Purification and kinetic characterization of 6-phosphofructo-1-kinase from the liver of gilthead sea bream (Sparus aurata)

6-phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P2) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P2 counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P2, whereas combination of activation by fru-2,6-P2 and increase in protein content may determine the long-term recovery of the enzyme activity.     

Fructose-2,6-bisphosphate in rat mesenteric lymph nodes

1. The fructose-2,6-bisphosphate (Fru-2,6-P2) content of mesenteric lymph nodes was measured in rats. 2. The effects of Fru-2,6-P2 on the activity of 6-phosphofructo-1-kinase (PFK-1) from rat mesenteric lymph nodes were also studied. 3. The affinity of the enzyme for fructose-6-phosphate was increased by Fru-2,6-P2 whereas the inhibition of the enzyme with high concentrations of ATP was released by Fru-2,6-P2. 4. The activity of lymphocyte PFK-1 was highly stimulated in a simultaneous presence of low concentrations of AMP and Fru-2,6-P2. 5. These results show that rat lymphocyte PFK-1 is highly regulated with Fru-2,6-P2 which means that glycolysis in rat lymphocytes is controlled by Fru-2,6-P2.     

Phosphofructokinases from Escherichia coli. Purification and characterization of the nonallosteric isozyme.

The main phosphofructokinase of Escherichia coli (PFK I) is an extensively studied allosteric enzyme specified by the pfkA gene. A nonallosteric phosphofructokinase was reported (Fraenkel, D.G., Kotlarz, D., and Bluc, H. (1973) J. Biol. Chem. 248, 4865-4866) in strains carrying the pfkB1 mutation, a suppressor of pfkA mutants, and very low levels of this enzyme have also been detected in strains not carrying the suppressor (i.e. pfkB+). The nonallosteric protein has now been prepared pure from three strains, one carrying pfkB1 and pfkA+, one carrying pfkB1 and completely deleted for pfkA, and one carrying pfkB+ and also deleted for pfkA. It is apparently the same enzyme (PFK II) in all three strains, which shows that pfkB1 is a mutation affecting the amount of a normally minor isozyme. PFK II is a tetramer of slightly larger subunit molecular weight than PFK I (36,000 and 34,000, respectively). No immunological cross-reactivity was detected between PFK II and PFK I. Unlike PFK I, PFK II does not show cooperative interactions with fructose-6-P, inhibition by P-enolpyruvate, or activation by ADP. Also unlike PFK I, PFK II is somewhat sensitive to inhibition by fructose-1,6-P2 and can use tagatose-6-P as substrate. Both enzymes can perform the reverse reaction, fructose-6-P + ATP from fructose-1,6-P2 + ADP in vitro, but not in vivo. The normal function of PFK II is not known.     

Purification and characterization of phosphofructokinase in bovine parotid gland.

1. Phosphofructokinase (PFK) was purified from bovine parotid gland to 750-fold with the specific activity of 67.5 units/mg protein by Cibacron Blue F3GA affinity chromatography, and TSK DEAE-5PW ion-exchange and TSK G4000SW size exclusion chromatographies on HPLC. 2. On gel-filtration, molecular weight of the native PFK was estimated to 400,000. 3. PFK was a heterotetramer composed of three kinds of subunit with molecular weights of 92,000 (C-type), 88,000 (M-type) and 86,000 (L-type), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densitometrically, relative amounts of C-, M- and L-type subunit were 1:1:2. 4. Under the physiological conditions of fructose 6-phosphate (Fru-6-P) and ATP concentrations and pH, PFK activity was suppressed and hardly detectable. 5. Fru-6-P relieved PFK from the ATP inhibition. 6. Fructose 2,6-bisphosphate (Fru-2,6-P2) and AMP activated PFK with a reduction of S0.5 for Fru-6-P and subunit cooperativity. Fru-2,6-P2 was more effective than AMP.     

