISCUSSION

The central finding of this report involves the complete loss of cooperativity for Mg when FBPase Gly-122 is mutated to alanine. It is known that mammalian FBPase is a divalent metal-requiring enzyme (8) and that the Hill coefficient is 2.0 at neutral pH (13, 14) but only 1.0 at alkaline pH(4, 13, 14) . Crystallographic data (10) suggest that two metal ions such as Mn and Zn are associated with the enzyme; however, with Mg, only one metal ion was found to bind FBPase.

Replacement of Gly-122 with alanine caused a 19-fold decrease in enzyme activity and a significant decrease of the enzyme's affinity for Mg. This is expected because the main chain NH group of Gly-122 is thought to act as a donor to form a bifurcated hydrogen bond with the O1 oxygen of the fructofuranoside ring and an oxygen of the 1-phosphoryl group of the substrate(10) . Therefore, this bifurcated hydrogen bond is believed to anchor the 1-phosphoryl group in the required position for both metal binding and catalysis(10) . In addition, the bifurcated hydrogen bond may contribute to catalysis by making the phosphoester bond weaker such that nucleophilic attack by OH on the 1-phosphorus atom would be facilitated.

A significant finding associated with this report is that the Hill coefficient of the G122A mutant FBPase for Mg is about 1.0. This conclusion is alluded to from kinetic data obtained in the absence of inhibitors. These results suggest that altering Gly-122 of FBPase to alanine essentially causes the complete loss of Mg cooperativity. Furthermore, the affinity of this mutant form of FBPase for metal ions decreased markedly. From these results, we conclude that the Gly-122 residue of mammalian FBPase is essential for both cooperativity and binding of Mg to the enzyme. An understanding of this important finding requires further investigations (e.g. solution of the crystal structure of G122A FBPase); however, modeling studies were undertaken to provide some insight into the effect of substituting alanine for glycine at position 122. Gly-122 is located in a 3-residue loop between strand B3 (residues 113-118) and helix H4 (residue 123-127). If one compares the various crystallographic complexes of FBPase, it is obvious that residues 121-127 are subject to conformational changes during catalysis(27) : root mean square deviation for backbone atoms of these residues ranges between 0.29 and 0.70 Å (between native FBPase and FBPase complexed with Fru-1,6-P(2), with alpha-methyl-Fru-1,6-P(2), or with Fru-6-P, respectively). Most interestingly, Gly-122 is at a hinge point between strand B3 and helix H4.

The active site region of FBPase, based on the crystal structure of the enzyme(10) , is shown in Fig. 4. If Gly-122 (Fig. 4A) is replaced by Ala-122 (Fig. 4B), the beta-methyl group is located in the Fru-1,6-P(2) binding site, close to the C1 carbon of the substrate or its analog. Ala-122 can reduce the flexibility of the 122-127 region or lock the enzyme in a conformation which suppresses cooperativity for Mg. In addition, binding of Mg causes repositioning of the 1-phosphoryl group of Fru-1,6-P(2) or its analog, alpha-methyl Fru-1,6-P(2)(27) . Based on these structural studies, the following events can be proposed: when the substrate binds, the 1-phosphoryl group adopts an initial conformation. The binding of Mg in the metal site moves the 1-phosphoryl group into a position that allows cleavage of the P-O bond, yielding the formation of Fru-6-P. The beta-methyl group of Ala-122 can lock the 1-phosphoryl group in a defined conformation when substrate binds and this affects the hinge movement of helix H4, disrupting some interactions that are required for signal transmission between monomers.


