Phosphoenolpyruvate synthetase inMethanobacterium thermoautotrophicum

The thermophilic autotrophMethanobacterium thermoautotrophicum assimilates CO₂ via a novel pathway rather than via the Calvin cycle. The central intermediate of this pathway is acetyl CoA which is reductively carboxylated to pyruvate. Cell extracts of the organism contained phosphoenolpyruvate synthetase with a specific activity of 100 nmol min^-1 mg^-1 protein (65°C). Pyruvate kinase and pyruvate, phosphate dikinase were not detected. Phosphoenolpyruvate synthetase was partially purified (50-fold) and the following reaction stoichiometry was established: $${\text{Pyruvate + ATP + H}}_{\text{2}} {\text{O }} \to {\text{ Phosphoenolpyruvate + AMP + P}}_{\text{i}} $$ The enzyme activity was depedent on free Mg²⁺ ions, NH ₄ ⁺ or K⁺ ions, and SH-groups. Mn²⁺, but not Ca²⁺, could partially substitute for Mg²⁺; Na⁺ could not substitute for K⁺ or NH ₄ ⁺ . The pH-optima,V _max-values and the apparentK _M-values for the substrates of the enzyme in both directions were determined. Thermodynamic, kinetic and regulatory features indicate that, in vivo, the enzyme functions in the direction of phosphoenolpyruvate synthesis from pyruvate. Not only is the synthesis of phosphoenolpyruvate via the PEP synthetase reaction energetically favorable; the enzyme also catalyzed this synthesis 100 times faster than the reverse reaction, the apparentK _M value for pyruvate (40 μM) being low and the apparentK _M value for phosphate (100 mM) being high. Furthermore, AMP, ADP, PP and α-ketoglutarate were inhibitors of PEP synthesis, indicating that the enzyme activity may be controlled in vivo. The role of phosphoenolpyruvate synthetase in autotrophic CO₂ assimilation pathway ofMethanobacterium, as expected from previous labelling studies, is confirmed.     

Evidence for the in vivo regulation of glucose 6-phosphate dehydrogenase activity in Hydrogenomonas eutropha H 16 from measurements of the intracellular concentrations of metabolic intermediates

The inhibition of fructose utilization by whole cells of Hydrogenomonas eutropha H 16, following the addition of hydrogen to the gas phase, has been explained as an inhibition of glucose 6-phosphate dehydrogenase (Blackkolb and Schlegel, 1968a, b). The intracellular concentrations of glucose 6-phosphate, 6-phosphogluconate, three inhibitors of the enzyme (NADH, ATP and phosphoenolpyruvate) and some related metabolites were measured in cells incubated in the presence and absence of hydrogen. Inhibition of glucose 6-phosphate dehydrogenase was confirmed by an increase in the glucose 6-phosphate pool and a decrease in the 6-phosphogluconate concentration. The regulatory control is apparently due to a threefold increase in the NADH concentration while the concentrations of the other two inhibitors fell slightly. When the measured intracellular concentrations of intermediates were used in the in vitro assay of glucose 6-phosphate dehydrogenase activity, an almost total inhibition of the dehydrogenase was observed, therefore further regulatory factors must be considered.     

Multiple forms of octopine dehydrogenase in Strombus luhuanus (Mollusca,Gastropoda, Strombidae): Genetic basis of polymorphism,properties of the enzymes,and relationship between the octopine dehydrogenase phenotype and the accumulation of anaerobic end products during exercise

Octopine dehydrogenase (ODH) is electrophoretically polymorphic in the gastropod mollusk Strombus luhuanus. The frequencies of the six electrophoretic phenotypes in the Heron Island population, together with the molecular weight values of 38,000 obtained for each of the three forms of the enzyme, demonstrate that the monomeric enzyme is encoded by three codominant alleles at a single locus. The purified allozymes are indistinguishable in terms of K _m values for substrates, product inhibition by octopine and NAD, pH optima, and substrate inhibition by pyruvate. No statistically significant correlations were found between the ODH phenotype and the maximum activities of ODH or alanopine dehydrogenase, the capacity for anaerobic muscle work, or the accumulation of octopine or strombine/alanopine during exercise. It would appear that the ODH allozymes may be functionally equivalent both in vitro and in vivo.     

Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties.

Procedures are described for isolating highly purified porcine liver pyruvate and α-ketoglutarate dehydrogenase complexes. Rabbit serum stabilized these enzyme complexes in mitochondrial extracts, apparently by inhibiting lysosomal proteases. The complexes were purified by a three-step procedure involving fractionation with polyethylene glycol, pelleting through 12.5% sucrose, and a second fractionation under altered conditions with polyethylene glycol. Sedimentation equilibrium studies gave a molecular weight of 7.2 × 10⁶ for the liver pyruvate dehydrogenase complex. Kinetic parameters are presented for the reaction catalyzed by the pyruvate dehydrogenase complex and for the regulatory reactions catalyzed by the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. For the overall catalytic reaction, the competitive K_i to K_m ratio for NADH versus NAD⁺ and acetyl CoA versus CoA were 4.7 and 5.2, respectively. Near maximal stimulations of pyruvate dehydrogenase kinase by NADH and acetyl CoA were observed at NADH:NAD⁺ and acetyl CoA:CoA ratios of 0.15 and 0.5, respectively. The much lower ratios required for enhanced inactivation of the complex by pyruvate dehydrogenase kinase than for product inhibition indicate that the level of activity of the regulatory enzyme is not directly determined by the relative affinity of substrates and products of catalytic sites in the pyruvate dehydrogenase complex. In the pyruvate dehydrogenase kinase reaction, K⁺ and NH⁺₄ decreased the K_m for ATP and the competitive inhibition constants for ADP and (β,γ-methylene)adenosine triphosphate. Thiamine pyrophosphate strongly inhibited kinase activity. A high concentration of ADP did not alter the degree of inhibition by thiamine pyrophosphate nor did it increase the concentration of thiamine pyrophosphate required for half-maximal inhibition.     

The effect of ferrous ions,tungstate and selenite on the level of formate dehydrogenase inClostridium formicoaceticum and formate synthesis from CO2 during pyruvate fermentation

In a medium containing a trace element solution and 10^-4 M ferrous ions the growth yield ofClostridium formicoaceticum on fructose was 5.5 g of weight per l; in the absence of metal ion solution it was 1 g per l. The specific activity of methyl viologen dependent formate dehydrogenase under both conditions was 0.28 and 0.03 units per mg of protein, respectively. It could be increased to 9.75 units when the growth medium contained 10^-4 M tungstate and 10^-5 M selenite in addition. Molybdate was only about 40% as effective as tungstate. Tungstate or molybdate could not be replaced by vanadate, selenite not by sulfide. The formate dehydrogenase catalyzed also the reduction of CO₂ to formate. The highest rate of formate synthesis was observed when pyruvate served as the reductant. No pyruvate: formate exchange but rapid pyruvate: CO₂ exchange could be observed with cell-free extracts ofC. formicoaceticum. Pyruvate is fermented byC. formicoaceticum to yield up to 1.16 mole acetate per mole of pyruvate. Resting cells accumulated some formate in addition to acetate.     

Substrate inhibition of soluble and bound lactate dehydrogenase (isoenzyme 5)

Substrate inhibition of chicken lactate dehydrogenase (EC 1.1.1.27) isoenzyme 5, was studied with the enzyme in the soluble phase and bound to muscle subcellular particulate structures. Inhibition studies were performed by incubating bound or soluble enzyme with NAD⁺ prior to measuring the reaction with a stopped-flow technique at 40 °C and a concentration of enzyme of 10^?7m. The value of V for soluble lactate dehydrogenase was 610 nmoles per sec, and for the bound enzyme it was 262. k_m (pyruvate) values were similar for both enzymes. Under our experimental conditions, up to 73% inhibition of the soluble enzyme was observed. On the other hand, there was no detectable inhibition of bound lactate dehydrogenase. It is suggested that the resistance to substrate inhibition of bound lactate dehydrogenase may possibly be due to the prevention of dissociation of the enzyme into monomeric or other subunits because of attachment to the particulate structures.     

