Presence Of Dihydroxyacetone Phosphate Research Articles

Structural and Functional Characterization of YdjI, an Aldolase of Unknown Specificity in Escherichia coli K12.

The ydj gene cluster is found in 80% of sequenced Escherichia coli genomes and other closely related species in the human microbiome. On the basis of the annotations of the enzymes located in this cluster, it is expected that together they catalyze the catabolism of an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the ydj gene cluster of E. coli K-12. It is predicted to be a class II aldolase of unknown function. Here we describe a structural and functional characterization of this enzyme. YdjI catalyzes the hydrogen/deuterium exchange of the pro-S hydrogen at C3 of dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol condensation with a variety of aldo sugars. YdjI shows a strong preference for higher-order (seven-, eight-, and nine-carbon) monosaccharides with specific hydroxyl stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best substrate is l-arabinuronic acid with an apparent kcat of 3.0 s-1. The product, l-glycero-l-galacto-octuluronate-1-phosphate, has a kcat/Km value of 2.1 × 103 M-1 s-1 in the retro-aldol reaction with YdjI. This is the first recorded synthesis of l-glycero-l-galacto-octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI, determined to a nominal resolution of 1.75 Å (Protein Data Bank entry 6OFU ), reveals unusual positions for two arginine residues located near the active site. Computational docking was utilized to distinguish preferable binding orientations for l-glycero-l-galacto-octuluronate-1-phosphate. These results indicate a possible alternative binding orientation for l-glycero-l-galacto-octuluronate-1-phosphate compared to that observed in other class II aldolases, which utilize shorter carbohydrate molecules.

Conversion of non-allosteric methylglyoxal synthase into a homotropic allosteric enzyme by C-terminal deletion

Abstract Our previous study revealed that the Hill coefficient of Escherichia coli methylglyoxal synthase (EMGS) is higher than what we have calculated for MGS from Thermus sp. GH5 (TMGS). Amino acid sequence alignment of EMGS and TMGS shows that key residues of allosteric pathways in EMGS exist in TMGS as well, except Arg150 which plays a crucial role in forming a salt bridge with Asp20 in the neighboring subunit and consequently transfers the allosteric signal between the subunits. To equalize allosteric pathway in EMGS with TMGS, ten amino acid residues, containing Arg150, are omitted from the EMGS C-terminal tail. The resulting recombinant enzyme (EMGS-ΔC) surprisingly shows homotropic cooperative behavior in presence of dihydroxyacetone phosphate. Structural studies and irreversible thermoinactivation data shows EMGS-ΔC is not only more flexible but also less stable compared to wild-type EMGS. These data suggest EMGS C-terminal tail may play an important role in allosteric behavior and stability of wild-type EMGS and thus indicating that the homotropic cooperativity is arisen by binding of the substrate which pushes the pre-existing equilibrium between the relatively inactive (R I ) and relatively active (R A ) conformations.

Substrate Effect on Catalytic Loop and Global Dynamics of Triosephosphate Isomerase

The opening/closure of the catalytic loop 6 over the active site in apo triosephosphate isomerase (TIM) has been previously shown to be driven by the global motions of the enzyme, specifically the counter-clockwise rotation of the subunits. In this work, the effect of the substrate dihydroxyacetone phosphate (DHAP) on TIM dynamics is assessed using two apo and two DHAP-bound molecular dynamics (MD) trajectories (each 60 ns long). Multiple events of catalytic loop opening/closure take place during 60 ns runs for both apo TIM and its DHAP-complex. However, counter-clockwise rotation observed in apo TIM is suppressed and bending-type motions are linked to loop dynamics in the presence of DHAP. Bound DHAP molecules also reduce the overall mobility of the enzyme and change the pattern of orientational cross-correlations, mostly those within each subunit. The fluctuations of pseudodihedral angles of the loop 6 residues are enhanced towards the C-terminus, when DHAP is bound at the active site.

