Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.     

Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe)from Escherichia coli K12. 2. Kinetic properties.

1. Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate. The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position. 3. The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM. 4. The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0. The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unaffected by the binding of phosphoenolpyruvate.     

Enzymatic Studies on the Conversion of Erythritol Fermentation to d-Mannitol Fermentation in n-Alkane-grown Yeast Candida zeylanoides

In the polyol fermentation by Candida zeylanoides KY6166, which occurred preferentially by keeping the pH of medium at acidic side (below 4.0), phosphate ion played a precise role in the conversion of erythritol fermentation to d-mannitol fermentation. Enzymatic studies on the conversion mechanism provided the following evidences.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphate ion.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphateion. From these results, the alteration from erythritol fermentation to mannitol fermentation by phosphate ion was explained as the result of the change in the level of erythrose 4-phosphate and fructose 6-phosphate which was caused by the inhibition of transketolase.     

The kinetics of a purified form of 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (tryptophan) from Neurospora crassa.

1. A method is described for the purification of a form of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) that probably differs from that of the native enzyme. 2. The kinetics of the reaction catalysed by 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) shows that the reaction proceeds via a ping-pong bi-bi mechanism, with activation by phosphoenolpyruvate (P-Prv), the first substrate, and inhibition by erythrose 4-phosphate (Ery-P) the second substrate. At low substrate concentrations, KP-Prv is 0.1 mM and KEry-P is 0.13 mM. 3. The substrates phosphoenolpyruvate and erythrose 4-phosphate and the product inorganic phosphate can protect the purified enzyme against heat denaturation, whereas the inhibitor, tryptophan, has no effect, although it binds to the enzyme in the absence of other ligands. 4. Product inhibition by inorganic phosphate is linear non-competitive with respect to phosphoenolpyruvate (Ki, slope = 22 mM and Ki, intercept = 54 mM) and substrate-linear competitive with respect to erythrose 4-phosphate (Ki, slope = 25 mM). 5. The enzyme has an activity optimum at pH 7.3 and a tryptophan inhibition optimum at pH 6.4, Trp 0.5 is 4 microM. Inhibition by tryptophan is non-competitive with respect to phosphoenolpyrovate and substrate-parabolic competitive with respect to erythrose 4-phosphate. 6. The role of the enzyme in metabolic regulation is discussed.     

Non-oxidative synthesis of pentose 5-phosphate from hexose 6-phosphate and triose phosphate by the L-type pentose pathway

1. Ribose 5-phosphate was non-oxidatively synthesized from glucose 6-phosphate and triose phosphate by an enzyme extract prepared from rat liver (RLEP). Analysis of the intermediates by GLC, ion-exchange chromatography and specific enzymatic analysis, revealed the presence of the following intermediates of the L-type pentose pathway: altro-heptulose 1,7-bisphosphate, arabinose 5-phosphate and D-glycero D-ido octulose 8-phosphate. 2. With either [1-14C] or [2-14C]glucose 6-phosphate as diagnostic substrates, the distribution of 14C in ribose 5-phosphate was determined. At early time intervals (0.5-8 hr), [1-14C]glucose 6-phosphate introduced 14C into C-1, C-3 and C-5 of ribose 5-phosphate, at 17 hr 14C was confined to C-1. With [2-14C]glucose 6-phosphate as substrate, 14C was confined to C-2, C-3 and C-5 of ribose 5-phosphate during early times (0.5-8 hr), while at 17 hr 14C was located in C-2. 3. The transketolase exchange reaction, [14C]ribose 5-phosphate + altro-heptulose 7-phosphate in equilibrium ribose 5-phosphate + [14C]altro-heptulose 7-phosphate, was demonstrated for the first time using purified transketolase, its activity was measured and it is proposed to play a major role in the relocation of 14C into C-3 and C-5 or ribose 5-phosphate during the prediction labelling experiments. 4. The coupled transketolase-transaldolase reactions, 2 fructose 6-phosphate in equilibrium altro-heptulose 7-phosphate + xylulose 5-phosphate and 2 altro-heptulose 7-phosphate in equilibrium fructose 6-phosphate + D-glycero D-altro octulose 8-phosphate were demonstrated with purified enzymes, but are concluded to play a minor role in the non-oxidative synthesis of pentose 5-phosphate and octulose phosphate by (RLEP). 5. The formation of gem diol and dimers of erythrose 4-phosphate is proposed to account in part for the failure to detect monomeric erythrose 4-phosphate in the carbon balance studies. 6. The equilibrium value for the pentose pathway acting by the reverse mode in vitro was measured and contrasted with the value for the pathway acting in the forward direction. The initial specific rates of the pentose pathway reactions in vitro for the reverse and forward directions are measured. 7. The study which includes carbon balance, time course changes and 14C prediction labelling experiments reports a comprehensive investigation of the mechanism of the pentose pathway acting reversibly.     