Citrate inhibition of rat-kidney cortex phosphofructokinase

The regulatory properties of citrate on the activity of phosphofructokinase (PFK) purified from rat-kidney cortex has been studied. Citrate produces increases in the K_0.5 for Fru-6-P and in the Hill coefficient as well as a decrease in the V_max of the reaction without affecting the kinetic parameters for ATP as substrate. ATP potentiates synergistically the effects of citrate as an inhibitor of the enzyme. Fru-2,6-P₂ and AMP at concentrations equal to K_a were not able to completely prevent citrate inhibition of the enzyme. Physiological concentrations of ATP and citrate produce a strong inhibition of renal PFK suggesting that may participate in the control of glycolysisin vivo.Abbreviations PFK 6-Phosphofructo-1-kinase (EC 2.7.1.11) - Fru-6-P Fructose 6-phosphate - Fru-2,6-P₂ Fructose 2,6-bisphosphate     

Purification and kinetic properties of 6-phosphofructo-1-kinase from gilthead sea bream muscle

The kinetic properties of 6-phosphofructo-1-kinase (PFK) from skeletal muscle (PFKM) of gilthead sea bream (Sparus aurata) were studied, after 10,900-fold purification to homogeneity. The native enzyme had an apparent molecular mass of 662 kDa and is composed of 81 kDa subunits, suggesting a homooctameric structure. At physiological pH, S. aurata PFKM exhibited sigmoidal kinetics for the substrates, fructose-6-phosphate (fru-6-P) and ATP. Fructose-2,6-bisphosphate (fru-2,6-P(2)) converted the saturation curves for fru-6-P to hyperbolic, activated PFKM synergistically with other positive effectors of the enzyme such as AMP and ADP, and counteracted ATP and citrate inhibition. The fish enzyme showed differences regarding other animal PFKs: it is active as a homooctamer, and fru-2,6-P(2) and pH affected affinity for ATP. By monitoring incorporation of (32)P from ATP, we show that fish PFKM is a substrate for the cAMP-dependent protein kinase. The mechanism involved in PFKM activation by phosphorylation contrasts with previous observations in other species: it increased V(max) and did not affect affinity for fru-6-P. Unlike the mammalian muscle enzyme, our findings support that phosphorylation of PFKM may exert a major role during starvation in fish muscle.     

Enzyme regulation in c(4) photosynthesis : identification and localization of activities catalyzing the synthesis and hydrolysis of fructose-2,6-bisphosphate in corn leaves

Activities catalyzing the synthesis of fructose-2,6-bisphosphate (fructose-6-phosphate,2-kinase or Fru-6-P,2K) and its breakdown (fructose-2,6-bisphosphatase or Fru-2,6-P₂ase) were identified in leaves of corn (Zea mays), a C₄ plant. Fru-6-P,2K and Fru-2,6-P₂ase were both localized mainly, if not entirely, in the leaf mesophyll cells. A partially purified preparation containing the two activities revealed that the kinase and phosphatase were regulated by metabolite effectors in a manner generally similar to their counterparts in C₃ species. Thus, corn Fru-6-P,2K was activated by inorganic phosphate (Pi) and fructose-6-phosphate, and was inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fru-2,6-P₂ase was inhibited by its products, fructose-6-phosphate and Pi. However, unlike its spinach equivalent, corn Fru-2,6-P₂ase was also inhibited by 3-phosphoglycerate and, less effectively, by dihydroxyacetone phosphate. The C₄ Fru-6-P,2K and Fru-2,6-P₂ase were also quite sensitive to inhibition by phosphoenolpyruvate, and each enzyme was also selectively inhibited by certain other metabolites.     

MgATP-dependent activation by phosphoenolpyruvate of the E187A mutant of Escherichia coli phosphofructokinase