Charge Stabilization and Entropy Reduction of Central Lysine Residues in Fructose-Bisphosphate Aldolase

Fructose-1,6-bisphosphate muscle aldolase is an essential glycolytic enzyme that catalyzes reversible carbon-carbon bond formation by cleaving fructose 1,6-bisphosphate to yield dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde phosphate. To elucidate the mechanistic role of conserved amino acid Asp-33, Asn-33 and Ser-33 mutants were examined by kinetic and structural analyses. The mutations significantly compromised enzymatic activity and carbanion oxidation in presence of DHAP. Detailed structural analysis demonstrated that, like native crystals, Asp-33 mutant crystals, soaked in DHAP solutions, trapped Schiff base-derived intermediates covalently attached to Lys-229. The mutant structures, however, exhibited an abridged conformational change with the helical region (34-65) flanking the active site as well as pK(a) reductions and increased side chain disorder by central lysine residues, Lys-107 and Lys-146. These changes directly affect their interaction with the C-terminal Tyr-363, consistent with the absence of active site binding by the C-terminal region in the presence of phosphate. Lys-146 pK(a) reduction and side chain disorder would further compromise charge stabilization during C-C bond cleavage and proton transfer during enamine formation. These mechanistic impediments explain diminished catalytic activity and a reduced level of carbanion oxidation and are consistent with rate-determining proton transfer observed in the Asn-33 mutant. Asp-33 reduces the entropic cost and augments the enthalpic gain during catalysis by rigidifying Lys-107 and Lys-146, stabilizing their protonated forms, and promoting a conformational change triggered by substrate or obligate product binding, which lower kinetic barriers in C-C bond cleavage and Schiff base-enamine interconversion.

Analysis of carbohydrate transport across the envelope of isolated cauliflower-bud amyloplasts.

Using isolated amyloplasts from cauliflower buds, we have characterized the interaction and transport of various carbohydrates across the envelope membrane of a heterotrophic plastid. According to our results, glucose 6-phosphate (Glc6P) and glucose 1-phosphate (Glc1P) do not share the same transport protein for uptake into cauliflower-bud amyloplasts. Glc6P-dependent starch synthesis is strongly inhibited in the presence of dihydroxyacetone phosphate (DHAP) or 4,4'-di-isothiocyano-2,2'- stilbenedisulphonic acid (DIDS), whereas Glc1P-dependent starch synthesis is hardly affected by these compounds. Analysis of the Glc6P uptake into proteoliposomes reconstituted from the envelope proteins of cauliflower-bud amyloplasts indicate that Glc6P is taken up in a counter-exchange mode with Pi, DHAP or Glc6P, whereas Glc1P does not act as a counter-exchange substrate. Pi is a strong competitive inhibitor of Glc6P uptake (Ki 0.8 mM) into proteoliposomes, whereas Glc1P does not significantly inhibit Glc6P transport. Beside a hexose-phosphate translocator, these amyloplasts possess an envelope protein mediating the transport of glucose across the membrane. This translocator exhibits an apparent Km for glucose of 2.2 mM and is inhibited by low concentrations of phloretin, known to be a specific inhibitor of glucose-transport proteins. Maltose inhibits the uptake of glucose (Ki 2.3 mM), indicating that both carbohydrates share the same translocator.

Molecular dynamics simulations of “loop closing” in the enzyme triose phosphate isomerase

We present molecular dynamics simulations on the active site region of dimeric triose phosphate isomerase (TIM) using the co-ordinates of native chicken muscle TIM as a starting point and performing simulations with no substrate, with dihydroxyacetone phosphate (DHAP), the natural substrate, and with dihydroxyacetone sulfate (DHAS), a substrate analog. Whereas most of the protein moves less than 1 Å during the simulation, some residues in the active site loop move more than 8 Å during the 10.5 picoseconds of dynamics for each of the simulations. Most interestingly, the nature of the loop motion depends on the substrate, with the largest motion found in the presence of DHAP, and only in the presence of DHAP does the loop move to “close off” the active site pocket. The final structure found for the DHAP-chicken TIM complex is qualitatively similar to that described by Alber et al. for DHAP-yeast TIM. Simulations on the monomeric protein gives insight into why the molecule is active only as a dimer.

CO2 Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays

Conditions for optimal CO(2) fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO(2) fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO(2) assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V(max) for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C(3) plants.

