Molecular orbital studies of enzyme activity: I: Charge relay system and tetrahedral intermediate in acylation of serine proteinases.
(17/935)
The charge relay ststem and its role in the acylation of serine proteinases is studied using the partial retention of diatomic differential overlap (PRDDO) technique to perform approximate ab initio molecular orbital calculations on a model of the enzyme-substrate complex. The aspartate in the charge relay system is seen to act as the ultimate proton acceptor during the charging of the serine nucleophile. A projection of the potential energy surface is obtained in a subspace corresponding to this charge transfer and to the coupled motions of active site residues and the substrate. These results together with extended basis set results for cruder models suggest that a concerted transfer of protons from Ser-195 to His-57 and from His-57 to Asp-102 occurs with an energy barrier of 20-25 kcal/mole (84-105 kJ/mole). The subsequent nucleophilic attack on the scissile peptide linkage by the charged serine is then seen to proceed energetically downhill to the tetrahedral intermediate. The formation of the tetrahedral intermediate from the Michaelis complex is calculated to be nearly thermoneutral. (+info)
Elucidation of enzymes in fermentation pathways used by Clostridium thermosuccinogenes growing on inulin.
(18/935)
Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by Clostridium thermosuccinogenes DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70 degrees C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58 degrees C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70 degrees C. No acetate kinase activity was observed at 70 degrees C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58 degrees C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70 degrees C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70 degrees C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature. (+info)
Extracellular oxidoreduction potential modifies carbon and electron flow in Escherichia coli.
(19/935)
Wild-type Escherichia coli K-12 ferments glucose to a mixture of ethanol and acetic, lactic, formic, and succinic acids. In anoxic chemostat culture at four dilution rates and two different oxidoreduction potentials (ORP), this strain generated a spectrum of products which depended on ORP. Whatever the dilution rate tested, in low reducing conditions (-100 mV), the production of formate, acetate, ethanol, and lactate was in molar proportions of approximately 2.5:1:1:0.3, and in high reducing conditions (-320 mV), the production was in molar proportions of 2:0.6:1:2. The modification of metabolic fluxes was due to an ORP effect on the synthesis or stability of some fermentation enzymes; thus, in high reducing conditions, lactate dehydrogenase-specific activity increased by a factor of 3 to 6. Those modifications were concomitant with a threefold decrease in acetyl-coenzyme A (CoA) needed for biomass synthesis and a 0.5- to 5-fold decrease in formate flux. Calculations of carbon and cofactor balances have shown that fermentation was balanced and that extracellular ORP did not modify the oxidoreduction state of cofactors. From this, it was concluded that extracellular ORP could regulate both some specific enzyme activities and the acetyl-CoA needed for biomass synthesis, which modifies metabolic fluxes and ATP yield, leading to variation in biomass synthesis. (+info)
Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange.
(20/935)
Pendred syndrome, characterized by congenital sensorineural hearing loss and goiter, is one of the most common forms of syndromic deafness. The gene causing Pendred syndrome (PDS) encodes a protein designated pendrin, which is expressed in the thyroid, kidney, and fetal cochlea. Pendrin functions as an iodide and chloride transporter, but its role in the development of hearing loss and goiter is unknown. In this study, we examined the mechanism of pendrin-mediated anion transport in Xenopus laevis oocytes. Unlabeled formate added to the uptake medium inhibited pendrin-mediated (36)Cl uptake in X. laevis oocytes. In addition, the uptake of [(14)C]formate was stimulated in oocytes injected with PDS cRNA compared with water-injected controls. These results indicate that formate is a substrate for pendrin. Furthermore, chloride stimulated the efflux of [(14)C]formate and formate stimulated the efflux of (36)Cl in oocytes expressing pendrin, results consistent with pendrin-mediated chloride/formate exchange. These data demonstrate that pendrin is functionally similar to the renal chloride/formate exchanger, which serves as an important mechanism of chloride transport in the proximal tubule. A similar process could participate in the development of ion gradients within the inner ear. (+info)
Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA.
(21/935)
Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N(10)-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C(1)-tetrahydrofolate (C(1)-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" parental strains. In spite of the absence of fMet-tRNA(fMet), the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of petite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion strain. (+info)
Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures.
