Km For PPi Research Articles

Characterization of the pyrophosphate-dependent 6-phosphofructokinase fromMethylococcus capsulatusBath

An active pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) from the thermotolerant methanotroph Methylococcus capsulatus Bath, containing a six-residue polyhistidine tag, was characterized. The enzyme was homodimeric (2 x 45 kDa), nonallosteric and most active at pH 7.0. PPi-PFK catalyzed reactions of PPi-dependent phosphorylation of fructose-6-phosphate (F-6-P) (K(m) 2.27 mM and V(max) 7.6 U mg(-1) of protein), sedoheptulose-7-phosphate (K(m) 0.027 mM and V(max) 31 U mg(-1)) and ribulose-5-phosphate. In the reaction with F-6-P, the apparent K(m) for PPi was 0.027 mM, while in the reverse reaction, K(m) for orthophosphate was 8.69 mM and that for fructose-1,6-bisphosphate 0.328 mM (V(max) 9.0 U mg(-1)). Phylogenetically, M. capsulatus PPi-PFK was most similar to PPi-PFKs from the lithoautotrophic ammonia oxidizers Nitrosomonas europaea (74.0%), Nitrosospira multiformis (73.6%) and Betaproteobacterial methylotroph Methylibium petroleiphilum PM1 (71.6% identity). Genes coding PPi-PFK and a putative V-type H(+)-translocating pyrophosphatase (H(+)-PPi-ase) were cotranscribed as an operon. The potential significance of the PPi-PFK for regulation of carbon and energy fluxes in M. capsulatus Bath is discussed.

Site-Directed Mutagenesis of the Fructose 6-Phosphate Binding Site of the Pyrophosphate-Dependent Phosphofructokinase of Entamoeba histolytica

Attempts to define the active site of pyrophosphate-dependent phosphofructokinase (PPi-PFK) using homology modeling based on the three-dimensional structure of the ATP-dependent PFKs from bacteria have been frustrated by low sequence identity between PPi- and ATP-PFKs in their carboxyl terminal halves. In the current study, alanine scanning mutagenesis of residues in the carboxyl terminal half of the PPi-PFK of Entamoeba histolytica coupled with comparative sequence analysis and computational modeling is used to identify residues that contribute to fructose 6-phosphate (fructose 6-P) binding. Of seven alanine mutants that were generated by site-directed mutagenesis, Arg377, Ser392, Arg405, Lys408, His415, His416, and Arg423, only the last mutant, Arg423Ala, was found to have dramatically lower affinity for fructose 6-P. Mutation of Arg 423 decreased kcat by 10,000-fold and decreased apparent affinity for fructose 6-P by 126-fold, while the Km for PPi increased only 4-fold. The second greatest effect was seen with Arg377Ala, which had a nearly 10-fold decrease in apparent affinity and an approximate 60-fold decrease in maximal activity. Another residue, Tyr420, was chosen for mutagenesis by its complete identity in all other PPi-PFK. This residue and its homologue in Escherichia coli ATP-PFK, His249, were mutated and shown to be very important for substrate binding in both enzymes.

Point mutations in the guanine phosphoribosyltransferase from Giardia lamblia modulate pyrophosphate binding and enzyme catalysis

Guanine phosphoribosyltransferase (GPRTase) from Giardia lamblia, an enzyme required for guanine salvage and necessary for the survival of this parasitic protozoan, has been kinetically characterized. Phosphoribosyltransfer proceeds through an ordered sequential mechanism common to many related purine phosphoribosyltransferases (PRTases) with α‐D‐5‐phosphoribosyl‐1‐pyrophosphate (PRPP) binding to the enzyme first and guanosine monophosphate (GMP) dissociating last. The enzyme is a highly unique purine PRTase, recognizing only guanine as its purine substrate (Km = 16.4 µm) but not hypoxanthine (Km > 200 µm) nor xanthine (no reaction). It also catalyzes both the forward (kcat = 76.7 s−1) and reverse (kcat = 5.8·s−1) reactions at significantly higher rates than all the other purine PRTases described to date. However, the relative catalytic efficiencies favor the forward reaction, which can be attributed to an unusually high Km for pyrophosphate (PPi) (323.9 µm) in the reverse reaction, comparable only with the high Km for PPi (165.5 µm) in Tritrichomonasfoetus HGXPRTase‐catalyzed reverse reaction. As the latter case was due to the substitution of threonine for a highly conserved lysine residue in the PPi‐binding loop [Munagala et al. (1998) Biochemistry37, 4045–4051], we identified a corresponding threonine residue in G. lamblia GPRTase at position 70 by sequence alignment, and then generated a T70K mutant of the enzyme. The mutant displays a 6.7‐fold lower Km for PPi with a twofold increase in the Km for PRPP. Further attempts to improve PPi binding led to the construction of a T70K/A72G double mutant, which displays an even lower Km of 7.9 µm for PPi. However, mutations of the nearby Gly71 to Glu, Arg, or Ala completely inactivate the GPRTase, suggesting the requirement of flexibility in the putative PPi‐binding loop for enzyme catalysis, which is apparently maintained by the glycine residue. We have thus tentatively identified the PPi‐binding loop in G. lamblia GPRTase, and attributed the relatively higher catalytic efficiency in the forward reaction to the unusual loop structure for poor PPi binding in the reverse reaction.

