Glucosephosphate isomerase from Trypanosoma brucei. Cloning and characterization of the gene and analysis of the enzyme

In Trypanosoma brucei the enzyme glucose-6-phosphate isomerase, like most other enzymes of the glycolytic pathway, resides in a microbody-like organelle, the glycosome. Here we report a detailed study of this enzyme, involving a determination of its kinetic properties and the cloning and sequence analysis of its gene. The gene codes for a polypeptide of 606 amino acids, with a calculated Mr of 67280. The protein predicted from the gene sequence has 54-58% positional identity with its yeast and mammalian counterparts. Compared to those other glucose-6-phosphate isomerases the trypanosomal enzyme contains an additional 38-49 amino acids in its N-terminal domain, as well as a number of small insertions and deletions. The additional amino acids are responsible for the 5-kDa-larger subunit mass of the T. brucei enzyme, as measured by gel electrophoresis. The glucose-6-phosphate isomerase of the trypanosome has no excess of positive residues and, consequently, no high isoelectric point, in contrast to the other glycolytic enzymes that are present in the glycosome. However, similar to other glycosomal proteins analyzed so far, specific clusters of positive residues can be recognized in the primary structure. Comparison of the kinetic properties of the T. brucei glucose-6-phosphate isomerase with those of the yeast and rabbit muscle enzymes did not reveal major differences. The three enzymes have very similar pH profiles. The affinity for the substrate fructose 6-phosphate (Km = 0.122 mM) and the inhibition constant for the competitive inhibitor gluconate 6-phosphate (Ki = 0.14 mM) are in the same range as those of the similar enzymes. The Km shows the same strong dependence on salt as the rabbit muscle enzyme, although somewhat less than the yeast glucose-6-phosphate isomerase. The trypanocidal drug suramin inhibits the T. brucei and yeast enzymes to the same extent (Ki = 0.29 and 0.36 mM, respectively), but it had no effect on the rabbit muscle enzyme. Agaricic acid, a potent inhibitor of various glycosomal enzymes of T. brucei, has also a strong, irreversible effect on glucose-6-phosphate isomerase, while leaving the yeast and mammalian enzymes relatively unaffected.     

Competitive inhibitors of yeast phosphoglucose isomerase: synthesis and evaluation of new types of phosphorylated sugars from the synthon D-arabinolactone-5-phosphate

Designed as competitive inhibitors of the isomerization reaction catalyzed by the potential chemotherapeutic target phosphoglucose isomerases (PGI), D-arabinonamide-5-phosphate and D-arabinohydrazine-5-phosphate were synthesized and fully characterized. These new types of phosphorylated sugar derivatives were easily and efficiently obtained in a one-step procedure from the promising synthon D-arabinono-1,4-lactone 5-phosphate. These two compounds proved to be new good competitive inhibitors of yeast PGI with the substrate D-fructose-6-phosphate, though not as strong as D-arabinohydroxamic acid-5-phosphate. Overall, our results are in accord with the postulated 1,2-cis-enediolate species as a probable high-energy intermediate of the PGI-catalyzed reaction.     

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

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

Kinetic properties of triose-phosphate isomerase from Trypanosoma brucei brucei. A comparison with the rabbit muscle and yeast enzymes

The kinetic properties of Trypanosoma brucei brucei triose-phosphate isomerase are compared with those of the commercially available rabbit muscle and yeast enzymes and with published data on the chicken muscle enzyme. With glyceraldehyde 3-phosphate as substrate Km = 0.25 +/- 0.05 mM and kcat = 3.7 X 10(5) min-1. With dihydroxyacetone phosphate as substrate Km = 1.2 +/- 0.1 mM and kcat = 6.5 X 10(4) min-1. The pH dependence of Km and Vmax at 0.1 M ionic strength is in agreement with the results published for the yeast and chicken muscle enzymes. At ionic strength below 0.05 M the effect of a charged group specific for the trypanosomal enzyme and absent from the yeast and rabbit muscle enzymes becomes detectable. This effect significantly increases Km whereas Vmax becomes slightly higher. Trypanosomal triose-phosphate isomerase is inhibited by sulphate, phosphate and arsenate ions, by 2-phosphoglycolate and a number of documented inhibitors in the same concentration range as are the other triose-phosphate isomerases. The trypanocidal drug, Suramin inhibits T. brucei and rabbit muscle triose-phosphate isomerase to the same extent while leaving the yeast enzyme relatively unaffected.     

Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts

Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.     

The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei

The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3'-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce D-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce D-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.     

Crystal structure of phosphoglucose isomerase from Trypanosoma brucei complexed with glucose-6-phosphate at 1.6 A resolution

Enzymes of glycolysis in Trypanosoma brucei have been identified as potential drug targets for African sleeping sickness because glycolysis is the only source of ATP for the bloodstream form of this parasite. Several inhibitors were previously reported to bind preferentially to trypanosomal phosphoglucose isomerase (PGI, the second enzyme in glycolysis) than to mammalian PGIs, which suggests that PGI might make a good target for species-specific drug design. Herein, we report recombinant expression, purification, crystallization and X-ray crystal structure determination of T. brucei PGI. One structure solved at 1.6 A resolution contains a substrate, D-glucose-6-phosphate, in an extended conformation in the active site. A second structure solved at 1.9 A resolution contains a citrate molecule in the active site. The structures are compared with the crystal structures of PGI from humans and from Leishmania mexicana. The availability of recombinant tPGI and its first high-resolution crystal structures are initial steps in considering this enzyme as a potential drug target.     

The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes

Crystals of triosephosphate isomerase from Trypanosoma brucei brucei have been used in binding studies with three competitive inhibitors of the enzyme's activity. Highly refined structures have been deduced for the complexes between trypanosomal triosephosphate isomerase and a substrate analogue (glycerol-3-phosphate to 2.2 A), a transition state analogue (3-phosphonopropionic acid to 2.6 A), and a compound structurally related to both (3-phosphoglycerate to 2.2 A). The active site structures of these complexes were compared with each other, and with two previously determined structures of triosephosphate isomerase either free from inhibitor or complexed with sulfate. The comparison reveals three conformations available to the "flexible loop" near the active site of triosephosphate isomerase: open (no ligand), almost closed (sulfate), and fully closed (phosphate/phosphonate complexes). Also seen to be sensitive to the nature of the active site ligand is the catalytic residue Glu-167. The side chain of this residue occupies one of two discrete conformations in each of the structures so far observed. A "swung out" conformation unsuitable for catalysis is observed when sulfate, 3-phosphoglycerate, or no ligand is bound, while a "swung in" conformation ideal for catalysis is observed in the complexes with glycerol-3-phosphate or 3-phosphonopropionate. The water structure of the active site is different in all five structures. The results are discussed with respect to the triosephosphate isomerase structure function relationship, and with respect to an on-going drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.     

Inhibition of type I and type II phosphomannose isomerases by the reaction intermediate analogue 5-phospho-D-arabinonohydroxamic acid supports a catalytic role for the metal cofactor

The phosphomannose isomerases (PMI) comprise three families of proteins: type I, type II, and type III PMIs. Members of all three families catalyze the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P) but share little or no sequence identity. Because (1) PMIs are essential for the survival of several microorganisms, including yeasts and bacteria, and (2) the PMI enzymes from several pathogens do not share significant sequence identity to the human protein, PMIs have been considered as potential therapeutic targets. Elucidation of the catalytic and regulatory mechanisms of the different types of PMIs is strongly needed for rational species-specific drug design. To date, inhibition and crystallographic studies of all PMIs are still largely unexplored. As part of our research program on aldose-ketose isomerases, we report in this paper the evaluation of two new inhibitors of type I and type II PMIs from baker's yeast and Pseudomonas aeruginosa, respectively. We found that 5-phospho-D-arabinonohydroxamic acid (5PAH), which is the most potent inhibitor of phosphoglucose isomerase (PGI), is by far the best inhibitor ever reported of both type I and type II PMI-catalyzed isomerization of M6P to F6P. 5PAH, which has an inhibition constant at least 3 orders of magnitude smaller than that of previously reported PMI inhibitors, may be the first high-energy intermediate analogue inhibitor of the enzymes. We also tested the related molecule 5-phospho-D-arabinonate (5PAA), which is a strong competitive inhibitor of PGI, and found that it does not inhibit either PMI. All together, our results are consistent with a catalytic role for the metal cofactor in PMI activity.     

