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The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.

The regulation of the enzymes involved in the operation of the glyoxylate cycle was studied in the yeast Saccharomyces cerevisiae. All enzymes showed an increase in specific activity under growth conditions where the glyoxylate cycle is needed as an anaplerotic pathway. In the presence of 1% glucose, the synthesis of all enzymes, except fumarase, was repressed. However, no indication for a specific regulation mechanism for the entire cycle could be found. Studies on the localization of the glyoxylate cycle enzymes in the yeast cell revealed that the key enzymes, isocitrate lyase and malate synthase, are located in the cytoplasm, whereas succinate dehydrogense was found only in the mitochondrial fraction. Activites of all other enzymes are found in the cytoplasm as well as in the mitochondira. In anaerobically grown cells, no mitochondria could be detected. Succinate dehydrogenase, isocritrate lyase, and malate synthase were absent. However, appreciable activities of citrate synthase, aconitase, fumarase, and malate dehydrogenase were found in the Cytoplasm. The significance of these observations for the operation of the glyoxylate cycle is discussed., The work was supported by Bundesministerium fur wissenschaftliche Forschung and Deutsche Forschungs- gemeinschaft.

The eukaryotic glyoxylate cycle has been previously hypothesized to occur in the peroxisomal compartment, which in the yeast Saccharomyces cerevisiae additionally represents the sole site for fatty acid β-oxidation. The subcellular location of the key glyoxylate-cycle enzyme malate synthase 1 (Mls1p), an SKL-terminated protein, was examined in yeast cells grown on different carbon sources. Immunoelectron microscopy in combination with cell fractionation showed that Mls1p was abundant in the peroxisomes of cells grown on oleic acid, whereas in ethanol-grown cells Mls1p was primarily cytosolic. This was reinforced using a green fluorescent protein (GFP)–Mls1p reporter, which entered peroxisomes solely in cells grown under oleic acid-medium conditions. Although growth of cells devoid of Mls1p on ethanol or acetate could be fully restored using a cytosolic Mls1p devoid of SKL, this construct could only partially alleviate the requirement for native Mls1p in cells grown on oleic acid. The combined results indicated that Mls1p remained in the cytosol of cells grown on ethanol, and that targeting of Mls1p to the peroxisomes was advantageous to cells grown on oleic acid as a sole carbon source.

Substrate and intermediate analogue inhibitors of enzymes were prepared in which the thioester oxygen of acyl-CoA substrates is replaced by hydrogen with formation of CoA-thioethers. Experiments performed with ATP citrate lyase and S-(3,4-dicarboxy-3-hydroxybutyl)-CoA are consistent with citryl-CoA but not with citryl-enzyme being the direct precursor of the products acetyl-CoA and oxaloacetate. Consistent with these results, a previously described isotopic exchange between acetyl-CoA and [3H]CoASH, indicating the formation of an acetyl-enzyme in the reaction pathway, could not be confirmed. Substrate analogue CoA-thioethers of malate synthase are inhibitors endowed with the affinity of the substrates. Acetyl carboxylase and fatty acid synthetase are not inhibited by the substrate analogue S-ethyl-CoA; S-carboxyethyl-CoA, which could substitute for malonyl-CoA, is likewise not inhibitory. An explanation is proposed. Previously suggested roles of S-carboxymethyl-CoA, an acetyl-CoA-related inhibitor of citrate synthase, are discussed in the light of new experimental data. S-Acetyl, S-propionyl and S-carboxymethyl derivatives of 1,N6-etheno-CoA loose the high affinity of their CoA-counterparts to citrate synthase, probably because the ethylene group prevents proper binding to the enzyme.

A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)-rich RNA of etiolated pumpkin cotyledons. Determination of the DNA sequence of the 1979-nucleotide cDNA revealed a 1698-nucleotide open reading frame that encodes a polypeptide of 64632 Da. The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide-sequence analyses of fragments generated by acid treatment of the purified enzyme. Northern blot analysis revealed that the probe hybridized to a single 2.3-kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons. In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively. Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed.

The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most alpha-proteobacterial genomes, and some should reside on long branches that reveal affinity to eubacterial rather than archaebacterial homologues, but no particular affinity for any specific eubacterial donor.

