Proc. Natl. Acad. Sci. USA Vol. 91, pp. 5242-5246, June 1994 Biochemistry The cooperativity and allosteric inhibition of Escherichia coli phosphofructokinase depend on the interaction between threonine-125 and ATP (phosphoryl transfer/site-directed mutgenesis/enzyme regulation) ISABELLE AUZAT, GIStLE LE BRAS, AND JEAN-RENAUD GAREL* Laboratoire d'Enzymologie du Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France Communicated by Robert L. Baldwin, February 14, 1994 ABSTRACT During the reaction catalyzed by the phos- 9), comparing the crystal structures of the R and T states of phofructokinase (EC 2.7.1.11) from Escherichia coli, the phos- PFK does not suggest a structural mechanism for cooper- phoryl group transferred from ATP interacts with Thr-125 ativity. The three-dimensional structure ofunliganded E. coli [Shir a, Y. & Evans, P. R. (1988) J. Mol. Biol. 204, PFK resembles that of the R state (10), indicating that 973-994]. The replacement of Thr-125 by serine changes the cooperativity (as well as activation by ADP or GDP) could saturation by fructose 6-phosphate from cooperative to hyper- involve only this R state. Also, the PFKs from E. coli and bolic and abolishes the allosteric inhibition by phospho- Bacillus stearothermophilus have similar three-dimensional enolpyruvate. The same changes, a saturation by fructose structures, and both undergo a transition from the R to the T 6-phosphate that is no longer cooperative and an activity that state upon binding PEP (5, 6, 11), but the two enzymes have is no longer inhibited by phosphoenolpyruvate, are observed different saturations by Fru-6P: extremely cooperative forE. with wild-type phosphofructokinase when adenosine 5'-[r coli PFK (3) and hyperbolic for B. stearothermophilus PFK thio]triphosphate is used instead of ATP as the phosphoryl (12). These results indicate that the cooperativity of PFK donor. These two perturbations of the ATP-Thr-125 interac- does not seem coupled to the transition between the crys- tion lead to the suppression of both the allosteric inhibition by tallographic R and T states. phosphoenolpyruvate and the cooperativity of fructose-6- In the x-ray structure ofthe R state of PFK with substrates phosphate saturation, as ifreplacing the neutral oxygen ofATP and/or products bound, the phosphoryl group transferred by sulfur or removing the methyl group of Thr-125 had from ATP to Fru-6P interacts with Thr-125 (5, 7, 13). The "locked" phosphofructokinase in its active conformation. The hydroxyl group of Thr-125 is important since the Thr-125 geometry ofthis ATP-Thr-125 interaction and/or the presence Ala mutation decreases the activity of PFK by three orders of the methyl group on the P-carbon of Thr-125 are crucial for of magnitude and abolishes the cooperativity of Fru-6P the regulatory properties of phosphofructokinase. This inter- saturation (13). In this work, we have replaced either Thr-125 action could be a hydrogen bond between the neutral oxygen of by a Ser (Thr-125 -- Ser mutant) or ATP by adenosine the rphosphate of ATP and- the hydroxyl group of Thr-125. 5'-[ythio]triphosphate (ATP[(yS]). The main result is that these two perturbations result in the same changes in the The regulation of many biological processes involves allo- regulatory properties ofE. coli PFK: the saturation by Fru-6P steric interactions between distinct sites of the same protein, is no longer cooperative but is hyperbolic, and the protein no such that ligand binding at one site modifies the functional longer undergoes a transition into the inactive T state upon properties at a distant site. Cooperativity is observed when PEP binding. This suggests that the interaction between positive interactions take place between identical sites (1). Thr-125 and ATP is important for the allosteric behavior of One ofthe "classic" cooperative enzymes is Escherichia coli PFK. The Thr-125 -) Ser mutant of PFK still has a hydroxyl phosphofructokinase (PFK; ATP:D-fructose-6-phosphate group, but the absence of the methyl group on the (3-carbon 1-phosphotransferase, EC 2.7.1.11) (2, 3), which shows a probably affects the interaction with ATP. Similarly, the markedly sigmoidal saturation by its substrate fructose replacement of a single oxygen atom by sulfur in ATP[YS] is 6-phosphate (Fru-6P), with a Hill cooperativity coefficient a "mutation" of the yphosphate that perturbs its interaction close to nH = 4 for four Fru-6P sites (3). This enzyme is also with Thr-125. These results indicate that the side chain of sensitive to allosteric effectors, which bind to a regulatory Thr-125 and its interaction with the noncooperative substrate site remote from the active site; PFK is activated by ADP (or ATP are essential for the coupling between