Competitive inhibitors of type B ribose 5-phosphate isomerases: design, synthesis and kinetic evaluation of new d-allose and d-allulose 6-phosphate derivatives

This study reports syntheses of d-allose 6-phosphate (All6P), d-allulose (or d-psicose) 6-phosphate (Allu6P), and seven d-ribose 5-phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichiacoli (EcRpiB). 5-Phospho-d-ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenophospho-d-ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step. Kinetic evaluation on Allpi activity of EcRpiB shows that two of these compounds, 5-phospho-d-ribonohydroxamic acid and N-(5-phospho-d-ribonoyl)-methylamine, indeed behave as new efficient inhibitors of EcRpiB; further, 5-phospho-d-ribonohydroxamic acid was demonstrated to have competitive inhibition. Kinetic evaluation on Rpi activity of both EcRpiB and RpiB from Mycobacteriumtuberculosis (MtRpiB) shows that several of the designed 6-carbon high-energy intermediate analogues are new competitive inhibitors of both RpiBs. One of them, 5-phospho-d-ribonate, not only appears as the strongest competitive inhibitor of a Rpi ever reported in the literature, with a K_i value of 9 μM for MtRpiB, but also displays specific inhibition of MtRpiB versus EcRpiB.     

Molecular analysis of the structural gene for yeast transaldolase

We have cloned the structural gene for yeast transaldolase. Transformants carrying the TAL1 gene on a multicopy plasmid over-produced transaldolase. A deletion mutant which was constructed using the cloned gene did not show any detectable transaldolase activity in vitro. Furthermore, both transaldolase isoenzymes which were detected in wild-type crude extracts by immunoblotting were missing in the deletion mutants. Thus, TAL1 is the only transaldolase structural gene in yeast. TAL1 is not an essential gene. Deletion of the transaldolase gene did not affect growth on complete media with different carbon sources or on synthetic media. However, the transaldolase-deficient strains accumulated sedoheptulose 7-phosphate, an intermediate of the pentose-phosphate pathway. Mutants lacking both transaldolase and phosphoglucose isomerase grew more slowly than the single mutants. They accumulated more sedoheptulose 7-phosphate on medium containing fructose than on glucose medium. This shows that fructose 6-phosphate and glyceraldehyde 3-phosphate, metabolites of glycolysis, can enter the nonoxidative part of the pentose-phosphate pathway.     

The crystal structure of phosphoglucose isomerase/autocrine motility factor/neuroleukin complexed with its carbohydrate phosphate inhibitors suggests its substrate/receptor recognition

Phosphoglucose isomerase catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. In addition, phosphoglucose isomerase has been shown to have functions equivalent to neuroleukin, autocrine motility factor, and maturation factor. Here we present the crystal structures of phosphoglucose isomerase complexed with 5-phospho-D-arabinonate and N-bromoacetylethanolamine phosphate at 2.5- and 2.3-A resolution, respectively. The inhibitors bind to a region within the domains' interface and interact with a histidine residue (His(306)) from the other subunit. We also demonstrated that the inhibitors not only affect the enzymatic activity of phosphoglucose isomerase, but can also inhibit the autocrine motility factor-induced cell motility of CT-26 mouse colon tumor cells. These results indicate that the substrate and the receptor binding sites of phosphoglucose isomerase and autocrine motility factor are located within close proximity to each other. Based on these two complex structures, together with biological and biochemical results, we propose a possible isomerization mechanism for phosphoglucose isomerase.     