Purification and properties of a fructose-1,6-diphosphate activated l-lactate dehydrogenase from Staphylococcus epidermidis

l-(+)-lactate dehydrogenase (LDH) from Staphylococcus epidermidis ATCC 14990 was purified by affinity chromatography. The purified enzyme was specifically activated by fructose-1,6-diphosphate (FDP). The concentration of FDP required for 50% maximal activity was about 0.15 mM. The enzyme activity was inhibited by adenosine diphosphate (ADP) and oxamate. The inhibition by ADP appeared to be competitive with respect to reduced nicotinamide adenine dinucleotide (NADH). The catalytic activity of the LDH for pyruvate reduction exhibited an optimum at pH 5.6. The enzyme is composed of four, probably identical, subunits. Sephadex gel filtration and sedimentation velocity at pH 5.6 yielded molecular weights of about 130000 and 126000 respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68000. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and sedimentation velocity at pH 2.0 or 8.5 revealed monomeric subunits with an approximate molecular weight of 36000. The thermostability of the heat labile enzyme was increased in the presence of FDP, NADH and pyruvate. The purified LDH exhibited an anomalous type of kinetic behavior. Plots of initial velocity vs. different concentrations of pyruvate, NADH or FDP led to saturation curves with intermediary plateau regions. As a consequence of these plateau regions the Hill coefficient alternated between lower and higher n-values. Some distinguishing properties of the S. epidermidis LDH and other LDHs activated by FDP are discussed.     

Structural states of the flexible catalytic loop of M. tuberculosis tyrosyl-tRNA synthetase in different enzyme–substrate complexes

Tyrosyl-tRNA synthetase from Mycobacterium tuberculosis (MtTyrRS) is an enzyme that belongs to class I of aminoacyl-tRNA synthetases, which catalyze the attachment of l-tyrosine to its cognate tRNATyr in the preribosomal step of protein synthesis. MtTyrRS is incapable of cross-recognition and aminoacylation of human cytoplasmic tRNATyr, so this enzyme may be a promising target for development of novel selective inhibitors as putative antituberculosis drugs. As a class I aminoacyl-tRNA synthetase, MtTyrRS contains the HIGH-like and KFGKS catalytic motifs that catalyze amino acid activation with ATP. In this study, the conformational mobility of MtTyrRS catalytic KFGKS loop was analyzed by 100-ns all-atoms molecular dynamics simulations of the free enzyme and its complexes with different substrates: tyrosine, ATP, and the tyrosyl–adenylate intermediate. It was shown that in the closed state of the active site, the KFGKS loop, readily adopts different stable conformations depending on the type of bound substrate. Molecular dynamics simulations revealed that the closed state of the loop is stabilized by dynamic formation of two antiparallel β-sheets at flanking ends which hold the KFGKS fragment inside the active center. Prevention of β-sheet formation by introducing point mutations in the loop sequence results in a rapid (<20 ns) transition of the loop from its functional “closed” M-like structure to an inactive “open” O-like structure, i.e. rapid diffusion of the catalytic loop outside the active site. The flexibility and rapid dynamics of the wild-type aaRS catalytic loop structure are crucial for formation of protein–substrate interactions and subsequently for overall enzyme functional activity.     

Catabolism of d-fructose and d-ribose by Pseudomonas doudoroffii

1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudomonas doudoroffii were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. The pH optima of these enzymes were 9.0 and 8.5, respectively.
2. When the concentrations of the substrates of the 1-PFK reaction were varied, Michaelis-Menten kinetics were observed. The Kms for d-fructose-1-P (F-1-P) and ATP were 3.03×10^-4 M and 3.39×10^-4 M, respectively. Variation of MgCl₂ at fixed concentrations of F-1-P and ATP resulted in sigmoidal kinetics; about 10 mM MgCl₂ was necessary for maximal activity. Activity of 1-PFK was inhibited when the ratio of ATP: Mg⁺⁺ was higher than 0.5, suggesting that ATP: 2Mg⁺⁺ was the substrate and that free ATP was inhibitory. Although an absolute requirement for K⁺ or NH ₊ ⁴ could not be demonstrated, these cations stimulated the rate of the reaction. Activity of 1-PFK was not significantly affected by 3 mM AMP, cyclic-AMP, P_i, d-fructose-6-P (F-6-P), ADP, P-enolpyruvate (PEP), pyruvate, citrate, or l-glutamate.
3. Sigmoidal kinetics were observed for 6-PFK when the concentration of F-6-P was increased and the level of ATP was kept constant. Activity of 6-PFK was increased by ADP, inhibited by PEP, and unaffected by 3 mM AMP, cyclic-AMP, P_i, F-1-P, pyruvate, or citrate.

