Reverse direction substrate kinetics and inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for ATP phosphoribosyltransferase were determined in the direction reverse to the biosynthetic reaction and are consistent with a sequential kinetic mechanism. Histidine inhibited the reverse reaction cooperatively and completely. Product and alternate product inhibition studies were conducted to elucidate binding order. The alternate product β,γ-methylene ATP was competitive with respect to N¹-phosphoribosyl-ATP and noncompetitive with respect to pyrophosphate. Phosphoribosylpyrophosphate was noncompetitive with respect to both substrates. These data and those of the biosynthetic direction reaction are in satisfactory quantitative agreement with the ordered Bi-Bi kinetic mechanism with ATP or phosphoribosyl-ATP binding to free enzyme.     

Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids: kinetics

A steady-state kinetic analysis has been performed on the pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria and castor bean (Ricinus communis L.) proplastids. Substrate interaction kinetics for all substrates gave parallel lines consistent with a multisite ping-pong mechanism. Product inhibition studies showed uncompetitive inhibition between acetyl-CoA and pyruvate and competitive inhibition between NADH and NAD⁺, both of which are also consistent with this mechanism. In the mitochondrial complex, acetyl-CoA showed noncompetitive inhibition versus CoA which suggests that the intermediate complex is kinetically important in the lipoamide transacetylase component of this complex. In contrast, the proplastid complex showed competitive inhibition in this interaction. NADH is a noncompetitive inhibitor versus CoA in both complexes indicating that these complexes, like the mammalian complex, may have protein-protein interactions between the second and third enzymes of the complex. Since NADH also shows noncompetitive inhibition versus pyruvate, this interaction may extend to all components of the complex. Acetyl-CoA shows noncompetitive inhibition versus NAD⁺ which may also be a result of interaction between the second and third enzymes of the complex. The limiting Michaelis constants for substrates and the inhibitor constants for both complexes were determined.     

The two 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition

Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 μM for phosphoenolpyruvate and 500 μM for erythrose 4-phosphate. The rate constant was calculated as 6 s^–1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (K _i of 0.9 μM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate. Received: 29 December 1997 / Accepted: 3 March 1998     

A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium

A one step procedure is presented for the preparation of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using pyruvate kinase. The reaction is carried out at chemical equilibrium and involves only an exchange of isotope between ATP and phosphoenolpyruvate. The initial phosphoenolpyruvate/ATP ratio in the reaction mixture determines the degree of ³²P incorporation into phosphoenolpyruvate when isotopic equilibrium is achieved.     

The disposition of citric acid cycle intermediates by isolated rat heart mitochondria

The mechanism of depletion of tricarboxylic acid cycle intermediates by isolated rat heart mitochondria was studied using hydroxymalonate (an inhibitor of malic enzymes) and mercaptopicolinate (an inhibitor of phosphoenolpyruvate carboxykinase) as tools. Hydroxymalonate inhibited the respiration rate of isolated mitochondria in state 3 by 40% when 2 mM malate was the only external substrate, but no inhibition was found with 2 mM malate plus 0.5 mM pyruvate as substrates. In the prescence od bicarbonate, arsenite and ATP, propionate was converted to pyruvate and malate at the rates of 14.0 ± 2.9 and 2.8 ± 1.8 nmol/mg protein in 5 min, respectively. Under these conditions, 0.1 mM mercaptopicolinate did not affect this conversion, but 2 mM hydroxymalonate inhibited pyruvate formation completely and resulted in an accumulation of malate up to 13.2 ± 2.9 nmol/mg protein. No accumulation of phosphoenolpyruvate was found under any condition tested. It is concluded that malic enzymes but not phosphoenolpyruvate carboxykinase, are involved in conversion of propionate to pyruvate in isolated rat heart mitochondria.     

Plant aspartate transcarbamylase: kinetic properties of the enzyme from mung bean (Phaseolus aureus) seedlings

Aspartate transcarbamylase (EC 2.1.3.2) catalyzes the bi substrate reaction—carbamyl phosphate+ L-aspartate ? carbamyl aspartate ? phosphate, The order of addition of substrates and release of products for the homogeneous aspartate transcarbamylase fromPhaseolus aureuss eedlings has been investigated by using the kinetic methods of analysis. p ]Initial velocity studies indicated that the mechanism might be a sequential one. Product inhibition studies showed that phosphate was a linear competitive inhibitor with respect to carbamyl phosphate and was anS (slope) andI (intercept) linear noncompetitive inhibitor with respect to aspartate. Carbamyl aspartate was a noncompetitive inhibitor with respect to both the substrates. These inhibition patterns agreed with an ordered mechanism of reaction with carbamyl phosphate as the leading substrate and phosphate as the last product to leave the enzyme surface. The presence of dead end complexes and the rapid equilibrium random mechanism were ruled out by the absence of inhibition by the substrate(s) and the linear replot slopevs. the inhibitor concentration. Acetyl phosphate, an analog ue of carbamyl phosphate was a non-competitive inhibitor with respect to aspartate. This result could be explained both in terms of an ordered as well as a random mechanism. On the other hand, succinate, an analog ue of aspartate was an uncompetitive inhibitor with respect to carbamyl phosphate, indicating that the mechanism was ordered. p ]The transition state analog ue, N-(phosphonoacetyl)-L-aspartate, binds much more tightly than either of the two substrates. This analog ue was a linear competitive inhibitor with respect to carbamyl phosphate and a linear noncompetitive inhibitor with respect to aspartate. These results are compatible with an ordered mechanism rather than a random one.     