Structure and mechanism of 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 A (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy.     

Phosphoenolpyruvate carboxykinase (guanosine 5'-triphosphate) from rat liver cytosol. Divalent cation involvement in the decarboxylation reactions

The presence of a divalent metal ion together with a catalytic amount of inosine 5'-diphosphate (IDP) is essential for the formation of pyruvate from oxalacetate catalyzed by purified rat liver cytosol phosphoenolpyruvate carboxykinase (PEPCK). With decreasing order of effectiveness, this pyruvate-forming activity was supported by micromolar levels of Cd2+, Zn2+, Mn2+, and Co2+. At the same concentrations, Mg2+ or Ca2+ was not effective. Combinations of Cd2+ with either Zn2+, Mn2+ or Co2+ were not additive with respect to the pyruvate-forming activity of PEPCK. Kinetic determination, with Cd2+ as the supporting cation, showed a 1:1 stoichiometry of interaction between each enzyme molecule and the nonconsumable substrate IDP. With 10 muM added Cd2+, the apparent Km for oxalacetate was 41 muM, and the apparent Ka for IDP was 0.25 muM. With Zn2+ or Mn2+, the apparent Ka for IDP was 0.2 or 0.13 muM, respectively. The effect of divalent transition-metal ions on PEPCK-catalyzed formation of phosphoenolpyruvate from oxalacetate was also investigated. Under steady-state conditions, the basal activity with MgITP was effectively enhanced with micromolar levels of Mn2+, Cd2+, or Co2+ included in the assay. The Vm increased 7- and 3.6-fold, and the apparent Km for MgITP changed by about a factor of 2 with the optimal concentrations of Mn2+ and Co2+, respectively. The most striking changes were in the apparent Km values for oxalacetate, which decreased to one-third and one-tenth when either Mn2+ or Co2+ was present in the assay together with Mg2+. The possible physiological importance of this kinetic effect is discussed.     

An enyzmic method for the analysis of D-erythrose 4-phosphate.

Two modifications of an enzymic method for the measurement of erythrose 4-phosphate, based on reactions catalyzed by transketolase, are described. Unlike the method generally employed, the new procedure is sensitive and highly specific and applicable to the assay of erythrose 4-phosphate in tissue extracts.     

Mechanistic implications of the pH independence of inhibition of phosphoglucose isomerase by neutral sugar phosphates.

In contrast to the strongly pH-dependent inhibition of phosphoglucose isomerase by substrate analogues with a free carboxyl group, inhibition of this enzyme by neutral sugar phosphates is essentially invariant between pH 7 and 9. Competitive inhibition constants for glucitol 6-phosphate (40 muM), arabinose 5-phosphate (50 muM), and erythritol 4-phosphate (100 muM) were found to be of the same order of magnitude as that reported previously for substrate binding constants (50 to 240 muM). The unique exception is erythrose 4-phosphate whose Ki (0.7 muM, independent of pH) reflects a tightness of binding similar to that found at pH values near or below neutrality for the transition state analogue 5-phosphorarabinonate. The pH independence of inhibition by erythrose 4-phosphate and other neutral sugar phosphates may reflect a mode and locus of binding to phosphoglucose isomerase different from that of the aldonate inhibitors.     

Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.     

Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus

The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus has been expressed in Escherichia coli. The expressed protein was insoluble but was partially solubilized as a dimer by the inclusion of 200 mM KCl in the cell lysis buffer. An effective two step purification procedure has been developed. The first step resulted in a high degree of purification and involved lysis by sonication at approximately 40 degrees C followed by a heat treatment at 70 degrees C. A continuous assay measuring the loss of PEP at 232 nm at elevated temperatures was also developed. Temperature, pH, and divalent metal ions all had an effect on the extinction coefficient of PEP. Purified recombinant P. furiosus DAH7PS is a dimer with a subunit Mr of 29,226 (determined by ESMS), shows resistance to denaturation by SDS, has activity over a broad pH range, and has an activation energy of 88 kJmol-1. The kinetic parameters are Km (PEP) 120 microM, Km (E4P) 28 microM, and kcat 1.5s-1, at 60 degrees C and pH 6.8. DAH7PS is not inhibited by phenylalanine, tyrosine, or tryptophan. EDTA inactivates the enzyme and enzyme activity is restored by a wide range of divalent metal ions including (in order of decreasing effectiveness): Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Ca2+, Hg2+, and Cu2+. This detailed characterization of the DAH7PS from P. furiosus raises the possibility that the subfamily Ibeta DAH7PS enzymes are metal ion dependent, contrary to previous predictions.     

Functional multiplicity of phosphoglucose isomerase from Lactobacillus casei

Phosphoglucose isomerase (D-glucose-6-phosphate ketolisomerase, EC 5.3.1.9), purified from Lactobacillus casei, showed multiplicity with respect to electrophoretic mobility, molecular weight, kinetic properties and responses to erythrose 4-phosphate. Among the three forms isolated, one having a dimeric conformation, was specific for glucose 6-phosphate. Erythrose 4-phosphate inhibited this preparation in a sigmoid fashion, while this compound activated the enzyme for isomerization of ribose 5-phosphate. In tetrameric conformation of the similar subunits, the enzyme was more specific for ribose 5-phosphate and the inhibition exerted by erythrose 4-phosphate was hyperbolic. The possible implications of these observations have been discussed.     

Engineering of Escherichia coli central metabolism for aromatic metabolite production with near theoretical yield.

Escherichia coli and many other microorganisms synthesize aromatic amino acids through the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP). It has been shown that overexpression of transketolase increases the production of DAHP in an aroB mutant strain (unable to further metabolize DAHP) with elevated DAHP synthase. However, the yield (percent conversion) of DAHP from glucose is still low. Stoichiometric analysis shows that many enzymes compete for intracellular PEP. In particular, the phosphotransferase system, responsible for glucose transport in E. coli, uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. This stoichiometric limitation greatly reduces the yield of aromatic metabolites. To relieve this limitation, we overexpressed PEP synthase in the presence of glucose and showed that it increased the final concentration and the yield of DAHP by almost twofold, to a near theoretical maximum. The PEP synthase effect is not observed without overproduced transketolase, suggesting that erythrose 4-phosphate is the first limiting metabolite. This result demonstrates the utility of pathway analysis and the limitation of central metabolites in the high-level overproduction of desired metabolites.     