The eukaryotic glyoxylate cycle has been previously hypothesized to occur in the peroxisomal compartment, which in the yeast Saccharomyces cerevisiae additionally represents the sole site for fatty acid beta-oxidation. The subcellular location of the key glyoxylate-cycle enzyme malate synthase 1 (Mls1p), an SKL-terminated protein, was examined in yeast cells grown on different carbon sources. Immunoelectron microscopy in combination with cell fractionation showed that Mls1p was abundant in the peroxisomes of cells grown on oleic acid, whereas in ethanol-grown cells Mls1p was primarily cytosolic. This was reinforced using a green fluorescent protein (GFP)-Mls1p reporter, which entered peroxisomes solely in cells grown under oleic acid-medium conditions. Although growth of cells devoid of Mls1p on ethanol or acetate could be fully restored using a cytosolic Mls1p devoid of SKL, this construct could only partially alleviate the requirement for native Mls1p in cells grown on oleic acid. The combined results indicated that Mls1p remained in the cytosol of cells grown on ethanol, and that targeting of Mls1p to the peroxisomes was advantageous to cells grown on oleic acid as a sole carbon source.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.

Two genes encoding the enzymes malate synthase G and glycolate oxidase, have been linked to locus glc (64.5 min), responsible for glycolate utilization in Escherichia coli. The gene encoding malate synthase G, for which we propose the notation glcB, has been cloned, sequenced and found to correspond to a 2262-nucleotide open-reading frame, which can encode a 723-amino-acid polypeptide, clearly different from the isoenzyme malate synthase A, which has 533 amino acids. Northern-blot experiments indicate that glcB was expressed as an apparently monocistronic transcript, inducible by glycolate. Malate synthase G was purified to near homogeneity. The molecular mass determined by gel filtration yielded a value of 82 kDa for the purified enzyme and the same value as for the crude extract enzyme, indicating a monomeric structure. Despite the lower sequence similarity between malate synthase G and the other reported malate synthases, three out of nine consensus boxes defined in most of these enzymes are conserved in addition to a cysteine residue that has been reported to be important for the catalytic mechanisms.

There is accumulating evidence that metabolic pathways are organized in vivo as multienzyme clusters or metabolons. To assess interactions between consecutive enzymes of a pathway in vitro, it is usually essential to modify the physical properties of water around the enzymes, e.g. by immobilizing the latter onto a solid support. Such immobilized enzyme preparations can be embedded in agarose gels and used for affinity electrophoresis [Beeckmans, S., Van Driessche, E. & Kanarek, L. (1989) Eur. J. Biochem. 183, 449–454; Beeckmans, S., Van Driessche, E. & Kanarek, L. (1990) J. Cell. Biochem. 43, 297–306]. In this study we use the aforementioned technique to investigate the association between two plant glyoxylic acid cycle enzymes, i.e. isocitrate lyase and malate synthase. A specific histochemical staining technique is described for both enzymes. Affinity electrophoresis using either isocitrate lyase or malate synthase as the immobilized enzyme clearly shows that associations are formed between both enzymes. Moreover, experiments with metabolically unrelated enzymes prove that the observed interaction is specific.

