Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae

Summary The structural gene PG11 coding for phosphoglucose isomerase was replaced by the LEU2 gene in the genome of Saccharomyces cerevisiae. Plasmids carrying the LEU2 gene between genomic regions flanking the PG11 gene were constructed and used to transform a PGI1/pgi1 diploid strain. Stable transformants lacking the PGI1 allele were isolated. Southern analysis of their meiotic products showed that haploid strains with a deletion of 1.6 kb within the 2.2 kb PG11 coding region were viable. Thus, the PGI1 gene is not essential in yeasts. However, unlike pgi1 mutants with residual phosphoglucose isomerase activity, no growth was detected in the pgi1 haploid strains when fructose was supplied as sole carbon source. The wild-type growth rate could be restored by adding 0.1% glucose to the medium. Furthermore, pgi1 mutants with residual enzymatic activity grew very slowly on fructose-supplemented media containing up to 2% glucose. Strains carrying the deletion allele, however, failed to grow at glucose concentrations higher than 0.5%. Also the pgi1 strains did not grow in glucose as sole carbon source. On the other hand pgi1/pgi1 diploid strains did not sporulate on the usual acetate medium. This defect could be alleviated by the addition of 0.05% glucose to the sporulation medium. Under these conditions the pgi1 mutants sporulated with an efficiency of 25% compared with the wild type. These results suggest that (a) the phosphoglucose isomerase reaction is the only step catalysing the interconversion of glucose-6-P and fructose-6-P, (b) glucose-6-P is essential in yeasts, and (c) the oxidation of glucose-6-P through the glucose-6-P dehydrogenase reaction is not sufficient to support growth in yeasts.     

Computationally designed prodrugs of statins based on Kirby’s enzyme model

DFT calculations at B3LYP/6-31G(d,p) for intramolecular proton transfer in Kirby’s enzyme models 1–7 demonstrated that the reaction rate is dependent on the distance between the two reacting centers, r_GM, and the hydrogen bonding angle, α, and the rate of the reaction is linearly correlated with r_GM and α. Based on these calculation results three simvastatin prodrugs were designed with the potential to provide simvastatin with higher bioavailability. For example, based on the calculated log EM for the three proposed prodrugs, the interconversion of simvastatin prodrug ProD 3 to simvastatin is predicted to be about 10 times faster than that of either simvastatin prodrug ProD 1 or simvastatin ProD 2. Hence, the rate by which the prodrug releases the statin drug can be determined according to the structural features of the promoiety (Kirby’s enzyme model).

Generation of C3- and C2-deuterated l-lactic acid by human erythrocytes exposed to d-[1-13C]glucose,d-[2-13c]glucose and d-[6-13C]glucose in the presence of D2O

• 1.1. The generation of C₂- and C₃-deuterated l-lactate was monitored by ¹³C NMR in human erythrocytes exposed to d-[1-¹³glucose, d-[2-¹³C]glucose or d-te-¹³C]glucose and incubated in a medium prepared in D₂O.
• 2.2. The results suggested that the deuteration of the C₁ of d-fructose 6-phosphate in the phosphoglucoisomerase reaction, the deuteration of the C₁ of d-glyceraldehyde-3-phosphate in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase and the deuteration of the C₃ of pyruvate in the reaction catalyzed by pyruvate kinase were all lower than expected from equilibration with D₂O.
• 3.3. Moreover, about 40% of the molecules of pyruvate generated by glycolysis apparently underwent deuteration on their C₃ during interconversion of the 2-keto acid and l-alanine in the reaction catalyzed by glutamate-pyruvate transaminase.
• 4.4. The occurrence of the latter process was also documented in cells exposed to exogenous [3-¹³C]pyruvate.
• 5.5. This methodological approach is proposed to provide a new tool to assess in intact cells the extent of back-and-forth interconversion of selected metabolic intermediates.

     

Uncoupling and isotope effects in γ-butyrobetaine hydroxylation

Replacement of unlabeled -butyrobetaine with -[2,3,4-²H₆]butyrobetaine has a profound effect on the stoichiometry between decarboxylation of 2-oxoglutarate and hydroxylation in the reaction catalyzed by human -butyrobetaine hydroxylase. The ratios between decarboxylation and hydroxylation are 1.16 with Unlabeled and 7.48 with deuterated -butyrobetaine as substrate. From these ratios an internal isotope effect of 41 has been calculated. DV in the overall reaction measured as 2- oxoglutarate decarboxylation is 2.5 and D_V/K is 1.0. For -butyrobetaine hydroxylase fromPseudomonas sp. AK 1, 2-oxoglutarate decarboxylation exceeds hydroxylation with 10% when deuterated -butyrobetaine is used. No excess was found with unlabeled substrate and no internal isotope effect could be calculated. D_V for the bacterial enzyme is 6.     