distant sites GDP) and inhibited by phosphoenolpyruvate (PEP) (3). responsible for cooperativity towards Fru-6P and allosteric The steady-state kinetics of E. coli PFK have been inter- inhibition by PEP of E. coli PFK. preted according to the concerted allosteric mechanism (4), in which the protein is in equilibrium between two states, an active R state, which binds Fru-6P and the activator ADP or MATERIALS AND METHODS GDP, and an inactive T state, which binds the inhibitor PEP The oligonucleotide 5'-GTCGATAGAGCCCGGGTA-3' (3). X-ray crystallography shows that PFK exists in two (Bioprobe Systems, Sous BQis, France) with a single mis- different conformations, R with activator and substrate (or match (underlined) was used to replace the ACT codon (Thr) products) bound (5) and T with inhibitor bound (6), which at position 125 by TCT (Ser). Site-directed mutagenesis was provides a structural explanation for the allosteric inhibition performed with the Amersham kit using Pvu I instead of Nci by PEP (6-8). However, in contrast with hemoglobin for contains an Nci I which the differences between the oxy and deoxy states I because the mutagenic oligonucleotide could largely explain the cooperativity of oxygen binding (8, Abbreviations: AMPPCP, adenosine 5'-[3,B,-methylene]triphos- phate; ATP[(yS], adenosine 5'-[ythio]triphosphate; Fru-6P, D-fruc- The publication costs of this article were defrayed in part by page charge tose 6-phosphate; PEP, phosphoenolpyruvate; PFK, phosphofruc- payment. This article must therefore be hereby marked "advertisement" tokinase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5242 Downloaded by guest on September 25, 2021 Biochemistry: Auzat et A Proc. Natl. Acad. Sci. USA 91 (1994) 5243 site. The modified gene was completely sequenced to verify Table 2. Comparison of the enzymatic properties of wild-type the lack of any other alteration. Mutated and wild-type pikA PFK and the Thr-125 -. Ser mutant measured in both the absence genes inserted into the pDR540 tac vector (Pharmacia) were and presence of the allosteric activator GDP at 2 mM expressed after transforming cells of E. coli strain DF1020, Property Wild type Thr-125 Ser which are deleted for both pJk genes (14). Wild-type and mutant PFKs were purified as described (14, Without GDP using a coupled assay (16) in kat, sec-1 120 45 15). PFK activity was measured [Fru-6P]o.s, MM 340 140 a buffer composed of 0.1 M Mes, 0.051 M N-ethylmorpho- Km for ATP, p.M 57 17 line, and 0.051 M diethanolamine at pH 8.2. The ATP- nH 3.9 0.9 regenerating system composed of creatine phosphate and Inhibition by 10 82 <10 creatine kinase was omitted when ATP[yS] was used as mM PEP, % substrate. With 2 mM GDP The same buffer containing 10 mM Mg2e ion was used to kct, sec-1 140 30 measure the changes at 340 nm with excitation at 295 nm in Km for Fru-6P, p.M 41 170 intrinsic fluorescence of PFK upon binding Fru-6P, adeno- Km for ATP, p.M 60 21 sine 5'-[(3, -t-methylene]triphosphate (AMPPCP), or PEP as 0.9 reported (17). nH 1.0 [Fru-6P]o.s, half-saturating Fru-6P concentration. RESULTS AND DISCUSSION ATP (7, 13). The half-saturating Fru-6P concentration in the Fluorescence Measurements of Ligand Binding to the Thr- absence of GDP is not drastically altered by replacement of 125 -& Ser Mutant. Like that of wild-type PFK (17, 18), the Thr-125, with a 2.5-fold decrease for the Thr-125 -+ Ser fluorescence of the Thr-125 -+ Ser mutant changes upon the mutant (Table 2) and a 2.5-fold increase for the Thr-125 -- Ala binding of ligands. The fluorescence is increased by the mutant (13). binding ofAMPPCP (a nonhydrolyzable ATP analog) or PEP The prominent features of the Thr-125 -* Ser (Fig. 1 and and decreased by the binding of Fru-6P (Table 1). Fru-6P Table 2) and Thr-125 -+ Ala (13) mutants are their hyperbolic binding to wild-type PFK is not cooperative as seen from saturations by Fru-6P. The complete absence of cooperativ- equilibrium dialysis (17) and fluorescence (17, 18) measure- ity of the saturation by Fru-6P of the Thr-125 -- Ser mutant ments, and the fluorescence changes of the Thr-125 -> Ser is shown by the value of nH = 0.9 obtained for the cooper- mutant upon binding Fru-6P also show no cooperativity. The ativity coefficient using the Hill equation (Table 2). The mutation Thr-125 -> Ser does not drastically affect the methyl group on the (-carbon that distinguishes wild-type equilibrium constants for the binding of Fru-6P, AMPPCP, and the Thr-125 -) Ser mutant PFKs appears essential for and PEP (Table 1). The absence of cooperativity for Fru-6P cooperativity, even though it contributes only marginally to saturation or of PEP inhibition of the Thr-125 -* Ser mutant substrate binding and catalysis (Tables 1 and 2).