1 Two specimens of stereospecifically tritiated 3-hydroxy-3-methylgutaric acid, respectively (2S,3S) + (2R,3R) [2-3H1] and (2R,3S+ (2S,3R) [2-3H1], were prepared by oxidation of corresponding 4-tritiated mevalonates. 2 After being mixed with 3-hydroxy-3-methyl[3-14C]glutaric acid these specimens were converted chemically into two specimens of 3-hydroxy-3-methylglutaryl-coenzyme A, in which the enzymically active components were respectively (2S, 3S,4R)-[2,4-3H2,3-14C] (specimen I) and (2R,3S,4S)-[2,4-3H2,3-14C](specimen II). 3 Each specimen of 3-hydroxy-3-methylglutaryl-coenzyme A was subjected, in a deuterium oxide medium, to the action of hydroxymethylglutaryl-CoA lyase. Glyoxylate and malate synthase were also present and they converted the liberated deuterio-tritio-acetyl-coenzyme A into 2S-malate, which was isolated. 4 The two specimens of 2S-malate were examined for loss of tritium on incubation with fumarase. Malate from specimen I lost 73% and malate from specimen II lost 23%, of their tritium. 5 It was concluded that chiral acetyl-coenzyme A (S from I and R from II) was generated by the action of the lyase which was therefore stereospecific and proceeded with inversion of configuration. 6 Each specimen of 3-hydroxy-3-methylglutaryl-coenzyme A was subjected to the action of methylglutaconyl-CoA hydratase. The 3-methylglutaconyl-CoA produced by this action was converted chemically into 2, 3-dibromo-3-methylglutaconic acid which was examined for loss of tritium. 7 It was found that the glutaconic acid from specimen I retained 95–97% of the tritium present in the precursor, whereas the glutaconic acid from specimen II retained 47–50% of precursor tritium. 8 It was concluded that the action of methylglutaconyl-CoA hydratase is stereospecific and that addition-elimination of water is syn, not anti. 9 Further evidence is produced that the 3-methylglutaconyl-CoA substrate for this enzyme is geometrically the E-from. It was shown that when E-3-methylglutaconyl-CoA is hydrolysed by cold dilute alkali the E-3-methylglutaconic acid produced has suffered no exchange of hydrogen at the C-2 and C-4 positions but that the accompanying Z-3-methylglutaconic acid has suffered extensive exchange at both positions. An explanation is proposed.

Two malate synthases are known to occur in Escherichia coli: one of them malate synthase A allows, with isocitritase, the growth on acetate, the other malate synthase G, the growth on glycolate as sole carbon sources. Mutants impaired in their growth on acetate (aceA, aceB) or on glycolate (glc) are characterized by their, growth rate on various carbon sources, by specific activities of isocitritase and malate synthase and by their relative content in each type of the two malate synthases. aceA and aceB mutants are deficient in isocitritase and malate synthase A respectively; mutant glc lacks the malate synthase G. The aceB mutation impairs growth on acetate only when present in a gle mutated strain. In strains of wild type for the genes concerned, growing on acetate, malate synthase A constitutes approximately 60% of the total activity of malate synthase, the rest being malate synthase G; on glycolate, the activity is nearly entirely represented by malate synthase G. Isocitritase and malate synthase A activities appear in similar relative amounts through different growth conditions, particularly, during growth on limited carbon source (chemostat); in such conditions, they allow the growth of a glucose negative strain (glu) even on glucose as sole carbon source. The location of aceA, aceB and glc mutations on the genetic map are established by sexual crosses and by phage 363 transductions. The frequencies of cotransduction between aceA or aceB and known loci (metB, glu-1, argH2, purD, metA, malB) completed by three point test analysis, lead to a detailed genetic map of this region (Fig. 3); aceA and aceB are located close together, between metA and malB. The glc mutation (deficiency in malate synthase G) happened in the strain HfrDV21 simultaneously with a translocation of the sexual factor of the parent strain Hfr 30SOMA4. The glc locus and the sexual factor of this new Hfr are located between the known loci argG and his, close to the marker serA with which glc is cotransducible. HfrDV21 transfers the genetic markers pheB, purC and his in the same order as the Hfr KL16 but approximately 8 minutes earlier. The results, together with the properties of the strain constitutive for isocitritase and malate synthase A, point to the occurrence of an ace operon (aceA, aceB) regulated by repression-derepression. Malate synthase G seems to be so easily inducible (by glyoxylate?) that it allows growth on acetate of a strain deficient in malate synthase A. The regulatory properties and physiological roles of the two isoenzymes suggest that the inducible malate synthase G would be more primitive than the repressible malate synthase A.

A procedure was developed for the isolation of membranes of glyoxysomes from the endosperm of germinating castor beans (Ricinus communis L. var. zanzibariensis). These membranes were prepared by osmotic-shock treatment of isolated glyoxysomes. In some cases this was followed by mild sonication. They were isolated by centrifugation on to a 57% (w/w) sucrose layer and purified by isopycnic sucrose gradient centrifugation. The membrane preparations were characterized by electronmicroscopy and assayed for enzyme activities. They consisted mainly of ghost-type vesicles and were found to contain a major part of fatty acid β-oxidation, malate synthase, citrate synthase and antimycin-A-insensitive NADH-cytochrome c reductase activities of the whole glyoxysomes. Malate dehydrogenase may also be bound to the membrane to a significant extent. A tentative scheme is postulated describing the structural organization of fatty acid β-oxidation and of the glyoxylate cycle in glyoxysomes.