Effects of ischaemia and reperfusion on NADH coenzyme Q reductase activity in rat liver

NADH coenzyme Q reductase (EC 1.6.5.3) has been suggested in the literature to be inactivated by ischaemia. In the present study, NADH coenzyme Q reductase activity was localized in unfixed cryostat sections of ischaemic rat livers and quantified using image analysis. In vitro ischaemia was induced by storage of rat liver fragments for 30, 60, and 120min at 37°C. In vivo ischaemia was provoked by clamping the afferent vessels of median and left lateral liver lobes for 60min followed by 30, 60 and 180min of reperfusion. NADH coenzyme Q reductase activity was demonstrated with the tetrazolium salt method in the presence of polyvinyl alcohol. Final reaction product was found in liver parenchymal cells and its distribution was homogeneous within liver lobules. Only low amounts of final reaction product were formed when the incubation was performed in the absence of the substrate NADH. A non-linear relation was found between the absorbance and incubation time when the reaction was performed in the presence of NADH. Therefore, the initial velocity was taken as the true rate of enzyme activity. A linear relationship was found for the initial velocity and section thickness up to 6µm followed by a levelling off. Electron microscopically, NADH coenzyme Q reductase activity was localized at the outer and inner membranes of mitochondria. In vitro ischaemia up to 120min did not affect NADH coenzyme Q reductase activity. At 30min reperfusion after in vivo ischaemia for 60min enzyme activity was slightly decreased in certain foci which also showed diminished lactate dehydrogenase activity. A further decrease of enzyme activities in foci was observed at 180min reperfusion after ischaemia. It is concluded that NADH coenzyme Q reductase activity is not sensitive to ischaemia. Furthermore, it is likely that the enzyme leaks from liver parenchymal cells into the circulation during reperfusion after ischaemia.     

Oligomerization reactions of deoxyribonucleotides on montmorillonite clay: The effect of mononucleotide structure,phosphate activation and montmorillonite composition on phosphodiester bond formation

2-d-5-GMP and 2-d-5-AMP bind 2 times more strongly to montmorillonite 22A than do 2-d-5-CMP and 5-TMP. The dinucleotide d(pG)₂ forms in 9.2% yield and the cyclic dinucleotide c(dpG)₂ in 5.4% yield in the reaction of 2-d-5-GMP with EDAC in the presence of montmorillonite 22A. The yield of d(pC)₂ (2.0%) is significantly lower but comparable to that obtained from 5-TMP. The yield of dimers which contain the phosphodiester bond decreases as the reaction medium is changed from 0.2 M NaCl to a mixture of 0.2 M NaCl and 0.075 M MgCl₂. A low yield of d(pA)₂ was observed in the condensation reaction of 5-ImdpA on montmorillonite 22A. The cyclic nucleotide (3,5-cdAMP) was obtained in 14% yield from 3-ImdpA. The yield of d(pA)₂ obtained when EDAC is used as the condensing agent increases with increasing iron content of the Na⁺-montmorillonite used as catalyst. Evidence is presented which shows that the acidity of the Na⁺-montmorillonite is a necessary but not sufficient factor for the montmorillonite catalysis of phosphodiester bond formation.     

Temporal asymmetry of sex interconversion in a strain of the homothallic fission yeastSchizosaccharomyces pombe

In the homothallic P/d sex interconversion system in a strain of a fission yeast (Schizosaccharomyces pombe), Pd is apparently twice as frequent as dP. This is interpreted to mean that Pd occurs before DNA replication, whereas dP occurs after. But the probabilities of their occurrence within a cell cycle are about the same (1 in 2⁷ cell divisions).     

Template-directed oligomerization of 3-isoadenosine 5′-phosphate

Summary Template-directed oligomerization of an activated derivative of 3-isoadenosine 5-phosphate (piA) on polyuridylic acid [poly(U)] was studied. The reaction of ImpiA is more efficient than the corresponding reaction of ImpA, and produces 3–5-linked oligomers while the reaction of ImpA gives only 2–5-linked oligomers. The base pairing between piA and poly(U) in this system is probably of the Hoogsteen type (involving the 6-amino group and N7 of 3-isoadenosine) rather than of the Watson-Crick type.     

