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PubMed1988

Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli.

The ilvC gene encodes acetohydroxy acid isomeroreductase (EC 1.1.1.89), the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Expression of the ilvC gene is induced by acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate. This substrate induction is mediated by a positive activator encoded by an adjacent gene, ilvY. The ilvY and ilvC genes are transcribed in opposite directions from promoters that are overlapping. In this paper we characterize the in vitro DNA binding properties of the ilvY-encoded activator protein. The ilvY product binds to two adjacent operator sites located in the divergent-overlapping ilvY and ilvC promoter region. One of these operators, designated O1 contains regions of dyad symmetry centered at position +17 relative to the ilvY transcriptional start site, and the second site, designated O2, contains an homologous inverted repeat sequence centered about the -35 region of the ilvC promoter. Binding of the ilvY product at the O1 and O2 operator sites is co-operative and this ilvY protein-DNA complex in the presence of acetohydroxy acid isomeroreductase substrate is a prerequisite for RNA polymerase binding to the ilvC promoter as detected by DNase I protection experiments. Additionally, chromosomal galK transcriptional fusion assays were performed to characterize the regulation of the ilvY and ilvC promoters in vivo. Transcription of the ilvC gene is maintained at a basal level of activity which is elevated as much as 15-fold in the presence of ilvY product and acetohydroxybutyrate. The ilvY product represses ilvY transcription in a manner that does not appear to be dependent on acetohydroxy acid isomeroreductase substrate. We discuss models in which activation of ilvC transcription results from a direct interaction of ilvY protein with RNA polymerase or an ilvY-mediated alteration of the DNA conformation of the ilvC -35 promoter region. Additionally, we discuss the role of acetohydroxybutyrate and acetolactate in ilvY transcriptional regulation.

2-Acetolactate Mutase↗
PubMed1996

Hemolytic anemias due to erythrocyte enzyme deficiencies.

Red blood cells can only fulfil their functions over the normal period of approximately 120 days with 1.7 x 10(5) circulatory cycles efficiently if they withstand external and internal loads. This requires ATP and redox equivalents, which have to be permanently regenerated by the energy and redox metabolism. These pathways are necessary to maintain the biconcave shape of the cells, their specific intracellular cation concentrations, the reduced state of hemoglobin with a divalent iron and the sulfhydryl groups of enzymes, glutathione and membrane components. If an enzyme deficiency of one of these metabolic pathways limits the ATP and/or NADPH production, distinct membrane alterations result causing a removal of the damaged cells by the monocyte-macrophage system. Most metabolic needs of erythrocytes are covered by glycolysis, the oxidative pentose phosphate pathway (OPPP), the glutathione cycle, nucleotide metabolism and MetHb reductase. Hereditary enzyme deficiencies of all these pathways have been identified; those that cause non-spherocytic hemolytic anemia are listed in Table 4. Their frequencies differ markedly both with respect to the affected enzyme and geographic distribution. Glucose-6-phosphate dehydrogenase enzymopathies (G6PD) are with more than 400 million cases by far the most common deficiency. The highest gene frequency has been found with 0.7 among Kurdish Jews. G6PD deficiencies are furthermore prevalent with frequencies of about 0.1 among Africans, Black Americans, and populations of Mediterranean countries and South East Asia. In Middle and Northern Europe the frequency of G6PD is much lower, and with approximately 0.0005, comparable with the frequency of pyruvate kinase (PK) enzymopathies, the most frequent enzyme deficiency in glycolysis in this area (Luzzatto, 1987; Beutler and Kuhl, 1990). The relationship between the degree of enzyme deficiency and the extent of metabolic dysfunction in red blood cells and other tissues depend on several factors: on the importance of the affected enzyme; its expression rate; the stability of the mutant enzyme against proteolytic degradation and functional abnormalities; the possibility to compensate the deficiency by an overexpression of the corresponding isoenzyme or by the use of an alternative metabolic pathway. Difficulties in estimating the quantitative degree of disorder in severe cases are due to the fact that these populations contain many reticulocytes, which generally have higher enzyme activities and concentrations of intermediates than erythrocytes. An alternative approach to predict metabolic changes is the analysis by mathematical modeling. Mathematical modeling of the main metabolic pathways of human erythrocytes has reached an advanced level (Rapoport et al., 1976; Holzhütter et al., 1985; Schuster et al., 1988). Models have been successfully employed to describe stationary and time-dependent metabolic states of the cell under normal conditions as well as in the presence of enzyme deficiencies. Figure 5 shows computational results of erythrocyte enzyme deficiencies. This analysis is based on the comprehensive mathematical model of the energy and redox metabolism for human erythrocyte presented in Fig. 6. Stationary states of the cell metabolism have been calculated by varying the activity of each of the participating enzymes by several orders of magnitude. To predict consequences of enzyme deficiencies a performance function has been introduced (Schuster and Holzhütter, 1995). It takes into account the homeostasis of three essential metabolic variables: the energetic state (ATP), the reductive capacity (reduced glutathione) and the osmotic state. From the data given in Fig. 5 one can conclude that generally the metabolic impairment resulting in deficiencies occurs earlier for enzymes with high control coefficients than for those catalyzing equilibrium reactions. On the other hand the flux curves of latter enzymes decrease more steeply below a critica

Anemia, Hemolytic↗
PubMed1982

Purification, characterization and inhibition by MK-401 of Fasciola hepatica phosphoglyceromutase.