Inhibited light activation of fructose and sedoheptulose bisphosphatase in spinach chloroplasts exposed to osmotic stress

The light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) was inhibited in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Decreases in the velocity and magnitude of light activation correlated with the overall reduction in CO2 fixation rates. Responses of osmotically stressed chloroplasts to both varying pH and exogeous dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerete (PGA) were examined. In the presence of DHAP, the absolute rate of CO2 fixation was increased and this increase was most pronounced at alkaline pH. Enhanced light activation of these enzymes was also observed under these conditions. However, in the presence of PGA, similar increases in photosynthetic rate and enzyme activation were not evident. Light-dependent stromal alkalization was unaffected by the stress treatments. Inhibition of light activation under hypertonic conditions is discussed in terms of substrate availability, possible alterations of the redox state of ferredoxin and associated electron carriers, and inhibited enzyme-enzyme or enzyme-substrate interactions involved in the light activation process.

Evidence for Endogenous Cyclic Photophosphorylation in Intact Chloroplasts

This study examines the capacity of intact spinach (Spinacia oleracea L.) chloroplasts to fix (14)CO(2) when supplied with Benson-Calvin cycle intermediates in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Under these conditions, substantial (14)CO(2) fixation occurred in the light but not in the dark when either dihydroxyacetone phosphate, ribulose 5-phosphate, fructose 6-phosphate, or fructose bisphosphate was added. The highest rate of (14)CO(2) fixation (20-40 micromoles per milligram chlorophyll per hour) was obtained with dihydroxyacetone phosphate. In contrast, no (14)CO(2) fixation occurred when 3-phosphoglycerate was used. (14)CO(2) fixation in the presence of dihydroxyacetone phosphate and DCMU was inhibited by carbonylcyanide m-chlorophenylhydrazone, dl-glyceraldehyde, and pyridoxal 5'-phosphate. Low concentrations of O(2) (25-50 micromolar) stimulated (14)CO(2) fixation, but the activity decreased with increasing O(2) concentrations. The fixation of (14)CO(2) in the presence of DCMU and dihydroxyacetone phosphate was also observed in maize bundle sheath cells. These results provide direct evidence for cyclic photophosphorylation in intact chloroplasts. The activity measured is adequate to support all the extra ATP requirements for maximum rates of photosynthesis in these intact chloroplasts.

Fructose-bisphosphate aldolase from Helix pomatia—I. Purification and catalytic properties

1. 1. Fructose 1,6-bisphosphate aldolase has been purified 450 fold from Helix pomatia (snail) muscle. 2. 2. The enzyme is inhibited by NaBH 4 in the presence of dihydroxyacetone phosphate and is therefore a class I aldolase. 3. 3. This aldolase has specific activity of 16 Units, V max = 2400, K mFDP = 0.3 μM, K mF-1-P = 1 mM, and FDP: F-1-P activity ratio = 20. Values for K m are the lowest yet observed for aldolase from any source. 4. 4. Carboxypeptidase A digestion of snail aldolase demonstrates that the carboxyl-terminal amino acid of the subunits is tyrosine, and that the tyrosines are not functionally identical.

Role of Orthophosphate and Other Factors in the Regulation of Starch Formation in Leaves and Isolated Chloroplasts

Starch synthesis in leaves was increased by phosphate starvation or by treatments which decreased cytoplasmic orthophosphate levels (such as mannose feeding). Usually less than 30% of the total carbon fixed during CO(2) assimilation was incorporated into starch in spinach (Spinacia oleracea L.), spinach beet (Beta vulgaris), and tobacco (Nicotiana tabacum) leaves.In isolated spinach chloroplasts, formation of starch from CO(2) was usually less than in leaves. In the absence of significant levels of 3-phosphoglycerate, concentrations of phosphate as low as 1 mm (in the medium) or 10 mm (in the stroma) almost completely inhibited starch synthesis. The inhibitory action of phosphate could be overcome by 3-phosphoglycerate. The controlling factor of starch synthesis appeared to be the ratio of phosphoglycerate to orthophosphate rather than the stromal hexose monophosphate concentration, and it is suggested that this control is exerted via the phosphate translocator and the known allosteric regulation of ADP-glucose pyrophosphorylase. Starch synthesis was also favored by the presence of dihydroxyacetone phosphate and by high light and high temperature. Oxygen was inhibitory, probably owing to carbon drain into glycolate. Starch formation by intact chloroplasts could not be promoted by added glucose or glucose 6-phosphate.Starch mobilization in the dark was promoted by orthophosphate and phosphate-dependent mobilization was inhibited by phosphoglycerate. The principal products of starch breakdown in the presence of phosphate were the transport metabolites dihydroxyacetone phosphate and 3-phosphoglycerate. Formation of these compounds from starch was stimulated by ATP or oxaloacetate. In a phosphate-independent reaction, starch was also converted to neutral products such as maltose and glucose. The rates of phosphate-dependent starch degradation phosphorolysis were very much higher than those of starch hydrolysis for which there was no phosphate requirement.