     

Initial steps in the degradation of n-alkane-1-sulphonates by Pseudomonas

The primary reaction in the degradation of n-alkane-1-sulfonates by Pseudomonas is the hydroxylation of the carbon atom bearing the sulfonate group. The 1-hydroxy-n-alkane-1-sulfonate (aldehyde-bisulfite adduct) formed easily hydrolyses to give the corresponding aldehyde and bisulfite. The enzyme catalysing the hydroxylation reaction depends for its action on the presence of molecular oxygen and NADH. The kinetics of this reaction and the substrate specificity of the enzyme were studied using a crude enzyme extract and a spectrophotometric assay method based on co-oxidation of NADH with the sulfonate.     

Simple kinetic models explaining critical phenomena in enzymatic reactions with isomerization of the enzyme and substrate

Kinetic models for enzyme reactions are considered which take into account enzyme and substrate isomerization. Application of graph-theoretic methods allows to reveal fragments in schemes which may induce multiple stead-states or concentrational selfoscillations. The role of substrate isomers in the inhibition of enzyme isomers to produce critical phenomena is considered. The boundaries of parameter domains for critical phenomena are estimated. It is shown that the controlled change in concentrations of substrate and enzyme isomers may be important in regulation of enzyme systems, if different enzyme isomers are inhibited mainly by different substrate isomers. The models are used for interpretation of possible critical phenomena in the open reaction catalyzed by lactate dehydrogenase. It is shown that lactate dehydrogenase may act as a trigger in carbohydrate metabolism by changing "critically" its activity in relation to changes in pH and pyruvate fluxes. Slow enzyme inhibition by enolpyruvate is suggested as a possible reason for glycolytic oscillations.     

The isozyme spectra of lactate dehydrogenase in the tissues of the raccoon dog <Emphasis Type="Italic">Nyctereutes procyonoides</Emphasis> in the autumn

An electrophoretic assay of lactate dehydrogenase (EC 1.1.1.27) isozymes in the tissue homogenates of cardiac and skeletal muscles, kidney, lungs, spleen, and liver of the raccoon dog Nyctereutes procyonoides from two different geographic zones, viz., northwestern Russia and Poland, as well as the Arctic blue fox Alopex lagopus L. and the red fox Vulpes vulpes L. was performed during the preparatory period to the winter season. Raccoon dogs, which hibernate under natural conditions, differ from other canids (the red fox and Arctic blue fox) to which they are close taxonomically by their body weight and by the higher proportion of aerobic H subunits of lactate dehydrogenase in all organs except for the heart. A higher content of “fast” anode fractions, lactate dehydrogenase-1 and lactate dehydrogenase-2, in the heart, kidney, lungs, liver, and spleen was detected in the raccoon dogs from the northern region compared to those from the southern geographic zone. The shift in the reaction catalyzed by lactate dehydrogenase towards the production of pyruvate indicates that this metabolite is necessary for the synthesis of fatty acids during lipogenesis in the autumn.     

The blue-light reaction in plasmodia of Physarum polycephalum is coupled to respiration

The influence of inhibitors of energy metabolism (2-deoxy-D-glucose, monoiodoacetate, KCN) as well as various substrates for respiration (sodium acetate, glycine, glutamine, α-ketoglutarate, pyruvate) were investigated with respect to the effect of blue light (450 nm) on contractile behaviour of plasmodial strands of Physarum polycephalum. When the energy metabolism is not experimentally modified, blue light induces a prolongation of the period of the contraction-relaxation cycle. This effect appears within 2–3 min and seems to represent the primary reaction of this organism to blue light. Inhibition of respiration by KCN completely abolished this response to blue-light irradiation. In contrast, an impediment of glycolysis enhanced the effect. This indicates that the reaction to blue light is related to respiration, i.e., to the function of mitochondria. Among different substrates for respiration only α-ketoglutarate combined with pyruvate and applied in the presence of inhibitors of glycolysis showed an enhancement of the photoresponse, i.e., a prolongation of the period and an increase of the amplitude of the force oscillations. This indicates that the pyruvate and α-ketoglutarate-dehydrogenase complexes functioning in mitochondrial respiration are involved in the primary blue-light reaction of plasmodia of Physarum polycephalum.     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