1 By chemical synthesis starting from phenylacetylene and utilizing stereospecific reactions, two specimens of acetic acid were prepared in which the following conditions were closely approached: (a) some of the methyl groups consisted of one hydrogen, one deuterium, and one tritium atom attached to carbon; (b) all such methyl groups in one specimen had the R configuration, and all such methyl groups in the other specimen had the S configuration, and the correct absolute configuration was assignable to each specimen from the method of synthesis used; (c) all methyl groups containing a tritium atom also contained a deuterium atom. 2 Each specimen of acetic acid was converted enzymically into acetyl-coenzyme A and (in the same incubation) this product was condensed with glyoxylic acid on malate synthase to form two specimens of S-malic acid. 3 Each specimen of malic acid was incubated with fumarase until the tritium content of the organic acids (fumarate + malate) had ceased to fall. 4 Malic acid synthesized from R-acetic acid retained 69% of its carbon-bound tritium (in fumarate + malate) on incubation with fumarase. Malic acid synthesised from S-acetic acid retained 31% of its carbon-bound tritium. 5 The enzymic assay described in paragraphs 2–4 constitutes a method for determining the absolute configuration of asymmetric methyl groups, provided that a substantial proportion of the tritium in the specimen examined is present in such groups. 6 The method thus established can be used to study the mechanism of enzymic reactions in which a methylene group is converted stereospecifically into a methyl group. With one logical proviso–that it is necessary to known whether protium or deuterium is preferentially removed by the enzyme from methyl groups also containing tritium–the method is also applicable to enzymic reactions in which a methyl group is converted stereospecifically into a methylene group. 7 Subject to the above proviso, it is indicated that on malate synthase the removal of hydrogen and the attachment of the glyoxylate residue occurs with inversion of configuration.

The kinetics of derepression of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, citrate synthase, fumarate hydratase, and malate dehydrogenase were studied following transfer of mycelium grown on a medium containing sucrose to one containing acetate as sole source of carbon. The maximum rate of isocitrate lyase synthesis was proportional to the number of isocitrate lyase structural genes. Comparisons of the repressed levels of isocitrate lyase and malate synthase on sucrose medium and the derepressed levels in acetate medium showed an over-all coordination between the levels of these two enzymes. A similar relationship existed between the levels of fumarate hydratase, citrate synthase and malate dehydrogenase. The Krebs cycle enzymes also showed coordinate repression on sucrose medium under conditions of enhanced catabolite repression. Other results, however, showed that neither for the glyoxylate shunt enzymes nor for the Krebs cycle enzymes was this coordination invariable. Under conditions in which some of the Krebs cycle enzymes assumed different roles no coordination was observed. It is suggested that this type of coordination, which is frequently observed in the pathways of central carbon metabolism in eucaryotes, results from the metabolic repressors of each enzyme being in equilibrium or in a constant ratio to one another. The coordination does not arise because the structural genes of each enzyme belong to the same operon.

A simple procedure is reported for the purification of the two malate synthases of Escherichia coli involved in the use of acetate (A) and glycolate (G), respectively, as carbon source. Both isoenzymes have been isolated simultaneously and in equally high amounts from a strain constitutive for the glyoxylate cycle, after growth on glycolate. After separation from each other by column chromatography on DEAE-cellulose, the malate synthases have been purified by heating, ammonium sulfate precipitation and molecular sieve chromatography on a Sephadex G-200 column, in the constant presence of their specific protectors, glycolate for malate synthase A, and oxalate for malate synthase G. The purified preparations so obtained appeared to be homogeneous on the basis of analytical ultracentrifugation and moving-boundary electrophoresis. The sedimentation coefficient at infinite dilution was 4.6 S for malate synthase A and 5.2 S for malate synthase G; their electrophoretic mobilities at pH 8 were 4.3 X 10−5 cm2 V−1 s−1 and 4.1 × 10−5 cm2 V−1 s−1, respectively. The apparent molecular weight of malate synthase A, estimated by Archibald's method, was 54000; that of malate synthase G, estimated by light scattering, was 52000. Threonine and serine have been found, by Sanger's method, to be N-terminal residues of malate synthases A and G, respectively.