Synthesis of some second-generation substrate analogues of early intermediates in the biosynthetic pathway of glycosylphosphatidylinositol membrane anchors

1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-2-O-octyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (23) and the corresponding 2-O-hexadecyl-D-myo-inositol compound 24 have been prepared as substrate analogues of an early intermediate in the biosynthetic pathway of glycosylphosphatidylinositol (GPI) membrane anchors. 1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-octyl-sn-glycerol 3-phosphate) has also been prepared as a substrate analogue. Biological evaluation of the analogues 23 and 24 revealed that they are neither substrates nor inhibitors of GPI biosynthetic enzymes in the human (HeLa) cell-free system but are potent inhibitors at different stages of GPI biosynthesis in the Trypanosoma brucei cell-free system.     

Expression of E. coli araBAD operon encoding enzymes for metabolizing L-arabinose in Saccharomyces cerevisiae

The Escherichia coli araBAD operon consists of three genes encoding three enzymes that convert L-arabinose to D-xylulose-5 phosphate. In this paper we report that the genes of the E. coli araBAD operon have been expressed in Saccharomyces cerevisiae using strong promoters from genes encoding S. cerevisiae glycolytic enzymes (pyruvate kinase, phosphoglucose isomerase, and phosphoglycerol kinase). The expression of these cloned genes in yeast was demonstrated by the presence of the active enzymes encoded by these cloned genes and by the presence of the corresponding mRNAs in the new host. The level of expression of L-ribulokinase (araB) and L-ribulose-5-phosphate 4-epimerase (araD) in S. cerevisiae was relatively high, with greater than 70% of the activity of the enzymes in wild type E. coli. On the other hand, the expression of L-arabinose isomerase (araA) reached only 10% of the activity of the same enzyme in wild type E. coli. Nevertheless, S. cerevisiae, bearing the cloned L-arabinose isomerase gene, converted L-arabinose to detectable levels of L-ribulose during fermentation. However, S. cerevisiae bearing all three genes (araA, araB, and araD) was not able to produce detectable amount of ethanol from L-arabinose. We speculate that factors such as pH, temperature, and competitive inhibition could reduce the activity of these enzymes to a lower level during fermentation compared to their activity measured in vitro. Thus, the ethanol produced from L-arabinose by recombinant yeast containing the expressed BAD genes is most likely totally consumed by the cell to maintain viability.     

Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites'' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.     

Inhibition of Escherichia coli glucosamine-6-phosphate synthase by reactive intermediate analogues. The role of the 2-amino function in catalysis

Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of D-glucosamine 6-phosphate from D-fructose 6-phosphate using L-glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. The most potent carbohydrate-based inhibitor of GlmS reported to date is 2-amino-2-deoxy-D-glucitol 6-phosphate, an analogue of the putative cis-enolamine intermediate formed during catalysis. The interaction of a series of structurally related cis-enolamine intermediate analogues with GlmS is described. Although arabinose oxime 5-phosphate is identified as a good competitive inhibitor of GlmS with an inhibition constant equal to 1. 2 (+/-0.3) mM, the presence of the amino function at the 2-position is shown to be important for potent inhibition. Comparison of the binding affinities of 2-deoxy-D-glucitol 6-phosphate and 2-amino-2-deoxy-D-glucitol 6-phosphate indicates that the amino function contributes -4.1 (+/-0.1) kcal/mol to the free energy of inhibitor binding. Similarly, comparison of the binding affinities of 2-deoxy-D-glucose 6-phosphate and D-glucosamine 6-phosphate indicates that the amino function contributes -3.0 (+/-0.1) kcal/mol to the free energy of product binding. Interactions between GlmS and the 2-amino function of its ligands contribute to the uniform binding of the product and the cis-enolamine intermediate as evidenced by the similar contribution of the amino group to the free energy of binding of D-glucosamine 6-phosphate and 2-amino-2-deoxy-D-glucitol 6-phosphate, respectively.     

