Mutants of Escherichia coli defective in membrane phospholipid synthesis. Phenotypic suppression of sn-glycerol-3-phosphate acyltransferase Km mutants by loss of feedback inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Revertants of Escherichia coli mutants defective in the first enzyme of membrane phospholipid synthesis, sn-glycerol-3-phosphate (glycerol-P) acyltransferase, were investigated. These glycerol-P acyltransferase mutants, selected as glycerol-P auxotrophs, contained membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than the parental activity. The glycerol-P acyltransferase activity was also more thermolabile in vitro than the parental activity. Most revertants no longer requiring glycerol-P for growth regained glycerol-P acyltransferase activity of normal thermolability and apparent Km for glycerol-P. However, two novel revertants were isolated which retained an abnormal glycerol-P acyltransferase activity. The glycerol-P dehydrogenase activities of these novel revertants were about 20-fold less sensitive to feedback inhibition by glycerol-P. The feedback-resistant glycerol-P dehydrogenase co-transduced with gpsA, the structural gene for the glycerol-P dehydrogenase. Further transduction experiments demonstrated that the feedback resistant glycerol-P dehydrogenase phenotypically suppressed the glycerol-P acyltransferase Km lesion. The existence of the class of glycerol-P auxotrophs which owe their phenotype to the glycerol-P acyltransferase Km lesion therefore depends on the feedback regulation of glycerol-P synthesis in E. coli.     

Triacylglycerol synthesis in isolated fat cells. Evidence that the sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities are dual catalytic functions of a single microsomal enzyme.

The acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol-P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 8 muM for glycerol-P with a Vmax of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 40 muM for DHAP with a Vmax of 9.7 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 7.2 muM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (Ki = 92 muM) of the glycerol-P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl-CoA chain length dependence, thermolability, and inactivation by N-ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol-P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol-P to DHAP acylation should be greater than 24:1.     

Efficiency of reconstitution of the membrane-associated sn-glycerol 3-phosphate acyltransferase of Escherichia coli

Membrane-associated enzymes are often solubilized with detergents, purified, and then reconstituted with phospholipid cofactors to regain function. Insofar as most purification and reconstitution procedures are not quantitative, the final reconstituted preparations could reflect a population of molecules ranging from fully functional to completely inactive. Quantitative studies on the efficiency of reconstitution of the Triton X-100-solubilized sn-glycerol 3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane were undertaken at each step of purification. Physical recovery of the 83,000 Mr polypeptide was quantitated in polyacrylamide gels using membranes from cells labeled with [3H]leucine. The 83,000 Mr polypeptide in such gels was demonstrated to consist exclusively of the glycerol-P acyltransferase peptide by V8 peptide mapping. Comparison between physical recovery of 83,000 Mr polypeptide and reconstituted activity allowed the efficiency of reconstitution to be determined. Unexpectedly, disproportionalities occurred during the purification. However, the final purification of reconstituted enzyme activity matched that of the 83,000 Mr polypeptide. This method also allowed measurement of the specific activities of the glycerol-P acyltransferase in membranes from a wild type E. coli strain and from plasmid-containing strains which express the plsB gene product to different extents. The physical amounts of the 83,000 Mr polypeptide and glycerol-P acyltransferase activity measured in membranes were not strictly proportional. In strains where the amount of 83,000 Mr polypeptide was enhanced, a larger proportion of latent activity was observed following solubilization and reconstitution. The results establish the suitability of the reconstituted preparations of glycerol-P acyltransferase for detailed kinetic analysis and permit inferences pertaining to regulation.     