The anaerobic metabolism of malate of Saccharomyces bailii and the partial purification and characterization of malic enzyme

1. The main pathway of the anaerobic metabolism of l-malate in Saccharomyces bailii is catalyzed by a l-malic enzyme.
2. The enzyme was purified more than 300-fold. During the purification procedure fumarase and pyruvate decarboxylase were removed completely, and malate dehydrogenase and oxalacetate decarboxylase were removed to a very large extent.
3. Manganese ions are not required for the reaction of malic enzyme of Saccharomyces bailii, but the activity of the enzyme is increased by manganese.
4. The reaction of l-malic enzyme proceeds with the coenzymes NAD and (to a lesser extent) NADP.
5. The K _m-values of the malic enzyme of Saccharomyces bailii were 10 mM for l-malate and 0.1 mM for NAD.
6. A model based on the activity and substrate affinity of malic enzyme, the intracellular concentration of malate and phosphate, and its action on fumarase, is proposed to explain the complete anaerobic degradation of malate in Saccharomyces bailii as compared with the partial decomposition of malate in Saccharomyces cerevisiae.

     

Metabolism of Round Spermatids: Pyruvate cannot Maintain the ATP Level

Round spermatids (steps 1–8) were isolated from rat testes and the effect of pyruvate on their intracellular ATP level was examined. Results showed that although the spermatids consumed a considerable amounts of pyruvate, this substrate alone did not maintain their ATP level. However, their ATP level was maintained in the presence of both pyruvate and α-ketovalerate or α-ketobutyrate. Maintenance of the ATP level by these substrates was associated with electron trasnport and oxidative phosphorylation. α-Ketoacid inhibited pyruvate reduction to lactate in the lactate dehydrogenase (LDH) reaction, but increased pyruvate oxidation to CO₂. The NADH level in spermatids was too low to be detectable, but the NAD level remained unchanged in the presence of pyruvate and α-ketovalerate. These results suggest that pyruvate by itself is not an adequate energy-yielding substrate for spermatids and that a high NADH/NAD ratio may be essential for maintenance of their ATP level.     

Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase

During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y_ATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y_ATP suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.In Escherichia coli as well as in other aerobic organisms, sugars such as glucose are metabolized in two separate steps: glycolysis, which converts glucose to pyruvate, and tricarboxylic acid (TCA) cycle enzymes, which oxidize acetyl coenzyme A (acetyl-CoA) to CO₂ (5, 9). The pyruvate dehydrogenase complex (PDH) connects the glycolytic reactions to TCA cycle enzymes by catalyzing the production of acetyl-CoA from pyruvate. Because of its unique central role in metabolism, PDH is regulated at both the genetic and the biochemical level (7, 12, 27, 33, 34). The NADH generated during the complete oxidation of sugar is reoxidized to NAD⁺ by O₂ through the respiratory electron transport pathway with accompanying energy production (11). Optimum coupling of these enzyme reactions helps to maintain the internal ratios of [NADH] to [NAD⁺] (redox balance) and of [ATP] to [ADP] plus [AMP] in order to support growth at the highest rate.The absence of O₂ or another external electron acceptor during the growth of E. coli (anaerobic conditions) forces the bacterium to minimize the contribution of the TCA cycle enzymes to biosynthesis from catabolism (4, 14). Under these conditions, pyruvate or acetyl-CoA derived from pyruvate serves as the electron acceptor (reduced to lactate or ethanol, respectively) to maintain the redox balance. The enzymes responsible for redox balance in anaerobic E. coli are pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH), and alcohol/aldehyde dehydrogenase (adhE; ADH-E). The main products of the fermentation of E. coli are a mixture of organic acids, such as acetate, lactate, and formate, in addition to ethanol (2, 4). Succinate, derived from phosphoenolpyruvate (PEP), is a minor product of fermentation and normally accounts for less than 5% of the total products produced from glucose by the culture.Anaerobic growth of E. coli, compared to aerobic growth, is also limited by energy, leading to an increase in glycolytic flux (19). The conversion of pyruvate to acetate and ethanol yields an additional ATP per glucose, suggesting that this would be the preferred route for pyruvate oxidation during anaerobic growth. This is accomplished by the PFL-dependent production of acetyl-CoA and further conversion to acetate (Fig. ​(Fig.1).1). This preference for PFL has been demonstrated `with several bacteria under carbon limitation conditions imposed either in a chemostat or in the presence of a poor carbon source (10, 20, 23). This additional ATP also elevates the ATP yield per glucose to 3, with an increase in the growth rate, and has been shown to be essential for the anaerobic growth of E. coli in xylose-mineral salts medium (13). The absence of this third ATP in a pfl mutant has been reported to increase glycolytic flux to lactate to compensate for this decrease in ATP yield per glucose (39). However, the flow of pyruvate carbon to acetate is tempered by the need to maintain redox balance, and this is achieved by the conversion of a second acetyl-CoA to ethanol by ADH-E. Under conditions of energy excess due to a declining growth rate, lactate production is expected to support redox balance maintenance without the additional ATP from the PFL-ADH-E pathway (Fig. ​(Fig.1).1). The production of this mixture of products in an appropriate ratio helps to maintain the redox balance under anaerobic conditions while also maximizing the ATP yield per glucose to support high growth rates and cell yields.Open in a separate windowFIG. 1.Anaerobic metabolic pathways of E. coli carrying the lpd101 mutation (PDH*).No PDH-based fermentation reaction to ethanol that can also help maintain cellular redox balance in an anaerobic cell has evolved in E. coli or other closely related bacteria. PDH activity is inhibited by NADH, normally found at higher levels in anaerobically growing cultures than in aerobic cultures (12, 18, 34, 35). Based on genome sequences available in GenBank, the genes encoding the components of PDH are not found in strictly anaerobic bacteria.We have recently described a mutation (lpd101) in the dihydrolipoamide dehydrogenase (LPD) of the PDH that allowed the enzyme to function in anaerobic cells (designated PDH* here) (17, 18). With this altered PDH*, an anaerobic cell can have three different pathways for pyruvate metabolism (Fig. ​(Fig.1).1). The three main enzymes that utilize pyruvate as a substrate, PDH*, PFL, and LDH, have different apparent K_m values for pyruvate (0.4, 2.0, and 7.2 mM, respectively) (1, 18, 37, 41). PDH requires NAD⁺ for activity (apparent K_m, 0.07 mM), while LDH is dependent on NADH (apparent K_m, 0.2 mM) as the second substrate (18, 37).The PDH* serves as the first enzyme in a pathway that oxidatively decarboxylates pyruvate to acetyl-CoA and NADH, followed by reduction of the acetyl-CoA by alcohol dehydrogenase to ethanol in a two-step process using 2 NADHs (Fig. ​(Fig.1).1). The NADH produced during the conversion of glucose to acetyl-CoA dictates that the acetyl-CoA generated by PDH be used for redox balance (ethanol) and not for ATP generation (acetate), unless some of the NADH is used for biosynthesis by the growing cell (17). PDH* and LDH serve essentially the same physiological role in the cell, oxidizing NADH to support continued operation of glycolysis, although it is not readily apparent with PDH*. Although PDH* contributes to an increase in NADH pool, the redox balance is still maintained by coupling PDH* to NADH-dependent reduction of acetyl-CoA to ethanol by ADH-E (Fig. ​(Fig.1).1). This potential competition between LDH and PDH has been eliminated in the wild type through inhibition of the activity of PDH by NADH (12, 18, 32). However, the in vivo role of PDH* in a mutant that has all three pathways has not been investigated, since the flow of pyruvate through any of the three reactions during growth and postgrowth fermentation of sugars to products is expected to be dependent on the redox state, the ATP requirement, and other physiological conditions of the anaerobic cell. Using a combination of metabolic flux analysis and mutations in one or more of the genes encoding these enzymes, we have evaluated the flow of pyruvate carbon among the three potential pathways. The results are presented in this communication.     