Steady-state kinetic studies of the metal ion-dependent decarboxylation of oxalacetate catalyzed by pyruvate kinase

Steady-state kinetic studies with differing divalent metals ions have been carried out on the pyruvate kinase-catalyzed, divalent cation-dependent decarboxylation of oxalacetate to probe the role of the divalent metal ion in this reaction. With either Mn2+ or Co2+, initial velocity patterns show that the divalent metal ion is bound to the enzyme in a rapid equilibrium prior to the addition of oxalacetate. Further, there is no change in the initial velocity patterns or the kinetic parameters in the presence or absence of K+, indicating that K+ is not required for oxalacetate decarboxylation. Dead-end inhibition of the decarboxylation reaction by the physiological substrate phosphoenolpyruvate indicates that phosphoenolpyruvate binds only to the enzyme-metal ion complex and not to free enzyme. The pKi values for both Mn2+ and Co2+ decrease below a pK of 7.0, and increase above a pK of 8.9. Since these pK values are the same for both ions, both of the observed pK values must be attributable to enzymatic residues. The pK of 7.0 is presumably that of a ligand to the metal ion, while the pK of 8.9 is probably that of the lysine involved in enolization of pyruvate in the normal physiological reaction. However, with Co2+ as divalent cation, the V for oxalacetate decreases above a pK of 8.0, the V/K decreases above two pK values averaging 7.8, and the pKi for oxalate decreases above a single pK of 7.3. These data indicate that metal-coordinated water is displaced during the binding of substrates or inhibitors and the other pK value observed in both V and V/K pH profiles (pK of 8.3 with Co2+ and 9.2 with Mg2+) is an enzymatic residue whose deprotonation disrupts the charge distribution in the active site and decreases activity.     

Partial purification and kinetic characterization of transaldolase fromDictyostelium discoideum

Transaldolase was purified 42-fold fromDictyostelium discoideum and the resulting preparation exhibited stoichiometry. Kinetic analyses consisted of initial velocity and product inhibition studies in both the forward and the reverse directions. The enzyme exhibited ping-pong kinetics with sedoheptulose 7-phosphate adding first and erythrose 4-phosphate releasing first. TheK_m values for sedoheptulose 7-phosphate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, and fructose 6-phosphate were 0.46, 0.072, 0.10, and 1.6 mM, respectively. TheK_i values for sedoheptulose 7-phosphate and erythrose 4-phosphate were 3.6 and 0.062 mM, respectively. Inorganic phosphate inhibited enzymatic activity and showed mixed-type inhibition when fructose 6-phosphate was varied. AK_i value of 35.2 mM was determined for inorganic phosphate.     

Substrate deactivation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by erythrose 4-phosphate.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 4.1.2.15) catalyzes the condensation of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to give DAH7P via an ordered sequential mechanism. In the absence of PEP (the first substrate to bind), E4P binds covalently to the phenylalanine-sensitive DAH7PS of Escherichia coli, DAH7PS(Phe), deactivating the enzyme. Activity is restored on addition of excess PEP but not if deactivation was carried out in the presence of sodium cyanoborohydride. Electrospray mass spectrometry indicates that a single E4P is bound to the protein. These data are consistent with a slow, reversible Schiff base reaction of the aldehydic functionality of E4P with a buried lysine. Molecular modeling indicates that Lys186, a residue at the base of the substrate-binding cavity involved in hydrogen bonding with PEP, is well placed to react with E4P forming an imine linkage that is substantially protected from solvent water.     

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in Spinacia oleracea and other higher plants: extreme substrate ambiguity and other properties