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Properties of wild type and Km defective sn-glycerol-3-phosphate acyltransferase activities.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase, the first enzyme of membrane phospholipid synthesis in Escherichia coli, was investigated in a wild type and a mutant strain defective in this activity. The mutant strain, selected as a glycerol-P auxotroph, was previously shown to contain a glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of its parent or revertants. The membranous mutant glycerol-P acyltransferase but did not appear to be thermolabile in vivo. Revertants no longer requiring glycerol-P for growth, showed glycerol-P acyltransferase activity with thermolability properties similar to the wild type. The second phospholipid biosynthetic enzyme, 1-acylglycerol-P acyltransferase, was not thermolabile in membranes containing a thermolabile glycerol-P acyltransferase activity. The pH optimum for the mutant acyltransferase was over 1 pH unit higher than that of the parental activity. Further, the mutant and wild type glycerol-P acyltransferase differed in their response to magnesium chloride and potassium chloride. The palmitoyl-CoA dependence of the wild type and mutant glycerol-P acyltransferase activities were different. The mutant glycerol-P acyltransferase activity was inhibited greater than 90% by Triton X-100 under conditions where the wild type activity was not affected. These experiments provide novel information about the wild type glycerol-P acyltransferase activity of E. coli and provide six additional lines of evidence for the mutant character of the glycerol-P acyltransferase in the mutant strains.     

Dual mechanisms for glucose 6-phosphate inhibition of human brain hexokinase.

Brain hexokinase (HKI) is inhibited potently by its product glucose 6-phosphate (G6P); however, the mechanism of inhibition is unsettled. Two hypotheses have been proposed to account for product inhibition of HKI. In one, G6P binds to the active site (the C-terminal half of HKI) and competes directly with ATP, whereas in the alternative suggestion the inhibitor binds to an allosteric site (the N-terminal half of HKI), which indirectly displaces ATP from the active site. Single mutations within G6P binding pockets, as defined by crystal structures, at either the N- or C-terminal half of HKI have no significant effect on G6P inhibition. On the other hand, the corresponding mutations eliminate product inhibition in a truncated form of HKI, consisting only of the C-terminal half of the enzyme. Only through combined mutations at the active and allosteric sites, using residues for which single mutations had little effect, was product inhibition eliminated in HKI. Evidently, potent inhibition of HKI by G6P can occur from both active and allosteric binding sites. Furthermore, kinetic data reported here, in conjunction with published equilibrium binding data, are consistent with inhibitory sites of comparable affinity linked by a mechanism of negative cooperativity.     

Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast.     

The triose-phosphate site of homogeneous reconstituted sn-glycerol-3-phosphate acyltransferase of Escherichia coli

The sn-glycerol-3-phosphate (glycerol-phosphate) acyltransferase of Escherichia coli was purified to near homogeneity and its activity reconstituted with phospholipids (Green, P.R., Merrill, A.M., Jr. and Bell, R.M. (1981) J. Biol. Chem. 256, 11151-11159). The competency of glycerol-P analogues to serve as inhibitors and as substrates was investigated. Dihydroxyacetone-P, ethyleneglycol-P, 1,3-propanediol-P, 3,4-dihydroxybutylphosphonate and DL-glyceraldehyde-3-P were inhibitors of the reconstituted purified glycerol-phosphate acyltransferase. The kinetics of inhibition, while formally of the mixed type, most closely resembled that of a simple competitive inhibition with respect to glycerol-3-P. Inorganic phosphate was also found to be a competitive inhibitor. All of the glycerol-3-P analogues except DL-glyceraldehyde-3-P were substrates. Of these, dihydroxyacetone-P proved to be the best substrate. The secondary hydroxyl was not necessary for activity. Glycerol-phosphate acyltransferase catalyzed the hydrolysis of palmitoyl-CoA in the presence of DL-, but not D-glyceraldehyde-3-P. This suggests that the gem diol of L-glyceraldehyde-3-P may be a substrate, and that the acylated adduct may be unstable. The enzyme was inactivated by phenylglyoxal and butanedione, suggesting that arginine may be at or near the active site.     

Hepatic sn-glycerol-3-phosphate acyltransferases: effect of monoacylglycerol analogs on mitochondrial and microsomal activities

The regulation of cellular diacylglycerol levels may have important consequences for protein kinase C activity. Because monoacylglycerols were said to inhibit the committed step of glycerolipid synthesis, the sn-glycerol-3-P acyltransferase (glycerol-P acyltransferase), we determined (1) whether both the mitochondrial and the microsomal glycerol-P acyltransferase isoenzymes were inhibited by 1- and 2-mono-18:1-glycerols, and their ether and amide analogs and (2) what the mechanism of inhibition was. 1- and 2-mono-18:1-glycerols, their ether and amide analogs, and 1-mono-18:1-glycerol 3-phosphate were all competitive inhibitors of the microsomal glycerol-P acyltransferase activity. The relative Ki values suggested that inhibition was strongest with the radyl group at the sn-1 position and that an oxygen bond is important at the sn-1 position. Although the monoacyl- and monoalkylglycerols were also competitive inhibitors of the mitochondrial glycerol-P acyltransferase, neither of the amide analogs was an inhibitor, suggesting that an oxygen bond is essential at both the sn-1 and sn-2 positions. Because monoradylglycerols inhibit several enzyme activities that contribute to the biosynthesis or the metabolism of diacylglycerol, these inhibitors may function within cells in part to regulate cellular diacylglycerol levels.     

Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo.

The glucose-6-phosphate (Glc6P) and 6-phosphogluconate (6PG) dehydrogenases of the amino-acid-producing bacterium Corynebacterium glutamicum were purified to homogeneity and kinetically characterized. The Glc6P dehydrogenase was a heteromultimeric complex, which consists of Zwf and OpcA subunits. The product inhibition pattern of the Glc6P dehydrogenase was consistent with an ordered bi-bi mechanism. The 6PG dehydrogenase was found to operate according to a Theorell-Chance ordered bi-ter mechanism. Both enzymes were inhibited by NADPH and the 6PG dehydrogenase additionally by ATP, fructose 1,6-bisphosphate (Fru1,6P2), D-glyceraldehyde 3-phosphate (Gra3P), erythrose 4-phosphate and ribulose 5-phosphate (Rib5P). The inhibition by NADPH was considered to be most important, with inhibition constants of around 25 microM for both enzymes. Intracellular metabolite concentrations were determined in two isogenic strains of C. glutamicum with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases. NADP+ and NADPH levels were between 130 microM and 290 microM, which is very much higher than the respective Km and Ki values. The Glc6P concentration was around 500 microM in both strains. The in vivo fluxes through the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified enzymes determined in vitro were in agreement with the same fluxes determined by NMR after 13C-labelling. From the derived kinetic model thus validated, it is concluded that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH and NADP+ concentrations and the specific enzyme activities of both dehydrogenases.     

Mechanism of substrate inhibition in Escherichia coli phosphofructokinase

Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites, which bind the substrates fructose-6-phosphate (Fru-6-P) and MgATP. In the presence of low concentrations of Fru-6-P, MgATP displays substrate inhibition. Previous proposals to explain this substrate inhibition have included both kinetic and allosteric mechanisms. We have isolated hybrid tetramers containing one wild type subunit and three mutated subunits (1:3). The mutated subunits contain mutations that decrease affinity for Fru-6-P (R243E) or MgATP (F76A/R77D/R82A) allowing us to systematically simplify the possible allosteric interactions between the two substrates. In the absence of a rate equation to explain the allosteric effects in a tetramer, the data have been compared to simulated data for an allosteric dimer. Since the apparent substrate inhibition caused by MgATP binding is not seen in hybrid tetramers with only a single native MgATP binding site, the proposed kinetic mechanism is not able to explain this phenomenon. The data presented are consistent with an allosteric antagonism between MgATP in one active site and Fru-6-P in a second active site.     

Active-site modification of glycerol-3-phosphate dehydrogenase by pyridoxal-5′-phosphate

Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from rabbit skeletal muscle is inhibited by pyridoxal-5′-phosphate. The inhibition observed in steady-state kinetic studies is competitive with respect to dihydroxyacetone phosphate and uncompetitive with respect to NADH. Similar inhibition was found for a series of related compounds which in order of increasing effectiveness of inhibition were: 4-deoxypyridoxine < pyridoxal < pyridoxic acid < pyridoxal-5′-phosphate < pyridoxine and pyridoxamine-5′-phosphate. Pyridoxal-5′-phosphate also reacts slowly with the enzyme to produce an adduct which upon treatment with sodium borohydride results in irreversible modification of the enzyme. The nature of the adduct was investigated by titration of the enzyme with pyridoxal-5′-phosphate, uv-visible and fluorescence spectroscopy, amino acid analysis, and peptide mapping. All such studies are consistent with a single, highly reactive lysyl residue on each enzyme subunit. Protection of the lysyl residue against modification was afforded by the presence of NADH. The modified enzyme, on the other hand, possessed kinetic properties similar to the native enzyme including a nearly identical inhibition constant for pyridoxal-5′-phosphate. Pyridoxal-5′-phosphate, therefore, seems to have two sites of interaction on the enzyme: a reversible binding site competitive with substrate and a Schiff-base site protected by NADH. These properties of glycerol-3-phosphate dehydrogenase set it apart from functionally similar enzymes.     