Biosynthesis and uptake of thiamine (vitamin B1) in bloodstream form Trypanosoma brucei brucei and interference of the vitamin with melarsen oxide activity

Bloodstream forms of Trypanosoma brucei brucei were cultivated in the presence and absence of thiamine (vitamin B1) and pyridoxine (vitamin B6). The vitamins do not change growth behaviour, indicating that Trypanosoma brucei is prototrophic for the two vitamins even though in silico no bona-fide thiamine-biosynthetic genes could be identified in the T. brucei genome. Intracellularly, thiamine is mainly present in its diphosphate form. We were unable to detect significant uptake of [3H]thiamine and structural thiamine analogues such as pyrithiamine, oxithiamine and amprolium were not toxic for the bloodstream forms of T. brucei, indicating that the organism does not have an efficient uptake system for thiamine and its analogues. We have previously shown that, in the fission yeast Saccharomyces pombe, the toxicity of melarsen oxide, the pharmacologically active derivative of the frontline sleeping sickness drug melarsoprol, is abolished by thiamine and the drug is taken up by a thiamine-regulated membrane protein which is responsible for the utilization of thiamine. We show here that thiamine also has weak effects on melarsen oxide-induced growth inhibition and lysis in T. brucei. These effects were consistent with a low affinity of thiamine for the P2 adenosine transporter that is responsible for uptake of melaminophenyl arsenicals in African trypanosomes.     

A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens.

We propose that arginine side chains often play a previously unappreciated general structural role in the maintenance of tertiary structure in proteins, wherein the positively charged guanidinium group forms multiple hydrogen bonds to backbone carbonyl oxygens. Using as a criterion for a "structural" arginine one that forms 4 or more hydrogen bonds to 3 or more backbone carbonyl oxygens, we have used molecular graphics to locate arginines of interest in 4 proteins: Arg 180 in Thermus thermophilus manganese superoxide dismutase, Arg 254 in human carbonic anhydrase II, Arg 31 in Streptomyces rubiginosus xylose isomerase, and Arg 313 in Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase. Arg 180 helps to mold the active site channel of superoxide dismutase, whereas in each of the other enzymes the structural arginine is buried in the "mantle" (i.e., inside, but near the surface) of the protein interior well removed from the active site, where it makes 5 hydrogen bonds to 4 backbone carbonyl oxygens. Using a more relaxed criterion of 3 or more hydrogen bonds to 2 or more backbone carbonyl oxygens, arginines that play a potentially important structural role were found in yeast enolase, Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase, bacteriophage T4 and human lysozymes, Enteromorpha prolifera plastocyanin, HIV-1 protease, Trypanosoma brucei brucei and yeast triosephosphate isomerases, and Escherichia coli trp aporepressor (but not trp repressor or the trp repressor/operator complex).(ABSTRACT TRUNCATED AT 250 WORDS)     

Analogues of thiolactomycin as potential anti-malarial and anti-trypanosomal agents

A series of analogues of the naturally occurring antibiotic thiolactomycin (TLM) have been synthesised and evaluated for their ability to inhibit the growth of the malaria parasite, Plasmodium falciparum. Thiolactomycin is an inhibitor of Type II fatty acid synthase which is found in plants and most prokaryotes, but not an inhibitor of Type I fatty acid synthase in mammals. A number of the analogues showed inhibition equal to or greater than TLM. The introduction of hydrophobic alkyl groups at the C3 and C5 positions of the thiolactone ring lead to increased inhibition, the best showing a fourteenfold increase in activity over TLM. In addition, some of the analogues showed activity when assayed against the parasitic protozoa, Trypanosoma cruzi and Trypanosoma brucei.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)