Using enzymatic assays and steady-state fluorescence emission, we performed a linkage analysis of the three-ligand interaction of fructose 6-phosphate (Fru-6-P), phosphoenolpyruvate (PEP), and MgATP on E187A mutant Escherichia coli phosphofructokinase (PFK). PEP allosterically inhibits Fru-6-P binding to E. coli PFK. The magnitude of antagonism is 90-fold in the absence and 60-fold in the presence of a saturating concentration of MgATP [Johnson, J. J., and Reinhart, G. D. (1997) Biochemistry 36, 12814-12822]. Substituting an alanine for the glutamate at position 187, located in the allosteric site (i.e., mutant E187A), activates Fru-6-P binding and inhibits the maximal rate of enzyme turnover [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. The allosteric action of PEP appears to depend on the presence of the cosubstrate MgATP. In the presence of a saturating concentration of MgATP, PEP enhances the binding of Fru-6-P to the enzyme by a modest 2-fold. Decreasing the concentration of MgATP mitigates the extent of activation. At MgATP concentrations approaching 25 microM, PEP becomes insensitive to the binding of Fru-6-P. At MgATP concentrations < 25 microM, PEP "crosses over" and becomes antagonistic toward substrate binding. The present study examines the role of Glu 187 at the allosteric site in the binding of Fru-6-P and offers a more complex explanation of the mechanism than that described by traditional allosteric mechanistic models.     

Pi和PGA对玉米叶片PFK-2/FBPase-2的作用

Fructose-6-phosphate 2-kinase and fructose 2,6-bisphosphatase have been partially purified from maize leaves by PEG fractionadon and by chromatography on TSK-DEAE ion exchanger and Blue-Sepharose 4B. Fructose-6-phosphate 2-kinase was activated by phosphate and inhibited by 3-pnosphoglycerate. Furctose 2,6-bisphosphatase was inhibited by inorganic phosphate and fructose-6-phosphate.The observed pattern of reguladon suggests that systhesis and degradation of Fru-2,6-P_2 respond to changes in the concentration of effectors. An increase in the level of glycerate-3-phosphate or dihydroxyacetonephosphate will result in a decrease in the level of Fru-2,6-P_2. Conversely a rise in Fru-6-P concentration will lead to an increase in the Fru-2, 6-P_2 concentration.     

Studies on the regulation of yeast phosphofructo-1-kinase: its role in aerobic and anaerobic glycolysis

The kinetics of yeast phosphofructo-1-kinase has been studied in vitro. Effector concentrations (Fru-6-P, ATP, ADP, AMP, Pi, Fru-1,6-P2, and Fru-2,6-P2) and pH were adjusted so as to mimic intracellular concentrations in yeast. Under these conditions we were able to reproduce the measured in vivo rate of PFK. In addition, by reconstituting the intracellular conditions existing during aerobic and anaerobic glycolysis, we were able to reproduce in vitro the changes in the rate of PFK observed under these conditions. Without the addition of the newly discovered effector Fru-2,6-P2, in vitro rates of PFK are much lower than its in vivo rate. Changes in Fru-2,6-P2, Fru-1,6-P2, ATP, AMP, Pi, and pH in going from aerobic to anaerobic conditions all contributed somewhat to the change in the rate of PFK observed during the Pasteur effect, with no contribution coming from ADP. These studies show that the control of PFK under the condition of the Pasteur effect cannot be ascribed to changes in any one particular effector but rather to contributions from a variety of effectors. Also, the net change in the rate of PFK in the switch from anaerobic to aerobic glycolysis is small compared with the change in its dependence upon its substrate Fru-6-P, indicating a compensation mechanism.     

Site-directed mutagenesis at the regulatory site of fructose 6-phosphate-1-kinase from Bacillus stearothermophilus

We have mutated Arg-25, Asp-59 and Arg-211 to alanine; and Asp-59 also to methionine, in fructose 6-phosphate-1-kinase from B. stearothermophilus (designated as RA25, DA59, RA211 and DM59 respectively). All four mutants did not change the affinity of the enzyme for ATP. RA25 has half the affinity for fructose 6-phosphate and exhibits sigmoidicity with respect to this substrate (Hill # = 2.0). DA59 has the same affinity for phosphoenolpyruvate (PEP) as the wild type whereas DM59 has 3-fold the affinity for this modulator and the inhibition is reversed by GDP. RA25 and RA211 are 100-fold less sensitive to PEP inhibition which is not relieved by GDP. It is concluded that Arg-25 and Arg-211, but not Asp-59, are involved in the direct binding of PEP and GDP.     

Phosphofructokinase 2 and glycolysis in HT29 human colon adenocarcinoma cell line. Regulation by insulin and phorbol esters.