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO 2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ⩾ 2.5 mM completely inhibits CO 2-dependent O 2 evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P i, but not by addition of 3-phosphoglycerate. In the absence of P i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the 14C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O 2 evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts. 2. Although O 2 evolution is completely suppressed by dihydroxyacetone phosphate, CO 2 fixation takes place in air with rates of up to 65μ mol · mg −1 chlorophyll · h −1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation. 3. Under anaerobic conditions, the rates of CO 2 fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg −1 chlorophyll · h −1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10 −7 M) reaching values of up to 60 μmol · mg −1 chlorophyll · h −1. As under these conditions the ATP necessary for CO 2 fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO 2 fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10 −7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO 2 fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO 2 fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation. 4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO 2 fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO 2 fixation in the light and dark

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 °C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit. 2. CO 2 fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3. Phosphoglycerate reduction, which is a partial reaction of CO 2 fixation, shows very little pH dependency. 3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO 2 fixation without affecting phosophoglycerate reduction. This inhibition of CO 2 fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP. 4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO 2 fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO 2 fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport. 5. It is shown that intact chloroplasts are able to fix CO 2 in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma. 6. Dark CO 2 fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO 2 fixation in the light. This demonstrates that CO 2 fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8. 7. It is shown that pH changes occurring in the stroma after illumination are sufficient to switch CO 2 fixation from zero to maximal activity. This offers a mechanism for light control of CO 2 fixation, avoiding wasteful CO 2 fixation in the dark.

D-Glucose 6-Phosphate Cycloaldolase: Inhibition Studies and Aldolase Function

d-Glucose 6-phosphate cycloaldolase is inhibited 83% by 0.66 mm EDTA and stimulated 1.7-fold by 0.6 mm KCl. Dihydroxyacetone phosphate, an analog of the last three carbons in the proposed intermediate, d-xylo-5-hexulose 6-phosphate, acts as a partially competitive inhibitor. Treatment with NaBH(4) in the presence of dihydroxyacetone phosphate does not cause permanent inactivation as would be expected if a Schiff base were being formed. In these properties it resembles a type II, metal-containing aldolase. Photooxidation in the presence of Rose Bengal inactivates this enzyme. NAD(+) partially protects against this photooxidation. Cells grown on medium lacking myoinositol had four times as much enzyme activity as cells grown on medium containing 100 mg of myoinositol per liter.

The Pathway of myo-Inositol Degradation in Aerobacter aerogenes: CONVERSION OF 2-DEOXY-5-KETO-d-GLUCONIC ACID TO GLYCOLYTIC INTERMEDIATES

Abstract The pathway of myo-inositol catabolism in Aerobacter aerogenes has been studied, beginning with the first noncyclic intermediate, 2-deoxy-5-keto-d-gluconic acid. A kinase acts on 2-deoxy-5-keto-d-gluconic acid to yield a monophosphorylated product. The isolation of the kinase product was rendered difficult by the presence, in both crude and 20-fold purified extracts, of the next enzyme in the pathway, an aldolase which catalyzes the cleavage of the kinase product to yield dihydroxyacetone phosphate and malonic semialdehyde. However, accumulation of the kinase product was accomplished by specific inactivation of the aldolase by reduction with sodium borohydride in the presence of dihydroxyacetone phosphate. The kinase product was identified as 2-deoxy-5-keto-d-gluconic acid 6-phosphate. Malonic semialdehyde is further metabolized by the last enzyme in the pathway, malonic semialdehyde oxidative decarboxylase. This enzyme was also partially purified. It catalyzes the conversion of malonic semialdehyde, coenzyme A, and NAD+ to acetyl-CoA, CO2, and NADH + H+. All three of the enzymes that have been identified in this study are induced by growth on myo-inositol, and are constitutive in a mutant which is constitutive for the first three enzymes of the inositol pathway. The operation of the pathway thus results in the conversion of myo-inositol to an equimolar mixture of three known metabolites: dihydroxyacetone phosphate, acetyl-CoA, and CO2.