Changes of H+ pumps of tonoplast vesicle from wheat roots in vivo and in vitro under aluminum treatment and effect of calcium

Wheat seedlings were treated with 0.1 mM Al for 7 days. Tonoplast vesicles were isolated from apical segments of roots and activities of H+‐ATPase and H+‐PPase measured. Compared with the control, 0.1 mM Al increased H+‐ATPase activity and decreased H+‐PPase activity. High external supply of calcium (Ca) (5 mM) diminished the extent of the stimulation of H+‐ATPase activity and alleviated the reduction of H+‐PPase activity under Al treatment. However, 0.1 mM Al treatment in vitro resulted in the inhibition of H+‐ATPase activity with the decrease of Vmax and Km for ATP. In vitro treatment with 0.1 mM Al also decreased the H+‐PPase activity, but increased the Km for PPi.

Steady-state kinetics of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus: the role of threonine-47.

Tritrichomonas foetus, an anaerobic flagellated protozoan, causes urogenital trichomoniasis in cattle. Hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRTase), an essential enzyme in T. foetus required for salvaging exogenous purine bases, has been regarded as a promising target for anti-tritrichomonial chemotherapy. The steady-state kinetic analyses of synthesis and pyrophosphorolysis of IMP, GMP, and XMP and product inhibition studies have been used to elucidate the reaction mechanisms. Double-reciprocal plots of initial velocities versus the varying concentrations of one substrate at a fixed concentration of the other show intersecting lines indicating a sequential mechanism for both the forward and the reverse reactions. In terms of the kcat/Km ratios, hypoxanthine is the most effective substrate whereas guanine and xanthine are converted equally well into their corresponding nucleotides. The minimum kinetic model from the data in product inhibition studies is an ordered bi-bi mechanism, where the substrates bind to the enzyme (first PRPP followed by the purine bases), and the products released (first PPi followed by purine nucleotide) in a defined order. The Kms for PPi in the T. foetus HGXPRTase-catalyzed reactions are unusually high, close to the millimolar range. Since the crystal structure of this enzyme [Somoza et al. (1996) Biochemistry 35, 7032-7040] suggests potential binding between the threonine-47 in a conserved cis-peptide loop and PPi whereas human HGPRTase has lysine-68 [Eads et al. (1994) Cell 78, 325-334] at the corresponding position, we prepared a T47K enzyme mutant and found in the T47K-catalyzed reaction a 4-10-fold decrease of Km for PPi. The lack of ionic interactions between Thr-47 and PPi and an increased distance between the loop and the active site as compared to the human HGPRTase are thus proposed to be responsible for the high Km for PPi in the T. foetus HGXPRTase-catalyzed reaction.

Identification of basic residues involved in substrate binding and catalysis by pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.

Despite overall low identity of sequence between ATP- and PPi-dependent phosphofructo-1-kinases (PFK), an alignment permits the tentative identification of catalytically important residues in PPi-dependent PFK. Seven basic residues of the PPi-dependent PFK of Propionibacterium freudenreichii, Arg-310, Lys-315, Arg-326, Arg-186, Arg-78, Arg-90, and Lys-148 were chosen on the basis of this alignment for site-directed mutagenesis to neutral residues. Mutants R186A, K315A, and R326A had substantial increases of 43-, 389- and 678-fold, respectively, in Km for fructose 6-phosphate relative to wild-type enzyme and relatively small effects on kcat. The modest kinetic effects of mutations at Arg-78, Arg-90, and Arg-310 were not considered to indicate important roles for these residues. On the other hand, mutant K148M had a Km for PPi that was increased by 132-fold and kcat lowered by a factor of 490. Cho and Cook (Cho, Y.-K., and Cook, P.F. (1988) Biochemistry 28, 4155-4160) concluded on the basis of kinetic and chemical modification data that the enzyme utilizes a proton shuttle mechanism and that two lysines are involved in substrate binding. The present data along with our previous data on mutant D151A support a double proton shuttle mechanism involving Asp-151 and Lys-148 with Lys-148, Arg-326, Lys-315, and perhaps Arg-186 being important for substrate binding.