A DFT investigation of conformational geometries and interconversion equilibria of phenylthiosemicarbazone and its complexation with zinc

The geometrical structures of phenylthiosemicarbazone (HAPhTSC) conformers have been obtained by geometry optimizations using density functional theory (DFT) calculations at the B3LYP/6-31G(d) and B3LYP/6-311G(d,p) levels of theory. Six thioamino and 24 thioimino tautomers of HAPhTSC have been found. Six tautomerization reactions between thioamino and thioimino tautomers occurring via transition states and their corresponding activation energies have been obtained. Conformational pathways for tautomerizations and interconversions of HAPhTSC conformers have been presented. Tautomerization between the most stable species of thioamino (Atttcc) and its thioimino (Itttcct) tautomer is an endothermic reaction, H⁰=18.17 kcal mol^–1 and its log K=–13.74, at 298.15 K. Thermodynamic quantities of tautomerizations, interconversions of HAPhTSC conformers and their equilibrium constants are reported. The geometry of the zinc complex with HAPhTSC, found as a Zn(HAPhTSC)₂Cl₂ structure, has been obtained using B3LYP/6-31G(d) calculations. Binding of the Zn(HAPhTSC)₂Cl₂ complex is an exothermic and spontaneous reaction.Figure Conformational notation defined as a name consisting of a letter A for a thioamino tautomer followed by c for cis or t for trans isomerism of five dihedral angles of (C4-C3-C2-N3), (C3-C2-N3-N2), (C2-N3-N2-C1), (N3-N2-C1-N1) and (N2-C1-N1-H2), serially, or a letter I for b thioimino tautomer followed by c for cis or t for trans isomerism of six dihedral angles of (C4-C3-C2-N3), (C3-C2-N3-N2), (C2-N3-N2-C1), (N3-N2-C1-N1), (N2-C1-N1-H2) and (N2-C1-S-H1), serially.Electronic Supplementary Material Supplementary material is available for this article at     

Histoenzymatic characterization of sub-types of Type I fibres in fast muscles of rats

Summary The histochemical activities of succinic dehydrogenase (SDH), myofibrillar Adenosine triphosphatase (ATPase) and alpha glycerophosphate dehydrogenase were studied in serial sections of rat vastus lateralis (red) (RVL), gastrocnemius and diaphragm muscles. Three main fibre-types were distinguished. The Type I fibres of RVL and gastrocnemius muscles fell into two distinct groups: one category-Type IA showed very low ATPase activity. The second category of Type IB fibres displayed moderate ATPase reaction. The Type IA fibres were divisible into two sub-groups when tested for SDH reaction. Type IA₁ fibres possessed a homogenous distribution of diformazan·granules throughout the fibre: Type IA₂ fibres displayed characteristic moth-eaten pattern of diformazan localization. The diaphragm muscle did not show either Type IB or Type IA₂ varieties. The great majority of Type I fibres were sub-type IA₁ in the three fast muscles studied. It is also demonstrated here that an inherent heterogeneity exists between Type I fibres of diaphragm and leg muscles in regard to -GPD localization. This histochemical data emphasizes the fact that subdivision of Type I striated muscle fibres of mammalian animals into two sub-types is only approximate and that a further subcategorization is possible.     

The trans action of HMRa in mating type interconversion

Summary HML and HMR are the sites of cryptic mating type genes in the yeast Saccharomyces cerevisiae. In the presence of the HO gene, the information from HML or HMR (an a or cassette) is transferred to the mating type locus (MAT). HML, HMR, and MAT are located on chromosome III, yet are widely separeted. Similarly, in other yeasts, at least some of the genes involved in mating type interconversion are linked to the mating type locus. We demonstrate here that a cassette donor (HMR) and the cassette target (MAT) need not be physically linked for successful mating type interconversion. In particular, we show that HMR a on one chromosome can donate an a cassette to the mating type locus on a homologous chromosome III.     

The ultrastructural localization of the enzymes related to steroid hormone metabolism in the guinea-pig testis

Synopsis A study of the ultrastructural localization of 3-hydroxysteroid dehydrogenase (3-HSD), 11-hydroxysteroid dehydrogenase (11-HSD), glucose-6-phosphate dehydrogenase (G-6-PD), -hydroxybutyrate dehydrogenase (-HBD), NADH diaphorase (NADH-D) and NADPH diaphorase (NADPH-D) in the guinea-pig testis is reported.The procedures employed included short immersion or perfusion fixation with aldehydes followed by incubation of small blocks in a tetrazolium salt or a ferricyanide medium. The effects of incubation conditions were investigated, and a reaction medium for the ultracytochemical demonstration of 11-HSD is described. Using suitable controls, evidence for the specificity of the cytochemical reactions is presented.It was found that all the enzymes studied were present in both the Leydig and Sertoli cells of the guinea-pig testis and that the intracellular distribution pattern for each enzyme was independent of the cell type. Using tetrazolium salt techniques, both 3-HSD and 11-HSD activities were localized on or in membranes of smooth endoplasmic reticulum and within the mitochondria. With the ferricyanide techniques, G-6-PD activity was found to be associated mainly with the smooth endoplasmic reticulum membranes, while -HBD activity was limited to mitochondria. With both the tetrazolium salt and ferricyanide techniques, the reaction products for NADH-D and NADPH-D activities showed localizations which were similar to those observed for the steroid dehydrogenases.     