Inhibition of phosphoenolpyruvate carboxylase by malate

Malate has been noted to be a `mixed' inhibitor of phosphoenolpyruvate (PEP) carboxylase. The competitive portion of this inhibition appears to be fairly constant regardless of the condition of the enzyme being measured, but the noncompetitive (V-type) inhibition is subject to variation depending on the source of the enzyme, its storage condition, the presence or absence of various ligands, and differences in pH. In the case of the maize (Zea mays L.) phosphoenolpyruvate carboxylase (PEPC), the V-type inhibition by malate is much less pronounced at pH 8 than at pH 7. Examination of the response of the maize PEPC to PEP concentration reveals a pronounced cooperativity at pH 8 which is not present at pH 7, and which results in the disappearance of the V-type inhibition at pH 8. The ability of high concentrations of PEP to convert PEPC from a form readily inhibited by malate to one resistant to malate inhibition has been previously demonstrated and we attribute the cooperativity shown at pH 8 to this response to high levels of PEP. Support for this proposal is provided by studies of the enzyme at pH 7 and pH 8 run in 20% glycerol. In this case there was no V-type inhibition of PEPC at either pH. Treatment with 20% glycerol has been shown to result in the aggregation of maize PEPC.     

Fluorescence Study of Chemical Modification of Phosphoenolpyruvate Carboxylase from Crassula argentea

The chemical modification of phosphoenolpyruvate carboxylase purified from Crassula argentea leaves was studied using the fluorescence of the extrinsic probe 8-anilino-1-naphalenesulfonate. The effects of ligands on kinetic parameters of phosphoenolpyruvate carboxylase activity, and its response to pH and metal cations, were associated with the binding of the ligands to the enzyme as measured by fluorescence. Binding of the ligands phosphoenolpyruvate, malate, and glucose-6-phosphate revealed by fluorescence measurements corresponds to competitive phenomena observed in kinetic studies. The fluorescence measurements also suggest the involvement of specific amino acids in the binding of a given ligand. Arginyl residues modified by 2,3-butanedione appear to be directly involved in the binding of phosphoenolpyruvate and malate to the active and the inhibition sites, respectively. A histidyl residue was involved in the binding of malate, accounting for the lack of inhibition by malate in kinetic studies of the enzyme treated with diethylpyrocarbonate. Although activity was lost, there was no decrease in the ability of the treated enzyme to bind phosphoenolpyruvate, suggesting that additional histidyl residues are essential for activity although not directly involved in the binding of phosphoenolpyruvate. The lysine reagent trinitrobenzenesulfonate caused a loss of activity and a reduction in malate inhibition and glucose-6-phosphate activation, but these modifications were not related to changes in the ability of the enzyme to bind any of the three ligands. This suggests that lysine residues were not directly involved in the binding of these ligands.     

STEADY STATE KINETICS OF RAT BRAIN PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX

Abstract— The overall steady state kinetic mechanism of pyruvate dehydrogenase multienzyme complex purified from rat brain has been investigated. Initial rate patterns were a series of parallel lines regardless of which substrate was varied at several fixed concentrations of other substrates. Product inhibition patterns showed that acetyl CoA is competitive vs CoA, that NADH is competitive vs NAD, and that both acetyl CoA and NADH are uncompetitive vs pyruvate. Both acetyl CoA and NADH are noncompetitive vs NAD and CoASH, respectively. These results are inconsistent with classical 'hexa uni' ping-pong mechanisms, but are consistent with a non-classical 3-site ping-pong mechanism.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase

The effect of 5-5′-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V_max is found to be independent of the presence of this thiol reagent. The K_m is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K_m (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K_m for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.     

Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of adenine nucleotides on the reaction catalyzed by pyruvate,orthophosphate dikinase in maize