Drosophila lactate dehydrogenase: Molecular and genetic aspects

(LDH) obtained from larvae of Drosophila melanogaster was purified to homogeneity by affinity chromatography on oxamate-Sepharose. The purification procedure is simple to operate and gives a homogeneous preparation in a good yield (34.86%) after only two steps. Utilizing the homogeneous LDH preparation, an attempt was made to characterize the LDH molecule. Thus, it was found that the N-terminal amino acid is isoleucine, and the enzyme is tetrameric and composed of four identical subunits of apparent molecular weight 38,000, suggesting that it is controlled by a single gene. Homogeneous LDH preparations exhibit one band on neutral acrylamide gels when the substrate is either dl-lactic acid or l-(+)-lactate. The optimum temperature is 45°C for the purified enzyme and 40°C for the crude homogenate. The K _m values for pyruvate and NADH are 0.154 and 0.027mm, respectively, while the K _m values for lactate and NAD are 29.4 and 1.33mm, respectively. A discontinuity in the E _a slope was observed at a transition temperature of 30°C. The E _a value between 20 and 30°C was calculated as 12.06 kcal/mol, while between 30 and 45°C the E _a value was 4.01 kcal/mol. This evidence, together with other observations reported in the literature, suggests that the LDHs of invertebrates and vertebrates have arisen by divergent evolution from a common ancestral gene.     

ATP-driven succinate oxidation in the catabolism of Desulfuromonas acetoxidans

The oxidation of succinate with elemental sulphur in Desulfuromonas acetoxidans was investigated using a membrane preparation of this bacterium. The following results were obtained:

1. The preparation catalyzed the oxidation of succinate with sulphur and NAD. These reactions were dependent on ATP and were abolished by the presence of protonophores or dicyclohexylcarbodiimide (DCCD).
2. The membrane preparation also catalyzed the reduction of fumarate with H₂S or with NADH. These activities were not dependent on ATP and were not affected by protonophores or DCCD.
3. By extraction-reincorporation experiments it could be shown that menaquinone is involved in electron transport between H₂S and fumarate and between NADH and fumarate.
4. The membrane fraction catalyzed the reduction of the water-soluble menaquinone-analogue dimethylnaphthoquinone (DMN) by succinate, H₂S, or NADH, and the oxidation of DMNH₂ by fumarate. These activities were not dependent on the presence of menaquinone and were not influenced by ATP.
5. The activities involving succinate oxidation or fumarate reduction were similarly sensitive to 2(n-nonyl)-4-hydroxyquinoline-N-oxide, while H₂S and NADH oxidation by DMN were not affected by the inhibitor.

It is concluded that the catabolism of D. acetoxidans involves the energy-driven oxidation of succinate with elemental sulphur or NAD as electron acceptors and that menaquinone is a component of the electron transport chain catalyzing these reactions.     

Properties of CAR-kinase: The enzyme that phosphorylates the cAMP chemotactic receptor ofD. discoideum

The cell surface cAMP chemotactic receptor ofD. discoideum can be phosphorylated in partially purified plasma membrane preparations in a ligand-dependent manner. CAR-kinase, the enzyme responsible for receptor phosphorylation, was shown to be an integral membrane protein. It could utilize either ATP or GTP to phosphorylate the receptor, although ATP was much more efficient. The apparent affinity constant for ATP was approximately 20–25 µM. Maximum CAR-kinase activity was observed betweenpH 6.5 andpH 7, and required the presence of Mg²⁺. Neither Mn²⁺ nor Ca²⁺ could substitute for that divalent cation. The enzyme was found to be sensitive to the ionic strength and temperature of the incubation reaction. Dephosphorylation of the receptor was not observed in the membrane preparations, indicating that the enhanced level of receptor phosphorylation that occurred upon ligand binding was not an indirect reflection of receptor dephosphorylation and subsequent incorporation of radiolabeled phosphate.