Properties of fructose-1,6-diphosphate aldolase from two thermophilic and a mesophilic clostridia

Frutose-1,6-diphosphate aldolase (fructose-1,6-diphosphate d-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) from the two thermophiles Clostridium tartarivorum and Clostridium thermosaccharolyticum and from the mesophile Clostridium pasteurianum has been purified about 55-fold. The partially purified proteins exhibited properties characteristic of Class II aldolases, i.e. they showed a requirement for a divalent cation best satisfied by Co 2+, were stimulated by K + or NH 4 +, exhibited a sharp pH optimum of 7.3–7.6, were not inactivated by carboxypeptidase or treatment with NaBH 4 in the presence of dihydroxyacetone phosphate and had a molecular weight of about 68000. The Michaelis constants for the cofactors and the substrate fructose 1,6-diphosphate were found to be similar for all three clostridial enzymes. The presence of cysteine or dithiothreitol in the absence of Co 2+ caused a 2-fold stimulation in activity. However, in the presence of Co 2+ the thiol compounds caused inhibition. Reaction with the sulfhydryl reagents p- chloromercuribenzoate and 5′,5′-dithiobis(2-nitrobenzoic acid) caused complete inactivation of the enzyme which could be reversed by the addition of 2-mercaptoethanol. Co 2+ but not fructose 1,6-diphosphate protected by enzyme from inactivation by the sulfhydryl reagents. C. pasteurianum aldolase exhibited maximum activity when assayed at 42° whereas the thermophile enzymes showed a maximum at 70–72°. The mesophile enzyme was completely inactivated within 5 min when incubated at 57°; the thermophiles aldolases remained stable for at least 4 h. Both the mesophile and thermophile enzymes were found to be equally susceptible to the denaturants urea and guanidine · HCl.

Adaptive alterations in Lactobacillus casei: II. Variation in the levels of aldolase in cells grown on glucose and ribose

The growth of homofermentative Laclobacillus casei on ribose caused reduction in aldol cleavage of fructose-1,6-diphosphate (FDP) while enhancing activities for condensation of triose phosphates and of fructose-1,6-diphosphatase. The purified preparation of aldolase from L. casei was not inhibited by EDTA and was not affected by bivalent metal ions (Fe 2+ and Zn 2+). The inactivation of the enzyme by sodium borohydride only in presence of dihydroxyacetone phosphate (DHAP) suggested formation of a Schiff-base intermediate. The enzyme could be resolved into three active components on DEAE-cellulose chromatography. These fractions showed distinct differences in properties with respect to cleavage of FDP and condensation of triose phosphates. The fractions having high cleavage activity gave low condensation reaction. This component was also active for transaldolase reaction. The other two fractions gave higher rate of condensation and lower cleavage values. The profiles obtained for aldolase prepared from ribose-grown cells of L. casei indicated that there was drastic reduction in the component having high cleavage activity. The results suggest that the equilibrium of the reaction toward cleavage or condensation was determined by isoenzymic composition of aldolase which appears to be adaptively altered by metabolic regulatory controls operative in glucose or ribose-grown cells of L. casei.