The effect of adenine nucleotides in pyruvate, orthophosphate dikinase (EC 2.7.9.1, ATP, pyruvate, orthophosphate phosphotransferase)_was studied with the enzyme furified from maize, and with the enzyme obtained from mesophyll chloroplast extracts during assay in the direction of pyruvate conversion to phosphoenolpyruvate. (1) In studies with the purified enzyme, the relationship of initial velocity to ATP concentrations follows Michaelis-Menten kinetics, and the K_m value for ATP was 22.8 μM (± 5.1 μM, n = 5). (2) AMP was a competitive inhibitor with respect to ATP, and its K_i value was 35.8 μM (± μM, n = 4). There was no inhibition of catalysis by ADP up to a concentration of 460 μM. (3) The theoretical response of the enzyme to change in the adenylate energy charge was calculated from the kinetic constants for ATP and AMP. The experimentally obtained values were similar to the theoretical response when varying energy charge was generated by addition of appropriate amounts of ATP, ADP and AMP in assays with the purified enzyme. The response of the enzyme to energy charge at different pH values (pH 7.0, 7.5, and 8.0) was similar, although the activity of the enzyme at pH 7.0 was about 40% of that at pH 8.0. (4) When mesophyll chloroplast extracts of maize, which contain high levels of adenylate kinase, were used as the source of the enzyme and the adenylate energy charge was generated by addition of different concentrations of ATP and AMP, the influence on catalysis was similar to that with the purified enzyme. (5) The data show that the effect of varying energy chage on the activity of the dikinase is not typical of a U-type enzyme, in contrast to phosphoglycerate kinase (EC 2.7.2.3, ATP: 3-phospho-D-glycerate 1-phosphotransferase), which is more strongly regulated. (6) Evidence is presented for competition between the dikinase and phosphoglycerate kinase for ATP in mesophyll chloroplast extracts of maize. (7) When the effect of adenylate energy charge on the state of activation and the direct effect on catalysis of the dikanase are combined, the total capacity for catalysis is very dependent on the energy charge.     

Electrophoretic pattern of cytosolic pyruvate kinase fractions A and B (type L and M2) from normal rat liver and Morris hepatoma 7777

Cytosolic pyruvate kinase fractions A and B obtained by salting out procedure from normal rat liver and Morris hepatoma 7777, purified by affinity chromatography on Blue Sepharose CL-6B, have shown similar electrophoretic patterns in polyacrylamide gel at pH 8.3, to previously studied pyruvate kinase extracts from chromatin of cell nuclei. Three variants (α₁, β₁, γ₁) from normal liver pyruvate kinase fraction A (type L) had the greatest electrophoretic mobility, showed sigmoidal kinetics in relation to 2-phosphoenolpyruvate (PEP), and sensitivity to ATP and fructose 1,6-diphosphate (FDP). The fraction A dominated over normal liver fraction B (type M₂), which in electrophoresis showed a slower y2 variant, similar to the fraction A of hepatoma. All variants from fractions B of normal liver and A of hepatoma had linear kinetics and were sensitive to ATP but not to FDP. The greatest differences showed pyruvate kinase fraction B from Morris hepatoma. Its all variants α₂, β₂, γ₃ were more cathodic and had linear kinetics in relation to PEP. They all were insensitive to normal signal molecules (ATP and FDP). The γ₃ alkaline variant acquired sensitivity to inhibition by l-cysteine. Showing several-fold higher activity, much greater affinity to the main substrate, and a lack of sensitivity to feed-back inhibition by ATP, it was responsible for a high rate of aerobic glycolysis and diminution of the Pasteur effect in metabolic studies. It was probably encoded during oncogene activation and plays a special role in different metabolic strategies of tumour cells.     

Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum

The assimilation of NH₄⁺ causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoenolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH₄⁺ assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.     

Reaction mechanism of aspartate transcarbamylase from mouse spleen

The reaction mechanism of aspartate transcarbamylase from mouse spleen has been determined, using steady-state kinetics, isotope-exchange experiments, inhibition studies with a transition-state analog, and product-inhibition studies. Intersecting reciprocal plots obtained when one substrate was varied against different concentrations of the second substrate indicate that the mechanism is sequential. The transition-state analog, N-(phosphonacetyl)-l-aspartate, was a powerful inhibitor of aspartate transcarbamylase, with an inhibition constant (K_i) of 2.6 × 10^?8m at 37 °C and pH 7.4 in 0.05 m Na HEPES buffer. PALA gave competitive inhibition with carbamyl phosphate and noncompetitive inhibition with l-aspartate, indicating that carbamyl phosphate must bind before aspartate for catalysis to occur. A ping-pong mechanism in which carbamyl phosphate binds first was excluded by isotope-exchange experiments, since [³²P]inorganic phosphate was not incorporated into carbamyl phosphate in the absence of aspartate. Product-inhibition studies showed that only inorganic phosphate and carbamyl phosphate gave a competitive pattern; all other combinations of substrate and product gave noncompetitive inhibition patterns when incubations were carried out at subsaturating concentrations of the second substrate. These inhibition patterns showed that carbamyl phosphate binds first, aspartate binds second, carbamyl aspartate dissociates first, and phosphate dissociates second.     

Regulation of phosphoenolpyruvate carboxylase in C4 plants: inhibition by pyrophosphate of the enzyme from Amaranthus viridis

Phosphoenolpyruvate carboxylase from the leaves of Amaranthus viridis was inhibited by pyrophosphate. The inhibition was competitive with respect to phosphoenolpyruvate (K_i 0.85 mm) and noncompetitive with respect to bicarbonate.     

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.