Figure 4: A, stereo model of the active site of FBPase as defined in the crystallographic study of porcine FBPase complexed with the substrate analogue alpha-methyl-Fru-1,6-P(2) and Mg(10) . For clarity the binding residues of the 6-phosphate group have been omitted. The backbone NH of Gly-122 is hydrogen bonded to oxygens O1 and O2 of the 1-phosphate group (2.8 and 2.5 Å, respectively). The backbone CO of Gly-122 interacts with the backbone NH of Ser-124 and Asn-125 and also with the side chain NH of Asn-125. The Mg ion is coordinated to the O2 oxygen of the 1-phosphate group and to residues Glu-97, Asp-118, and Glu-280. B, stereo model of the catalytic site showing the effect of the alanine substitution at position 122. If the position of backbone atoms are kept as in the crystallographic complex shown in A, the beta-methyl group of Ala-122 is only 3.4 Å from the C1 carbon of alpha-methyl-Fru-1,6-P(2) and only 2.4 Å from the side chain of Asn-125. Slightly different positions of residues 122-125 are required in order to accommodate the presence of a beta-methyl group in residue 122. The energy-minimized model (B) shows that interactions of the backbone NH of Ala-122 with the oxygen O1 and O2 of the 1-phosphate group of the substrate analog are maintained. The beta-methyl group of Ala-122 is 4 Å from the C1 carbon of the substrate analog and 3.8 Å from the side chain of Asn-125.



It is well documented that the mechanism of regulation of FBPase involves AMP and Fru-2,6-P(2), which are potent synergistic inhibitors of the enzyme(28, 29, 30, 31) . Fromm and co-workers (4, 7, 25) have shown that AMP and metal ions are mutually exclusive in their binding to the enzyme. X-ray diffraction studies suggested that the metal binding sites are far from the allosteric sites for AMP(32) . The data shown in Table 1indicate that the K(i) for AMP of the G122A mutant is much higher than that of the wild-type enzyme. It is obvious that converting Gly-122 to alanine not only affects the enzyme's affinity for divalent metal ions, it also affects the enzyme's affinity for AMP. These results are consistent with previous suggestions that the metal binding sites and the allosteric sites for AMP can somehow communicate with each other(4, 7, 28) . When AMP binds to the enzyme, the coordination sphere of Mg is affected as follows; side chains of residues Glu-97, Asp-118, and Asp-121 belonging to strand B3 move from their R state conformation 1.79 Å (OE1), 1.71 Å (OD2), and 4.10 Å (OD1). Residues Lys-274 and Glu-280 are also affected upon binding(27) . From our results, Gly-122, located in the hinge region between strand B3 and helix H4, seems to be directly involved in the signal transmission pathway for communication between metal binding sites and allosteric sites.

The binding of AMP to wild-type FBPase exhibits cooperativity(28) . Site-directed mutagenesis at the AMP binding site led to a total loss of cooperativity for AMP with FBPase(28) . The kinetic data of AMP inhibition with G122A FBPase gave excellent fits to a cooperative model. This finding suggests that the Gly-122 residue may not be involved in the cooperativity of AMP, although it is absolutely required for Mg cooperativity.

It is believed that Fru-2,6-P(2) has two functions in regulating FBPase. One is that it is a potent competitive inhibitor of the substrate and competes with Fru-1,6-P(2) at the enzyme's active site(24, 25, 31, 33) . The second function is to enhance the effect of AMP by making AMP ``stickier'' to the enzyme(7) . AMP is known to be a potent competitive inhibitor of Mg(28) and to prevent divalent metal ion binding to the enzyme, i.e. the two ligands are mutually exclusive in their binding to FBPase.