Kinetic properties of phosphofructokinase 2 (PFK2) and regulation of glycolysis by phorbol 12-myristate 13-acetate (PMA) and insulin were investigated in highly glycolytic HT29 colon cancer cells. PFK2 was found to be inhibited by citrate and, to a lesser extent, by phosphoenolpyruvate and ADP, but to be insensitive to inhibition by sn-glycerol phosphate. From these kinetic data, PFK2 from HT29 cells appears different from the liver form, but resembles somewhat the heart isoenzyme. Fructose 2,6-bisphosphate (Fru-2,6-P2) levels, glucose consumption and lactate production are increased in a dose-dependent manner in HT29 cells treated with PMA or insulin. The increase in Fru-2,6-P2 can be related to an increase in the Vmax. of PFK2, persisting after the enzyme has been precipitated with poly(ethylene glycol), without change in the Km for fructose 6-phosphate. The most striking effects of PMA and insulin on Fru-2,6-P2 production are observed after long-term treatment (24 h) and are abolished by actinomycin, cycloheximide and puromycin, suggesting that protein synthesis is involved. Furthermore, the effects of insulin and PMA on glucose consumption, lactate production, Fru-2,6-P2 levels and PFK2 activity are additive, and the effect of insulin on Fru-2,6-P2 production is not altered by pre-treatment of the cells with the phorbol ester. This suggests that these effects are exerted by separate mechanisms.     

The mechanism of activation of heart fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Partially purified fructose-6-P,2-kinase:fructose-2,6-bisphosphatase from beef heart was phosphorylated by cAMP protein kinase. The phosphorylated fructose-6-P,2-kinase shows lower Km for Fru-6-P (43 versus 105 microM) and for ATP (0.55 versus 1.3 mM) but no change in the Vmax, compared to those for unphosphorylated enzyme. There was no detectable change in Km or Vmax of fructose-2,6-bisphosphatase activity by the phosphorylation. These changes in heart fructose-6-P,2-kinase were in direct contrast to previous results for the liver isozyme in which phosphorylation led to inhibition of the kinase activity and activation of the phosphatase activity.     

Role of Ser530, Arg292, and His662 in the allosteric behavior of rabbit muscle phosphofructokinase.

Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is a potent allosteric activator of the ATP-dependent phosphofructokinase (PFK) in eukaryotes. Based on the sequence homology between rabbit muscle PFK and two bacterial PFKs and the crystal structures of the latter, Ser(530), Arg(292) and His(662) of the rabbit enzyme are implicated as binding sites for Fru-2,6-P(2). We report here the effects of three mutations, S530D, R292A, and H662A on the activation of rabbit muscle PFK by Fru-2,6-P(2). At pH 7.0 and the inhibitory concentrations of ATP, the native enzyme gives a classic sigmoidal response to changes in Fru-6-P concentration in the absence of Fru-2,6-P(2) and a nearly hyperbolic response in the presence of the activator. Under the same conditions, no activation was seen for S530D. On the other hand, H662A can be activated but requires a 10-fold or higher concentration of Fru-2,6-P(2). Limited activation was observed for mutant R292A. A model illustrating the sites for recognition of Fru-2,6-P(2) in rabbit muscle PFK as well as the mechanism of allosteric activation is proposed.     

MgATP and fructose 6-phosphate interactions with phosphofructokinase from Escherichia coli.

A thermodynamic linked-function analysis is presented of the interactions of MgATP and fructose 6-phosphate (Fru-6-P) with phosphofructokinase (PFK) from Escherichia coli in the absence of allosteric effectors. MgATP and Fru-6-P are shown to bind in random fashion by product inhibition of the back-reaction as well as by the kinetically competent binding of each ligand individually as monitored by the consequent changes in the intrinsic fluorescence of E. coli PFK. When Fru-6-P is saturating, the dissociation of MgATP is sufficiently slow that it cannot achieve a binding equilibrium in the steady state, causing the observed Km (49 microM) to significantly exceed the Kd (1.7 microM) deduced from a thermodynamic linkage analysis. The following features distinguish the interactions of MgATP and Fru-6-P with E. coli PFK: MgATP and Fru-6-P antagonize each other's binding to the enzyme in a saturable manner with an overall apparent coupling free energy equal to +2.5 kcal/mol at 25 degrees C; MgATP induces positive cooperativity in the Fru-6-P binding profile, with the Hill coefficient calculated from the Fru-6-P binding curves reaching a maximum of 3.6 when MgATP is saturating; and MgATP exhibits substrate inhibition at low concentrations of Fru-6-P. Simulations based upon the rate equation pertaining to a two-active-site, two-substrate dimer indicate that these features can all result from two independent couplings: an antagonistic MgATP-Fru-6-P coupling extending at least in part between active sites and a MgATP-induced Fru-6-P-Fru-6-P coupling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.     