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS-Co: EC 4.1.2.15) in Spinacia oleracea, Solanum tubersosum and many other higher plants was found to use a diversity of substrates. Diose (glycolaldehyde), triose (D-glyceraldehyde, L-glyceraldehyde and DL-glyceraldehyde 3-phosphate), tetrose (D-erythrose, L-erythrose, D-erythrose 4-phosphate, D-threose and L-threose), and pentose (D-ribose 5-phosphate and D-arabinose 5-phosphate) were utilized in combination with phosphoenolpyruvate (PEP) to make the corresponding 2-keto-3-deoxy sugar acids. Glyoxylate was also utilized by DS-Co. Glycoladehyde exhibited the highest reaction velocity when substrates were tested at 3 mM concentrations. Pentoses were poor substrates except when phsophorylated, an effect which is probably due to an increased fraction of the molecules being in the open-chain form. Little stereoselective discrimination exists since comparable velocities were demonstrated with the D and L isomers of glyceraldehyde, erythrose or threose. The enzyme is not a reversible aldolase since pyruvate failed to substitute for PEP. The use of D-erythrose 4-phsophate or glycolaldehyde resulted in K_m values of 1.95 mM and 8.60 mM, respectively. However, glycolaldehyde exhibited the largest V_maxK_m ratio, suggesting a greater catalytic efficiency for this substrate. Glycolaldehyde is an ideal substrate for inexpensive assays of DS-Co that are absolutely selective in the presence of two other plant enzymes which also utilize erythrose 4-phosphate and PEP. The spinach DS-Co enzymes required divalent metals for activity. The presence of 20 mM Mg²⁺, 1 mM Co²⁺ and 1 mM Mn²⁺ yielded relative activities of 100, 70 and 15, respectively. The pH optimum was 9.5 and temperature optimum for activity was 49°C. The molecular masses of DS-Co from spinach, tobacco and pea were all in the range of 400 kDa. The possible roles of DS-Co in biosynthesis of α-ketoglutarate and aromatic amino acids, in biosynthesis of components of cell wall and phytotoxin, and in acting as a sink for 2-and 3-carbon sugars are discussed.     

Purification and properties of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (phenylalanine-inhibitable) of Saccharomyces cerevisiae

The phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonate-7-phosphate (dHp1P) synthase from Saccharomyces cerevisiae has been purified to apparent homogeneity by a 1250-fold enrichment of the enzyme activity present in wild-type crude extracts, employing an overproducing strain. The estimated molecular mass of 42 kDa corresponds to the calculated molecular mass of 42.13 kDa deduced from the previously determined primary sequence. Gel filtration indicates that the active enzyme is a monomer. The enzyme is an Fe protein and is inactivated by EDTA in a reaction which is reversible by several bivalent metal ions. The Michaelis constant of the enzyme is 18 microM for phosphoenolpyruvate (P-pyruvate) and 130 microM for erythrose 4-phosphate (Ery4P) and the rate constant was calculated as 10 s-1. Inhibition by phenylalanine is competitive with respect to erythrose 4-phosphate and non-competitive to phosphoenolpyruvate, with a Ki of 10 microM.     

Isotope effect studies of chicken liver NADP malic enzyme: role of the metal ion and viscosity dependence

The role of the metal ion in the oxidative decarboxylation of malate by chicken liver NADP malic enzyme and details of the reaction mechanism have been investigated by 13C isotope effects. With saturating NADP and the indicated metal ion at a total concentration 10-fold higher than its Km, the following primary 13C kinetic isotope effects at C4 of malate [13(V/Kmal)] were observed at pH 8.0: Mg2+, 1.0336; Mn2+, 1.0365; Cd2+, 1.0366; Zn2+, 1.0337; Co2+, 1.0283; Ni2+, 1.025. Knowing the partitioning of the intermediate oxalacetate between decarboxylation to pyuvate and reduction to malate allows calculation of the intrinsic carbon isotope effect for decarboxylation. For Mg2+ as activator, this was 1.049 with NADP and 1.046 with 3-acetylpyridine adenine dinucleotide phosphate, although the intrinsic primary deuterium isotope effects on dehydrogenation were 5.6 and 4.2, and the partition ratios of the oxalacetate intermediate for decarboxylation as opposed to hydride transfer were 0.11 and 3.96 (the result of the different redox potentials of NADP and the acetylpyridine analogue). The close agreement of the intrinsic 13C isotope effects with each other and with the 13C isotope effect for the Mg2+-catalyzed nonenzymatic decarboxylation of oxalacetate of 1.0489 [Grissom, C. B., & Cleland, W. W. (1986) J. Am. Chem. Soc. 108, 5582] indicates a similarity of transition states for these reactions. It was not possible to calculate reasonable intrinsic carbon isotope effects with the other metal ions by use of the partitioning ratio of oxalacetate because of decarboxylation by another mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

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