sn-glycerol-3-phosphate acyltransferase tubule formation is dependent upon heat shock proteins (htpR)

Overexpression of the Escherichia coli sn-glycerol-3-phosphate (glycerol-P) acyltransferase, an integral membrane protein, causes formation of ordered arrays of the enzyme in vitro. The formation of these tubular structures did not occur in an E. coli strain bearing a mutation in the htpR gene, the regulatory gene for the heat shock response. The htpR165 mutation was shown by genetic analysis to be the lesion responsible for blockage of tubule formation. Similar amounts of glycerol-P acyltransferase were produced in isogenic htpR+ and htpR165 strains, ruling out an effect of htpR165 on expression of glycerol-P acyltransferase. Further, phospholipid metabolism was not altered in either strain after induction of glycerol-P acyltransferase synthesis. Increased glycerol-P acyltransferase synthesis caused a partial induction of the heat shock response which was dependent upon a wild type htpR gene. The heat shock proteins induced were identified as the groEL and dnaK gene products on two-dimensional gels. These two proteins have been implicated in the assembly of bacteriophage coats. These heat shock proteins appear essential for tubule formation.     

Kinetics and mechanism of action of aldehyde reductase from pig kidney.

An improved procedure for purifying aldehyde reductase is described. Utilization of Blue Dextran--Sepharose 4B and elimination of hydroxyapatite chromatography greatly improves the yield and ease of purification. Starting with 340 g of kidney tissue (two pig kidneys) approx. 50 mg of purified reductase may be routinely and reproducibly obtained. The purified reductase was used to establish the kinetic reaction mechanism of the enzyme. Initial-velocity analysis and product-inhibition data revealed that pig kidney aldehyde reductase follows an Ordered Bi Bi reaction mechanism in which NADPH binds first before D-glyceraldehyde. The limiting Michaelis constants for D-glyceraldehyde and NADPH were 4.8 +/- 0.7 mM and 9.1 +/- 2.1 micrometer respectively. The mechanism is similar to that of another monomeric oxidoreductase, octopine dehydrogenase, towards which aldehyde reductase exhibits several similarities, but differs from that of other aldehyde reductases. Phenobarbital is a potent inhibitor of aldehyde reductase, inhibiting both substrate and cofactor non-competitively (Ki = 80.4 +/- 10.5 micrometer and 66.9 +/- 1.6 micrometer respectively). Barbiturate inhibition seems to be a common property of NADPH-dependent aldehyde reductases.     

Asymmetric reconstitution of homogeneous Escherichia coli sn-glycerol-3-phosphate acyltransferase into phospholipid vesicles

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane was purified in Triton X-100 (Green, P. R., Merrill, A. H., Jr., and Bell, R. M. (1981) J. Biol. Chem. 256, 11151-11159) and incorporated into mixed micelles containing Triton X-100, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and beta-octyl glucoside. Enzyme activity was quantitatively reconstituted from the mixed micelle into single-walled phospholipid vesicles by chromatography over Sephadex G-50. Activity coeluted with vesicles of 90-nm average diameter on columns of Sepharose CL-4B and Sephacryl S-1000. These vesicles contained less than 2 Triton X-100 and 5 beta-octyl glucoside molecules/100 phospholipid molecules. Calculations suggested that up to eight 91,260-dalton glycerol-P acyltransferase polypeptides were incorporated per 90-nm vesicle. The pH dependence and apparent Km values for glycerol-P and palmitoyl-CoA of the glycerol-P acyltransferase reconstituted into vesicles were similar to those observed upon reconstitution by mixing of the enzyme in Triton X-100 with a 20-fold molar excess of sonicated phosphatidylethanolamine:phosphatidylglycerol:cardiolipin, 6:1:1. The integrity of vesicles containing glycerol-P acyltransferase was established by trapping 5,5'-dithiobis-(2-nitrobenzoic acid). Chymotrypsin inactivated greater than 95% of the glycerol-P acyltransferase in intact vesicles and cleaved the 91,260-dalton polypeptide into several vesicle-bound and several released peptides, indicating that critical domains of the enzyme are accessible in intact vesicles. Trinitrobenzene sulfonate and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene caused greater than 90% loss of glycerol-P acyltransferase in vesicles. Disruption of vesicles with Triton X-100 did not reveal significant latent activity. These data strongly suggest that the glycerol-P acyltransferase was reconstituted asymmetrically into the vesicles with its active site facing outward.     