The findings of this report demonstrate that Gly-122 is essential for the well established cooperativity and divalent metal binding affinity of porcine liver FBPase. The x-ray diffraction investigations of FBPase by the Lipscomb group (34) has provided a basis for AMP cooperativity. The enzyme functions as a dimer of dimers in which R and T states exist. In the case of AMP binding, the T state is induced, whereas the substrate adds to the R state of the enzyme. This model provides a rational explanation for a Hill coefficient of 2 for AMP. On the other hand, an explanation of divalent metal ion binding seems to be less well understood. Two binding sites for either Mn or Zn have been demonstrated; however, the x-ray diffraction studies can pinpoint only a single Mg ion per monomer. In addition, kinetic studies at pH 9.6 (4) accord with the latter finding, i.e. only 1 Mg per monomer and no cooperativity. It is well established that at neutral pH, Mg binding is cooperative and the Hill coefficient is 2(11, 13, 14, 15) . The data shown in Fig. 3confirm this finding. The origin of Mg binding cooperativity remains unclear even though the metal binding sites of FBPase were markedly altered by site-directed mutagenesis(16) . Although the k of the mutant FBPase was decreased by at least 3 orders of magnitude relative to the wild-type enzyme, the cooperativity of metal ions did not show significant alteration(16) . These results suggest, but do not provide conclusive proof, that divalent metal ion cooperativity is inter- rather than intrasubunit. The results of the present study may lead to a better understanding of the basis of the cooperativity phenomenon for metal ion binding to FBPase at the molecular level when the three-dimensional structure of the Gly-122 mutant becomes available.


FOOTNOTES

*
This research was supported in part by Research Grants NS 10546 and GM06920 from the National Institutes of Health, United States Public Health Service and by Grant MCB-9218763 from the National Science Foundation. This is Journal Paper J-15979 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project 2575. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed.

(^1)
The abbreviations used are: FBPase, fructose-1,6-bisphosphatase; Fru-1,6-P(2), fructose 1,6-bisphosphate; Fru-6-P, fructose 6-phosphate; Fru-2,6-P(2), fructose 2,6-bisphosphate.

(^2)
was alluded to on the basis of stochastic considerations. The curves shown in Fig. 2and Fig. 3converge at a common point to the left of the 1/velocity axis, respectively. At pH 7.5 Cd is a competitive inhibitor of Mg and a noncompetitive inhibitor of Fru-1,6-P(2), and Fru-2,6-P(2) is a competitive inhibitor of Fru-1,6-P(2) and a noncompetitive inhibitor of Mg. Similar results were obtained at pH 9.6(4) .


ACKNOWLEDGEMENTS

We thank Professor William N. Lipscomb for informative and helpful discussions.