1 d-(1R)-(1-2H1)[1-3H] Fructose 6-phosphate was prepared by the action of glucosephosphate isomerase on d-(1-2H)glucose 6-phosphate in tritiated water. Using the same enzyme but interchanging the labeling pattern of the substrate d-(1S)-(1-2H)[1-3H] fructose 6-phosphate was also obtained. 2 The doubly labeled samples of d-fructose 6-phophate were converted into chiral acetylphosphate on phosphoketolase from Bifidium globosum (ATCC 25864), and these were reacted in situ with phosphotransacetylase and malate synthase in the presence of coenzyme A and glyoxylate. 3 The steric distributin of the tritium in the samples of malate, as shown by the action of fumarase, revealed that d-(1R)-and d-(1S)-(1-2H1)[1-3H] fructose 6-phosphate was converted into (r) and (S)-(2H1)[3H] acetylphosphate, repectively. 4 The results are discussed in terms of the mechanism of action of phosphoketolase.

1. R-Acetate was generated enzymically from R-acetate in the sequence acetate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase kH/k2H was determined as 3.7 +/- 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate leads to citrate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3S-[2-2H1,3-2H1]malate were prepared from 2S-[2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R-[2-2H1,3-2H1,3H1]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3H1]acetate and R-[2H1,3H1]acetate, respectively. 5. The isolated R-[2H1,3H1]acetate and S-[2H1,3H1]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 +/- 0.7% and 20.3 +/- 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50% value found with [3H1]acetate strengthens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase kH/k2H was determined from the data of this study as 3.9 +/- 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to kH/k2H=3.8 +/- 0.1. It was concluded that the configurational analysis of stereochemically pure R-[2H1,3H1]acetate and S-[2H1,3H1]acetate yields malates which in the presence of fumarase retain 79 +/- 2% and 21 +/- 2%, respectively, of their total tritium content. Hence, a deviation of 29 +/- 2% from the 50% value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale.

In light/dark synchronized cultures of Euglena gracilis Klebs Z the enzymes malate synthase, isocitrate lyase and acetate thiokinase were induced upon addition of acetate at all stages of the cell cycle. Cycloheximide and p-fluorophenylalanine inhibited the development of enzyme activity, showing that induction was dependent on protein synthesis. The maximum rate of induction for all three enzymes was constant for much of the cell cycle but doubles in a single step during the period of DNA replication. Although these data indicate that enzyme potential was regulated by gene dosage and that the structural gene for each enzyme was continuously available for transcription during the cell-cycle it was not possible by using inhibitors of RNA synthesis, to demonstrate concurrent transcription during enzyme induction.

Malate synthase from baker's yeast has been investigated in solution by the small-angle X-ray scattering technique. Size, shape and structure of the native substrate-free enzyme and of various enzyme-substrate complexes have been determined. As the enzyme was found to be rather unstable against X-rays, several precautions as well as sophisticated evaluation procedures had to be adopted to make sure that the results were not influenced by radiation damage. These included use of low primary intensity, short time of measurement, the presence of high concentrations of dithiothreitol, combined use of the conventional slit-collimation system and the new cone-collimation system. 1. For the native substrate-free enzyme the following molecular parameters could be established: radius of gyration R = 3.96 +/- 0.02 nm, maximum particle diameter D = 11.2 +/- 0.6 nm, radius of gyration of the thickness Rt = 1.04 +/- 0.04 nm, molecular weight Mr = 187000 +/- 3000, correlation volume Vc = 338 +/- 5 nm3, hydration x = 0.35 +/- 0.02 g/g, mean intersection length - l = 5.0 +/- 0.2 nm. Comparison of the experimental scattering curve with theoretical curves for various models showed that the enzyme is equivalent in scattering to an oblate ellipsoid of revolution rather than to a circular cylinder. The semiaxes of this ellipsoid are a = b = 6.06 nm and c = 2.21 nm. Thus with an axial ratio of about 1:0.36 the enzyme is of very anisometric shape. 2. Binding of the substrates (acetyl-CoA, glyoxylate) or the substrate analogue pyruvate causes slight structural changes of the enzyme. These changes are reflected mainly by a slight decrease of the radius of gyration (0.3--1.3%, as established both with the slit-smeared and the desmeared curves). Concomitantly there occurs a decrease of the maximum particle diameter and an increase of the radius of gyration of the thickness. These changes imply an increase of the axial ratio by 2.2--6.9%, i.e. substrate binding induces a decrease of anisometry. While the particle volume appears to be unchanged on binding glyoxylate or its analogue pyruvate, binding of acetyl-CoA causes slight changes of this parameter. In a similar manner the binding of acetyl-CoA leads to a slight enhancement of the molecular weight; this increase corresponds to the binding of 2.7 +/- 1 molecules of acetyl-CoA.