Kinetic studies on the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes.

Steady-state kinetic techniques have been used to investigate each of the reactions catalyzed by the bifunctional enzyme, chorismate mutase-prephenate dehydrogenase, from Aerobacter aerogenes. The results of steady-state velocity studies in the absence of products, as well as product and dead-end inhibition studies, suggest that the prephenate dehydrogenase reaction conforms to a rapid equilibrium random mechanism which involes the formation of two dead-end complexes, viz, enzyme-NADH-prephenate and enzyme-NAD+-hydroxyphenylpyruvate. Chorismate functions as an activator of the dehydrogenase while both prephenate and hydroxyphenylpyruvate acted as competitive inhibitors in the mutase reaction. By contrast. bpth NAD+ and NADH function as activators of the mutase. Values of the kinetic parameters associated with the mutase and dehydrogenase reactions have been determined and the results discussed in terms of possible relationships between the catalytic sites for the two reactions. The data appear to be consistent with the enzyme having either a single site at which both reactions occur or two separate sites which possess similar kinetic properties.     

Kinetic studies of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle

Initial rate studies at pH 7.6 with three aldehydes, product inhibition patterns with NADH and dead-end inhibition with adenosine diphosphoribose show that the kinetic mechanism of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle cannot be ordered, and support an enzyme-substitution mechanism. Deviations from Michaelis-Menten behaviour are consistent with negative interactions in the binding of NAD+ and instability of the species E(NAD)3 and E(NAD)4. Inhibition with large concentrations of phosphate and arsenate indicates competition for a binding site for glyceraldehyde 3-phosphate, and is not found with glyceraldehyde as substrate.     

Maize phosphoenolpyruvate carboxylase. Mutations at the putative binding site for glucose 6-phosphate caused desensitization and abolished responsiveness to regulatory phosphorylation

Phosphoenolpyruvate carboxylases (PEPC, EC 4.1.1.31) from higher plants are regulated by both allosteric effects and reversible phosphorylation. Previous x-ray crystallographic analysis of Zea mays PEPC has revealed a binding site for sulfate ion, speculated to be the site for an allosteric activator, glucose 6-phosphate (Glc-6-P) (Matsumura, H., Xie, Y., Shirakata, S., Inoue, T., Yoshinaga, T., Ueno, Y., Izui, K., and Kai, Y. (2002) Structure (Lond.) 10, 1721-1730). Because kinetic experiments have also supported this notion, each of the four basic residues (Arg-183, -184, -231, and -372' on the adjacent subunit) located at or near the binding site was replaced by Gln, and the kinetic properties of recombinant mutant enzymes were investigated. Complete desensitization to Glc-6-P was observed for R183Q, R184Q, R183Q/R184Q (double mutant), and R372Q, as was a marked decrease in the sensitivity for R231Q. The heterotropic effect of Glc-6-P on an allosteric inhibitor, l-malate, was also abolished, but sensitivity to Gly, another allosteric activator of monocot PEPC, was essentially not affected, suggesting the distinctness of their binding sites. Considering the kinetic and structural data, Arg-183 and Arg-231 were suggested to be involved directly in the binding with phosphate group of Glc-6-P, and the residues Arg-184 and Arg-372 were thought to be involved in making up the site for Glc-6-P and/or in the transmission of an allosteric regulatory signal. Most unexpectedly, the mutant enzymes had almost lost responsiveness to regulatory phosphorylation at Ser-15. An apparent lack of kinetic competition between the phosphate groups of Glc-6-P and of phospho-Ser at 15 suggested the distinctness of their binding sites. The possible roles of these Arg residues are discussed.