A switch in the kinase domain of rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase plays an essential role in the regulation of glucose metabolism by both producing and degrading Fru-2,6-P(2) via its distinct catalytic activities. The 6-PF-2-K and Fru-2,6-P(2)ase active sites are located in separate domains of the enzyme. The kinase domain is structurally related to the superfamily of mononucleotide binding proteins that includes adenylate kinase and the G-proteins. We have determined three new structures of the enzymatic monomer, each with a different ligand in the ATP binding site of the 6-PF-2-K domain (AMP-PNP, PO(4), and water). A comparison of these three new structures with the ATPgammaS-bound 6-PF-2-K domain reveals a rearrangement of a helix that is dependent on the ligand bound in the ATP binding site of the enzyme. This helix motion dramatically alters the position of a catalytic residue (Lys172). This catalytic cation is analogous to the Arg residue donated by the rasGAP protein, and the Arg residue at the core of the GTP or GDP sensing switch motion seen in the heterotrimeric G-proteins. In addition, a succinate molecule is observed in the Fru-6-P binding site. Kinetic analysis of succinate inhibition of the 6-PF-2-K reaction is consistent with the structural findings, and suggests a mechanism for feedback inhibition of glycolysis by citric acid cycle intermediates. Alterations in the 6-PF-2-K kinetics of several proteins mutated near both the switch and the succinate binding site suggest a mode of communication between the ATP- and F6P binding sites. Together with these kinetic data, these new structures provide insights into the mechanism of the 6-PF-2-K activity of this important bifunctional enzyme.     

Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase.

We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a Mr of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced Mr of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,2-kinase were KmFru 6-P = 85 microM and KmATP = 270 microM, and those of fructose 2,6-bisphosphatase were KmFru 2,6-P2 = 21 microM and KiFru 6-P = 3.4 microM. The enzyme was phosphorylated by Fru-2,6[2-32P]P2 and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.     

A direct substrate-substrate interaction found in the kinase domain of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

To understand the molecular basis of a phosphoryl transfer reaction catalyzed by the 6-phosphofructo-2-kinase domain of the hypoxia-inducible bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), the crystal structures of PFKFB3AMPPCPfructose-6-phosphate and PFKFB3ADPphosphoenolpyruvate complexes were determined to 2.7 A and 2.25 A resolution, respectively. Kinetic studies on the wild-type and site-directed mutant proteins were carried out to confirm the structural observations. The experimentally varied liganding states in the active pocket cause no significant conformational changes. In the pseudo-substrate complex, a strong direct interaction between AMPPCP and fructose-6-phosphate (Fru-6-P) is found. By virtue of this direct substrate-substrate interaction, Fru-6-P is aligned with AMPPCP in an orientation and proximity most suitable for a direct transfer of the gamma-phosphate moiety to 2-OH of Fru-6-P. The three key atoms involved in the phosphoryl transfer, the beta,gamma-phosphate bridge oxygen atom, the gamma-phosphorus atom, and the 2-OH group are positioned in a single line, suggesting a direct phosphoryl transfer without formation of a phosphoenzyme intermediate. In addition, the distance between 2-OH and gamma-phosphorus allows the gamma-phosphate oxygen atoms to serve as a general base catalyst to induce an "associative" phosphoryl transfer mechanism. The site-directed mutant study and inhibition kinetics suggest that this reaction will be catalyzed most efficiently by the protein when the substrates bind to the active pocket in an ordered manner in which ATP binds first.