1. Malate synthase from baker's yeast (5 kg) was purified 400--500-fold to homogeneity. About 50--200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell-free crude extracts. The enzyme, pI = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies. 2. Sedimentation and diffusion coefficients were determined: S 0 20,w = 8.26 +/- 0.05 S, D 0 20,w = 4.5 +/- 0.1 X 10(-7) cm2 s-1. The molecular weight of the synthase was found to be 175 000 +/- 10 000 and 180 000 +/- 10 000 by sedimentation/diffusion and by high-speed sedimentation equilibrium respectively. It was concluded from these and other results that malate synthase has a molecular mass of 180 000 +/- 10 000. 3. The synthase on sodium dodecylsulfate gel electrophoresis was dissociated to yield a single specimen of Mr about 66 000. The result indicates a composition of the native enzyme from three subunits of identical or nearly identical mass. 4. The binding of acetyl-coenzyme A to the synthase is independent of Mg2+ but that of glyoxylate is strictly dependent on the presence of Mg2+. Kinetic studies indicate that the malate synthase reaction follows a sequential random mechanism. 5. The intermolecular isotopic effect, kH:k2H = 1.4, was determined with acetyl-coenzyme A and [2H3]acetyl-coenzyme A under several different experimental conditions and was shown to reflect different maximal velocities of the two substrates. An enzymic procedure for the preparation of doubly labelled [3H, 14C]acetyl-coenzyme A is also presented.

The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.

1 A preparation of malic enzyme was made from chicken liver. 2 The enzyme was incubated, in tritiated buffer containing NADP, with 2S, 3S-[2,3-2H2]malate. A third malate, 2S, 3R-[3-3H1]malate, was incubated with the enzyme, NADP, and unlabelled buffer. Conditions were chosen so that the initially formed pyruvate was converted into lactate, which was isolated. 3 The lactate from each of the three incubations was oxidized chemically to acetate, which was purified. 4 Each specimen of acetate was assayed for chirality by the previously established method of conversion into S-malate by condensation with glyoxylate on malate synthase, and measuring the fractional loss of tritium on incubation of the S-malate with fumarate hydratase. 5 In this way, the acetate derived from 2S, 3R-[3-2H1]malate was shown to have S chirality and the acetate derived from 2S, 3S-[2,3-2H2]malate was shown to have R chirality. As expected, the acetate from 2S, 3R-[3-3H1]malate was found non-chiral. 6 It is deduced that when S malate is decarboxylated on malic enzyme, carboxyl is replaced by hydrogen without net change of configuration. This confirms a result obtained by Rose in 1970 by different methods.

1 The labelling pattern of a β-hydroxyacid, synthesized on nucleophilic enzymatic reaction of acetyl-CoA with an α-oxoacid in H218O, yields information about the mechanism of this type of enzyme action. Two related reactions catalyzed by citrate synthase and malate synthase were investigated in H218O. Malate was formed as the product in both cases. 2 A micro-scale procedure is described for the stepwise and specific removal of the oxygen groups present in malate, by enzymatic degradation. 3 Mass spectrometric analyses of the enzymatically obtained 18O-labelled malates and of their degradation products revealed the labelling of one oxygen atom of one carboxyl group of malate. The significance of this result is discussed.