(22/935)
Lactic streptococci, classically regarded as homolactic fermenters of glucose and lactose, became heterolactic when grown with limiting carbohydrate concentrations in a chemostat. At high dilution rates (D) with excess glucose present, about 95% of the fermented sugar was converted to l-lactate. However, as D was lowered and glucose became limiting, five of the six strains tested changed to a heterolactic fermentation such that at D = 0.1 h(-1) as little as 1% of the glucose was converted to l-lactate. The products formed after this phenotypic change in fermentation pattern were formate, acetate, and ethanol. The level of lactate dehydrogenase, which is dependent upon ketohexose diphosphate for activity, decreased as fermentation became heterolactic with Streptococcus lactis ML(3). Transfer of heterolactic cells from the chemostat to buffer containing glucose resulted in the nongrowing cells converting nearly 80% of the glucose to l-lactate, indicating that fine control of enzyme activity is an important factor in the fermentation change. These nongrowing cells metabolizing glucose had elevated (ca. twofold) intracellular fructose 1,6-diphosphate concentrations ([FDP](in)) compared with those in the glucose-limited heterolactic cells in the chemostat. [FDP](in) was monitored during the change in fermentation pattern observed in the chemostat when glucose became limiting. Cells converting 95 and 1% of the glucose to l-lactate contained 25 and 10 mM [FDP](in), respectively. It is suggested that factors involved in the change to heterolactic fermentation include both [FDP](in) and the level of lactate dehydrogenase. (+info)
In vitro fermentation pattern of D-tagatose is affected by adaptation of the microbiota from the gastrointestinal tract of pigs.
(23/935)
Knowledge of the fermentation pattern of D-tagatose is important for the assessment of energy value and compliance of D-tagatose. In vitro fermentation experiments with pig intestinal contents and bacteria harvested from the gastrointestinal tract of pigs were used to investigate the degradation of D-tagatose and the formation of fermentation products. Two groups of eight pigs were fed either a control diet containing 150 g/kg sucrose or a diet which had 100 g/kg of the sucrose replaced by D-tagatose. After 18 d the pigs were killed and the gastrointestinal contents collected for in vitro studies. No microbial fermentation of D-tagatose occurred in the stomach or in the small intestine, whereas the sugar was fermented in the cecum and colon. Formate, acetate, propionate, butyrate, valerate, caproate and some heptanoate were produced by the microbial fermentation of D-tagatose by gut microbiota. Hydrogen and methane were also produced. The population of D-tagatose-degrading bacteria in fecal samples and the capacity of bacteria from the hindgut to degrade D-tagatose were higher in the pigs adapted to D-tagatose compared with unadapted pigs. In unadapted pigs, the major fermentation product from D-tagatose was acetic acid. Much more butyric and valeric acids were produced from D-tagatose by bacterial slurries of tagatose-adapted pigs compared with unadapted pigs; this was especially the case for samples from the colon. We conclude that D-tagatose is not fermented in the upper gastrointestinal tract, and the ability of the large intestinal microbiota to ferment D-tagatose is dependent on adaptation. (+info)
Studies on tyrosine phenol lyase. Modification of essential histidyl residues by diethylpyrocarbonate.
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Tyrosine phenol-lyase of Escherichia intermedia is inactivated by treatment with diethylpyrocarbonate at pH 6.0 AND 4 degrees. Spectrophotometric studies show that the inactivation is stoichiometric, with a modification of 2 histidyl residues per molecule of the enzyme. Finding that this inactivation is largely reversed by treatment with hydroxylamine indicates that the inactivation is mainly due to modification of the histidyl residues. No changes in the sulfhydryl content or in the aromatic amino acids are observed as a result of this modification. The modified tyrosine phenol-lyase retains most of its ability to form a nearly normal complex with its coenzyme, pyridoxal phosphate. This has been shown by studies of its absorption, by the determination of pyridoxal phosphate, and by reduction of the holoenzyme with tritiated sodium borohydride. The modified enzyme also appears to form a Schiff base intermediate with L-alanine. The modified holoenzyme fails to catalyze the exchange of the alpha-hydrogen of L-alanine with tritium from tritiated water. This is consistent with a catalytic role for modified histidyl residues at the active site of the enzyme; this role is the removal of the alpha-hydrogen of substrates. (+info)