The biochemistry of cestode mitochondria, i. aerobic metabolism of mitochoindria from Taenia taeniaeformis

1. 1. The oxidative capacity of mitochondrial fractions isolated from larval and adult Taenia taeniaeformis was assayed polarographically in the presence of various substrates and inhibitors. α-Glycerophosphate was the substrate most readily oxidized. Malate and NADH were oxidized at substantially lower rates; other tricarboxylic acid cycle intermediates, amino-acids, and fatty acids were either less effective or inert as substrates. 2. 2. Dihydroxyacetone phosphate was the product of α-glycerophosphate oxidation. Hydrogen peroxide was not detected, but the presence of an active catalasc may have prevented its identification. 3. 3. α-Glycerophosphate oxidation in the mitochondrial fractions was not mediated by pyridine nucleotide, but was stimulated by vitamin Kj. The cytoplasm of the parasite contained an extramitochondrial α-glycerophosphate dehydrogenase that catalysed the oxidation of NADH in the presence of dihydroxyacetone phosphate. This pair of enzymes provides the essential components of the a-glycerophosphate cycle. 4. 4. The activity of the mitochondrial a-glycerophosphate oxidase was inhibited by dicoumarol. Antimycin and cyanide were less effective, and there was little definitive evidence of a classic cytochrome system participating in the terminal electron transport. 5. 5. Oxidation of a-glycerophosphate was inhibited by several metal-chelating agents. These observations, together with the finding of substantial amounts of non-haem relative to haem iron in the mitochondrial preparations, suggest that non-haem iron has a functional role in the tapeworm respiratory chain. 6. 6. Both oxidized and reduced pyridine nucleotide were found in these preparations, and inhibition of malate oxidation was demonstrated with rotenone or Amytal (amylobarbitone). 7. 7. The respiratory chain of T. taeniaeformis appears to contain the following components: flavoprotein dehydrogenases, non-haem iron, pyridine nucleotide, and a branched cytochrome system, one portion sensitive to cyanide and the other insensitive. 8. 8. The presence of high endogenous ATPase activity in the mitochondrial fractions may indicate that the organelles were functionally altered during isolation with respect to the coupled phosphorylation reactions, which were not demonstrable.

Fructose diphosphate aldolase from Fusarium oxysporum f. lycopersici. 3. Studies on the mechanism of reaction.

Frnctose-1,6-diphosphate aldolase from Fusarium oxysporum f. lycopersici is inhibited by acetylimidazole. The inhibition is reduced in the presence of fructose diphosphate or DL-glyceraldehyde-3-phosphate, but not in the presence of dihydroxyacetone phosphate. Under conditions of almost total inactivation, as measured by the rate of cleavage of fructose diphosphate, the decrease in the rate of exchange is only 18%. These results indicate that the amino acid residues which are altered under these conditions are concerned with the D-glyceraldehyde-3-phosphate rather than the dihydroxyacetone phosphate active site.The rate of cleavage of fructose diphosphate in D2O decreases, with increasing pH, more rapidly than the rate in H2O and resembles the decreased rate of tritium exchange into dihydroxyacetone phosphate with increasing pH. These results suggest that a correlation exists between the isolated proton exchange and cleavage reactions and that the decreased rate of exchange and cleavage with increasing pH is due to a decreased rate of proton neutralization of the enzyme – dihydroxyacetone phosphate anion. The addition of acetaldehyde to the aldolase assays at pH 8.0 and pH 9.0 stimulates the cleavage rate of fructose diphosphate to a level normally obtained at pH 7.5, confirming the supposition that the release of dihydroxyacetone phosphate becomes rate limiting with increasing pH. The results obtained by following the cleavage in D2O also indicate that general base catalysis is involved in the proton neutralization step and that the pK of the general base is less than pH 6.0. The evidence suggests that the general base is either a β-COOH of aspartate or a γ-COOH of glutamate.

The Cyanide Adduct of the Aldolase Dihydroxyacetone Phosphate Imine

Abstract Aldolase was slowly inhibited in the presence of dihydroxyacetone phosphate and hydrogen cyanide. Activity was recovered on removal of these reagents. Neither dihydroxyacetone phosphate or hydrogen cyanide separately nor dihydroxyacetone with cyanide inhibited aldolase. The formation of an inactive aminonitrile enzyme derivative by addition of cyanide to an enzyme-substrate imine (Schiff base) is postulated. The rate and equilibrium constants for inhibition were marginally lower for carboxypeptidase A-treated aldolase than for native aldolase.