A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii.

An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homogeneity. The molecular mass estimated by gelfiltration was 139 +/- 7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an alpha 2 beta 2 oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri- and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg2+ was required for activity. The pH optimum was 8 at 1 mM PPi and 5 mM Mg2+. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent Km for PPi of 0.1 mM in the presence of 5 mM Mg2+. The Vmax was 590 mumol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a Kcat of 1400 per second. The enzyme was found in the soluble enzyme fraction after ultracentrifugation, when cells were disrupted by French Press. Upto 5% of the pyrophosphatase was associated with the membrane fraction, when gentle lysis procedure were applied.

Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase.

The crystal structure of a fully biologically active monomeric form of Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990) J. Mol. Biol. 216, 411-424), revealing details of the active site of the enzyme, including the location of amino acid residues potentially involved in substrate binding. In the present paper, the role of 3 active site residues in interaction with methionine, ATP, and tRNA(fMet) and in catalysis of methionyl-adenylate has been explored using site-directed mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala produced a 14-fold increase in the Km for ATP with only a small (2-3-fold) change in the other kinetic parameters, indicating that the major role of this residue is in formation of the initial complex with ATP and/or in stabilization of the methionyl-adenylate reaction intermediate. Mutation of the adjacent residue Tyr358 to Ala had no effect on the Km values for methionine or ATP but produced nearly a 2000-fold decrease in the rate of ATP-PPi exchange. This mutation also dramatically reduced the rate of pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of pyrophosphate without increasing the Km for PPi. None of the mutations affected the Km for tRNAfMet in the aminoacylation reaction. The results suggest that Tyr358 may enhance the rate of methionyl-adenylate formation by binding to the alpha-phosphate of ATP in the transition state. Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction requires a significant change in the conformation of this region of the active site compared to the structure found in the MetRS.ATP complex. Such a shift is consistent with an induced-fit mechanism for methionine activation. Primary sequence comparisons of methionine-specific enzymes from yeast and bacterial sources reveals that Tyr358 is conserved in all of the known MetRS sequences.

Characterization of the pyrophosphate‐dependent proton transport in microsomal membranes from maize roots

Cleared maize (Zea mays L. cv. LG 11) root homogenates were prepared and layered on the top of sucrose step gradients (10, 35 and 45%). The ATP‐ and pyrophosphate (PPi)‐dependent proton‐pumping activities were recovered almost completely at the 10%/35% interface, corresponding to the microsomal fraction (Golgi, tonoplast and endoplasmic reticulum). The PPi‐dependent proton pump was characterized by the fluorescence quenching of quenching of quinacrine. The pH optimum was 7 to 8. The H+‐PPase was Mg2+‐dependent and the Km for PPi (in the presence of 3 mM MgSO4) was 28 μM. The pump was electrogenic, K+‐dependent and a permeant anion was necessary to dissipate the membrane potential (NO3−= I− >Br− > Cl−). No activity was detected in the presence of electroneutral proton inonophores or, when valinomycin was added, with electrogenic ionophores. The H+‐PPase was insensitive to vanadate, oligomycin and molybdate. ‐Diethylstilbestrol (DES) and N,N′‐dicyclohexylcarbodiimide (DCCD) were strongly inhibitory at 100 μM.

Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus.

An inorganic pyrophosphatase was purified over 600-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme is a tetramer of Mr = 84,000, has a sedimentation coefficient of 5.8S, a Stokes radius of 3.5 nm, and an isoelectric point of 5.7. Like the enzyme of Escherichia coli, the pyrophosphatase appears to be made constitutively. The pH and temperature optima are 8.3 and 80 degrees C, respectively. The Km for PPi is 0.6 mM. A divalent cation is essential, with Mg2+ preferred. The enzyme uses only PPi as a substrate.