Lipase-catalyzed synthesis of β-methylglucoside esters containing an α-hydroxy acid

Esterification of -methylglucoside with an -hydroxy acid (glycolic, lactic or malic acid) was carried out using Novozym 435 (Lipase B from Candida antarctica). 2-Methyl-2-butanol was more efficient as solvent for the reaction than ethyl acetate, 1,4-dioxane, n-hexane, acetonitrile or acetone. The molar ratio of -methylglucoside:-hydroxy acid which gave the quickest reaction rate was 1:10 and the highest conversion (75%) was with malic acid.     

Anionic Tetrahedral Complexes as Serine Protease Inhibitors

Potent inhibitors of proteases are constantly sought because of their potential as new therapeutic lead compounds. In this paper we report a simple computational methodology for obtaining new ideas for functional groups that may act as effective inhibitors. We relate this study to serine proteases. We have analyzed all of the factors that operate in the enzyme-substrate interactions and govern the free energy for the transformation of the Michaelis complex (MC) to the anionic covalent tetrahedral complex (TC). The free energy of this transformation ( G_MC-TC ) is the quantitative criterion that differentiates between the catalytic and inhibitory processes in proteases. The catalytic TC is shifted upwards (G_MC-TC > 0) relative to the MC in the free energy profile of the reaction, whereas the inhibitory tetrahedral species is shifted downward (G_MC-TC < 0). Therefore, the more stable the TC, the more effective it should be as an inhibitor. We conclude that the dominant contribution to the superstabilization of an anionic TC for transition state analog inhibitors originates from the formation of a -covalent bond between the reactive centers of the enzyme and its inhibitor. This energetic effect is a quantitative value obtained in ab initio calculations and provides an estimate as to whether a functional group is feasible as potent inhibitor or not. To support our methodology, we describe several examples where good agreement is shown between modeled ab initio quantum chemical calculations and experimental results extracted from the literature.     

Dual anomeric specificity of phosphomannoisomerase assessed by 2D phase sensitive 13C EXSY NMR

The reversible conversion between D-mannose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast phosphomannoisomerase was studied by phase sensitive 2D ¹³C-¹H EXSY NMR spectroscopy at 100.623 MHz, using ¹³C enriched substrates in the C₂ position of the D-hexose 6-phosphates. The unique pair of isomerization cross-peaks observed in the 2D EXSY map correlates the ¹³C₂ resonances of the -anomers of both D-[2-¹³C]-mannose-6-phosphate and D-[2¹³C]-fructose 6-phosphate. This indicates that phosphomannoisomerase specifically catalyzes the reversible conversion between -D-mannose 6-phosphate and -D-fructose 6-phosphate. Since phosphoglucoisomerase was recently found to catalyze specifically the interconversion of -D-glucose 6-phosphate and -D-fructose 6-phosphate, the -anomer of the ketohexose ester could be directly channeled in a multi-enzyme system involving phosphoglucoisomerase, phosphomannoisomerase and phosphofructokinase.     

Alternative bases in the RNA world: The prebiotic synthesis of urazole and its ribosides

Urazole is five-membered heterocyclic compound which is isosteric with uracil's hydrogen-bonding segment. Urazole reacts spontaneously with ribose (and other aldoses) to give mixture of four ribosides: and pyranosides and furanosides. This reaction occurs in aqueous solution at mild temperatures. Thermodynamic and kinetic parameters for the reaction of urazole with ribose were determined. In contrast, uracil is completely unreactive with ribose under these conditions. Urazole's unusual reactivity is ascribed to the hydrazine portion of the molecule. Urazole can be synthesized from biuret and hydrazine under prebiotic conditions. The prebiotic synthesis of guanazole, which is isosteric in part to diaminopyrimidine and cytosine, is accomplished from dicyandiamide and hydrazine. Kinetic parameters for both prebiotic reactions were measured. Urazole and guanazole are transparent in the UV, which would be favorable property in the absence of an ozone layer on the early Earth. Urazole makes hydrogen bonds with adenine in DMSO similar to those of uracil, as established by IH NMR. All of these properties make urazole an attractive potential precursor to uracil and guanazole a potential precursor to cytosine in the RNA or pre-RNA world.Correspondence to: S.L. Miller 0805Presented at the ISSOL Meeting in Barcelona, 5 July 1993 V.M. Kolb is on sabbatical leave from the University of Wisconsin-Parkside, Kenosha, WI 53141     