REFERENCES

  1. Underwood, A. H., and Newsholme, E. A. (1965) Biochem. J. 95, 767-774 [Medline] [Order article via Infotrieve]
  2. McGilvay, R. W. (1964) in Fructose-1,6-diphosphatase and Its Role in Gluconeogenesis (McGilvay, R. W., and Pogell, B. M., eds) pp. 3-10, Port City Press Inc., Baltimore
  3. Krebs, H. A. (1963) in Advances in Enzyme Regulation (Weber, G., ed) Vol. 1, pp. 385-400, Pergamon Press Ltd., London
  4. Liu, F., and Fromm, H. J. (1990) J. Biol. Chem. 265, 7401-7406 [Abstract/Free Full Text]
  5. Hers, H.-G., and Van Schaftingen, E. (1982) Biochem. J. 206, 1-12 [Medline] [Order article via Infotrieve]
  6. Scheffler, J. E., and Fromm, H. J. (1986) Biochemistry 25, 6659-6665 [Medline] [Order article via Infotrieve]
  7. Liu, F., and Fromm, H. J. (1988) J. Biol. Chem. 263, 9122-9128 [Abstract/Free Full Text]
  8. Gomori, G. (1943) J. Biol. Chem. 148, 139-149
  9. Hubert, E., Villanueva, J., Gonzalez, A. M., and Marcus, F. (1970) Arch. Biochem. Biophys. 138, 590-597 [Medline] [Order article via Infotrieve]
  10. Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W. N. (1993) Biochemistry 32, 1844-1857 [Medline] [Order article via Infotrieve]
  11. Benkovic, S. J., and deMaine, M. M. (1982) Adv. Enzymol. Relat. Areas Mol. Biol. 53, 45-82 [Medline] [Order article via Infotrieve]
  12. Benkovic, P. A., Caperelli, C. A., deMaine, M. M., and Benkovic, S. J. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2185-2189 [Abstract]
  13. Nimmo, H. G., and Tipton, K. F. (1975) Eur. J. Biochem. 58, 567-574 [Abstract]
  14. Nimmo, H. G., and Tipton, K. F. (1975) Eur. J. Biochem. 58, 575-585 [Abstract]
  15. Tejwani, G. (1983) Adv. Enzymol. Relat. Areas Mol. Biol. 54, 121-193 [Medline] [Order article via Infotrieve]
  16. Chen, L., Remesh, H., Chen, M., and Fromm, H. J. (1993) Arch. Biochem. Biophys. 307, 350-354 [CrossRef][Medline] [Order article via Infotrieve]
  17. El-Maghrabi, M. R., Gidh-Jain, M., Austin, L. R., and Pilkis, S. J. (1993) J. Biol. Chem. 268, 9466-9472 [Abstract/Free Full Text]
  18. Marcus, F., Edelstein, I., Reardon, I., and Heinrikson, R. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7161-7165 [Abstract]
  19. Burton, V. A., Chen, M., Ong, W. C., Ling, T., Fromm, H. J., and Stayton, M. M. (1993) Biochem. Biophys. Res. Commun. 192, 511-517 [CrossRef][Medline] [Order article via Infotrieve]
  20. Nakamaye, K., and Eckstein, F. (1986) Nucleic Acids Res. 14, 9679-9898 [Abstract]
  21. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  22. Pontremodi, S., and Traniello, S. (1975) Methods Enzymol. 42, 369-374 [Medline] [Order article via Infotrieve]
  23. Leatherbarrow, R. J. (1987) Enzfitter: A Non-Linear Regression Data Analysis Program for the IBMPC , pp. 13-75, Elsevier Science Publishers BV, Cambridge, United Kingdom
  24. Ke, H., Thorpe, C. M., Seaton, B. A., Marcus, F., and Lipcomb, W. N. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1475-1479 [Abstract]
  25. Ke, H., Thorpe, C. M., Seaton, B. A., Lipscomb, W. N., and Marcus, F. (1990) J. Mol. Biol. 212, 513-539 [Medline] [Order article via Infotrieve]
  26. Scheffer, J. E., and Fromm, H. J. (1986) Biochemistry 25, 6659-6665 [Medline] [Order article via Infotrieve]
  27. Liang, J.-Y., Zhang, Y., Huang, S., Ke, H., and Lipscomb, W. N. (1992) Regulation of Protein by Ligands: Proceedings of the Robert A. Welch Foundation Conference on Chemical Research , October 26-27, 1992 , Houston, Texas
  28. Chen, M., Chen, L., and Fromm, H. J. (1994) J. Biol. Chem. 269, 5554-5558 [Abstract/Free Full Text]
  29. Van Schaftingen, E., Hue, L., and Hers, H. G. (1980) Biochem. J. 192, 887-895 [Medline] [Order article via Infotrieve]
  30. Van Schaftingen, E., Hue, L., and Hers, H. G. (1980) Biochem. J. 192, 897-901 [Medline] [Order article via Infotrieve]
  31. Van Schaftingen, E., and Hers, H. G. (1980) Biochem. Biophys. Res. Commun. 96, 1524-1531 [Medline] [Order article via Infotrieve]
  32. Pilkis, S. J., El-Maghrabi, M. R., Pilkis, J., and Claus, T. (1981) J. Biol. Chem. 256, 3619-3622 [Abstract/Free Full Text]
  33. Ke, H., Zhang, Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5243-5247 [Abstract]
  34. Ganson, N. J., and Fromm, H. J (1982) Biochem. Biophys. Res. Commun. 108, 233-239 [Medline] [Order article via Infotrieve]
  35. Ke, H., Liang, J. Y., Zhang, Y., and Lipscomb, W. N. (1991) Biochemistry 30, 4412-4420 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.