Enzymatic Phosphorylation of Serine

Abstract The occurrence of a pyrophosphate:l-serine phosphotransferase in extracts of Propionibacterium shermanii is reported. A 102-fold purification, with 15% recovery, was obtained by successive use of protamine sulfate precipitation, acidification, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and on Bio-Gel P-150 columns. This purification eliminates inorganic pyrophosphatase and serine phosphatase activity present in the crude extract, but a serine-serine phosphate exchange activity remains. Evidence is presented that this exchange is catalyzed by a second enzyme. The enzyme is stable at room temperature, but rapidly loses activity above 50°. In the presence of l-serine the enzyme exhibits a somewhat higher lability to preliminary incubation at elevated temperatures. The Km for PPi is 1.0 x 10-4 m; the Km for l-serine is 1.9 x 10-3 m. The enzyme is specific for both substrates. ATP is not a substrate. Tripolyphosphate can substitute to some extent for PPi. d-Serine and l-cysteine are not phosphorylated. dl-Homoserine is phosphorylated to a slight extent, as is l-threonine. Mg++ greatly stimulates the reaction. Some stimulation is obtained with Mn++ and with Co++. The Km for Mg++ at 1 mm PPi is 1.3 x 10-4 m, very close to the Km for PPi at 1 mm Mg++. Kinetic evidence suggests that the enzyme operates by a random equilibrium mechanism, involving a ternary complex of enzyme and substrates. The reaction has an apparent equilibrium constant of 480 at pH 6.7 and of 950 at pH 7.7, and an Arrhenius activation energy of 14.7 kcal per mole at pH 7.8.

Inorganic Pyrophosphatase Activity of Dog Aorta

SummaryDog aortas have been found to contain a Mg2+-stimulated inorganic pyrophosphate phosphohydrolase activity. The enzyme is maximally active at pH 7.5 and has a Km for PPi of 3 μM at this pH. This activity is extremely sensitive to inhibition by Ca2+. A role for this latter phenomenon in a postulated mechanism for the inhibition of calcification of the aorta is suggested.

Enzymatic Synthesis of Glycerol 1-Phosphate (D-α-Glycerophosphate): INORGANIC PYROPHOSPHATE-GLYCEROL PHOSPHOTRANSFERASE AND INORGANIC PYROPHOSPHATE-GLUCOSE PHOSPHOTRANSFERASE COMPARED

Abstract Microsomal particulate fractions of rat organ homogenates are capable of catalyzing the synthesis of glycerol 1-phosphate1 from inorganic pyrophosphate and glycerol. The product has been isolated and characterized by elementary analysis, specific rotation, and chemical and enzymatic studies. PPi-glycerol phosphotransferase is presumably specific for the primary carbinol group of glycerol opposite to that which is phosphorylated by ATP-glycerol phosphotransferase (glycerol kinase). The distribution and some of the kinetic properties of PPi-glycerol phosphotransferase have been studied and compared with those of PPi-glucose phosphotransferase, which catalyzes the synthesis of glucose 6-phosphate. The two activities are roughly parallel in liver, kidney, and intestinal mucosa, and glucose is a competitive inhibitor of the synthesis of glycerol 1-phosphate by these organ preparations. Spleen, brain, and lung preparations catalyze the phosphorylation of glycerol from PPi while they are completely incapable of glucose 6-phosphate synthesis. In these organs glucose does not inhibit the synthesis of glycerol 1-phosphate. This suggests that there are at least two transferase enzymes which phosphorylate glycerol, one of which may be the same as the liver PPi-glucose phosphotransferase that is probably identical with glucose 6-phosphatase. Liver PPi-glycerol phosphotransferase has a pH optimum of about 5.0, is activated by preliminary treatment with deoxycholate or by NH4OH at pH 9.5, has no requirement for Mg++ or other added cofactor, and has a Km for PPi of 5 x 10-3 and for glycerol of about 3 m. The rate of hydrolysis of inorganic pyrophosphate, which proceeds simultaneously with synthetic transferase activity, is inhibited by glucose and accelerated by glycerol. This phenonemon is observed also when studied as a function of pH or of PPi, glucose, or glycerol concentration. Cytidine triphosphate or glucose-6-P can replace PPi as an efficient donor, while ATP is relatively inactive in the synthesis of glycerol 1-phosphate by the liver microsomal enzyme. Glycerol 1-phosphate is hydrolyzed at a much faster rate than is glycerol 3-phosphate by the liver microsomal preparations. This enzymatic synthesis provides a convenient method for the preparation of glycerol 1-phosphate (D-α-glycerophosphate).