Test of the extended two-state model for the kinetic intermediates observed in the folding transition of ribonuclease A

A test has been made of the proposal that: (a) the extended two-state model describes the kinetic intermediates seen in the folding transition of RNAase A, i.e. that the only species present in folding experiments are the native protein and multiple forms of the completely unfolded protein; and (b) that the interconversion between the two known unfolded forms of RNAase A (the U₁

U₂ reaction) is described solely by the cis-trans isomerization of the proline residues. The test is to measure the rate of the U₁

U₂ reaction in a wide range of refolding conditions and to compare these data with the kinetic properties of proline isomerization.The main results are as follows. (1) The activation enthalpy of the U₁

U₂ reaction in refolding conditions (pH 6, 20 ° to 40 °C) is less than 5 kcal/mol. This is much too small to be explained as proline isomerization. (2) Both the rate and the activation enthalpy change sharply at guanidine hydrochloride concentrations below 2 m. There appear to be two pathways for the U₁

U₂ reaction in refolding conditions, and the slower pathway is favored by adding guanidine hydrochloride. (3) The rate and activation enthalpy for proline isomerization in l-alanyl-l-proline are unaffected by 2 m-guanidine hydrochloride.The results show that the proline isomerization hypothesis and the extended two-state model cannot both be correct for RNAase A. They suggest that partial folding occurs rapidly in refolding conditions and that the extended two-state model is invalid. They leave open the question of whether or not proline isomerization is the rate-limiting step in the U₁

U₂ reaction.Another possible source of slow configurational reactions in the unfolded state is mentioned. The three major, overlapping, disulfide-bonded loops of RNAase A can exist in two isomeric configurations. Interconversion of these isomers requires pulling one loop, or one end of the polypeptide chain, through a second loop and this is likely to be a slow process.In some conditions, heat-unfolded but not guanidine-unfolded RNAase A shows a second slow-refolding process. It may result from aggregates of the heatunfolded protein which are formed and broken up slowly. Conditions are given for eliminating this reaction.     

An alpha-L-fucosidase from Thermus sp. with unusually broad specificity

An -L-fucosidase (E.C. 3.2.1.51) exhibiting a wide aglycon specificity expressed in ability of cleaving 1 6-, 1 3-, 1 4-, and 1 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The -L-fucosidase hydrolyzes p-nitrophenyl -L-fucopyranoside with V _max of 12.0 ± 0.1 M/min/mg and K _m = 0.20 ± 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 ± 10 kDa consisting of four identical subunits with a molecular mass of 61.0 ± 0.5 kDa. The N-terminal sequence showed homology to some -L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl -L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the -L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl -L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.     

Transport of auxin (indoleacetic acid) through lipid bilayer membranes

Summary Diffusion of auxin (indole-3-acetic acid) through planar lipid bilayer membranes was studied as a function of pH and auxin concentration. Membranes were made of egg or soybean lecithin or phosphatidyl serine inn-decane (25–35 mg/ml). Tracer and electrical techniques were used to estimate the permeabilities to nonionized (HA) and ionized (A^–) auxin. The auxin tracer flux is unstirred layer limited at low pH and membrane limited at high pH, i.e., when [A^–][HA]. The tracer flux is not affected by the transmembrane voltage and is much higher than the flux predicted from the membrane conductance. Thus, only nonionized auxin crosses the membrane at a significant rate. Auxin transport shows saturation kinetics, but this is due entirely to unstirred layer effects rather than to the existence of an auxin carrier in the membrane. A rapid interconversion of A^– and HA at the membrane surface allows A^– to facilitate the auxin flux through the unstirred layer. Thus, the total flux is higher than that expected for the simple diffusion of HA alone. The relation between flux (J _A), concentrations and permeabilities is: 1/J _A=1/P ^UL([A^–]+[HA])+1/P _HA ^M [HA]. By fitting this equation to our data we find thatP ^UL=6.9×10^–4 cm/sec andP _HA ^M =3.3×10^–3 cm/sec for egg lecithin-decane bilayers. Similar membrane permeabilities were observed with phosphatidyl serine or soybean lipids. Thus, auxin permeability is not affected by a net surface charge on the membrane. Our model describing diffusion and reaction in the unstirred layers can explain the anomolous relationship between pH and weak acid (or weak base) uptake observed in many plant cells.