Phosphoglyceromutase (EC 2.7.5.3) of Fasciola hepatica was purified 1390-fold to homogeneity. The enzyme had a molecular weight of 120 000 and was a tetramer composed of identical 30 000 molecular weight subunits. The enzyme was 2,3-dephosphoglyceric acid dependent, possessed reactive sulfhydryl groups and was inhibited irreversibly by iodoacetamide, and N-ethylmaleimide and reversibly by p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid). Initial velocity studies suggest that reaction occurred via a sequential mechanism and that MK-401 was a competitive inhibitor versus both 3-phosphoglyceric acid and 2,3-diphosphoglyceric acid.

2,3-Diphosphoglycerate↗
PubMed1984

Biochemical and immunological arguments for homology between red cell and liver phosphoglyceromutase isozymes.

Phosphoglyceromutase (2,3-bisphospho-D-glycerate:2- phospho-D-glycerate phosphotransferase, EC 2.7.5.3) from the red cell is compared with the muscle and the liver forms of the enzyme obtained from rabbits. Partially purified samples were used for this study. The electrophoretic mobility of red cell phosphoglyceromutase is completely different from that of muscle and similar to that of liver. The muscle isozyme can hybridize with liver or red blood cell phosphoglyceromutase. N-Ethylmaleimide, an SH group reagent, completely inhibits muscle phosphoglyceromutase activity and slightly inhibits the activity of the enzyme from liver and from red cells. Both liver and red cell isozymes are completely inactivated by heating at 60 degrees C for 15 min, whereas a 25% decrease in inactivity is noted for the muscle enzyme. Finally, chicken antibody directed against human red cell phosphoglyceromutase reacts with the enzyme isolated from rabbit red cells and liver, but not with that obtained from muscle.

Animals↗
PubMed1984

Neurochemical enzyme changes in Alzheimer's and Pick's disease.

Neurochemical investigations of the whole temporal lobe of cases with Alzheimer's disease (n = 15); 80.7 +/- 1.7 yr), Pick's disease (n = 3; 65 +/- 1.7 yr), and age-matched controls (n = 18; 74.7 +/- 2.6 yr), demonstrate that Alzheimer's and Pick's disease are primary degenerative brain diseases. The activities of glycolytic enzymes, ATPases, carbonic anhydrase, acetylcholinesterase and protein kinase were significantly lower in Alzheimer's and in Pick's disease than in age-matched controls. Pick's disease is characterised by a more pronounced reduction of the enzymes investigated, which is considered to be an expression of a more dramatic degenerative process. The differences between Alzheimer's disease and Pick's disease are quantitative.

Acetylcholinesterase↗
PubMed1992

Molecular design and catalytic function of a B12-enzyme mimetic.

Developing artificial holoenzymes requires attention to the molecular design, not only of the active site, but also of its microenvironment, which in the naturally occurring enzyme is provided by the apoprotein. Microenvironmental effects play a major role in determining the catalytic performance of an enzyme. Using the development of a B12-dependent holoenzyme mimetic as an example, the important factors and concepts underlying the approaches to developing an artificial holoenzyme are discussed.

Amino Acid Isomerases↗
PubMed1990

Fitting abnormal oxygen equilibrium curves of hemoglobin.

A growing number of oxygen equilibrium curves for hemoglobin (Hb) mutants, post-translational modifications, or the binding of potent new effectors of Hb cannot be fitted adequately with the two-state model. Examples are curves showing double maxima in the derivative of the Hill plot, or slopes of less than unity. We present such examples of modified hemoglobins and strong effectors in this study and calculate at which substate level the two-state model differs from the data. Analysis of hemoglobin oxygen equilibrium curves is reconsidered using the two-state model extended to allow variation of the individual substate probabilities. In this way the effect on the equilibrium due to perturbations in energy of each substate can be studied as a diagnostic tool.

Allosteric Regulation↗
PubMed1975

Mathematical analysis of multienzyme systems. I. Modelling of the glycolysis of human erythrocytes.

A mathematical model for the glycolysis of human erythrocytes is presented which takes into account ATP-synthesis and -consumption. A set of three differential equations describes the steady states and the time-dependent changes of the metabolite concentrations under blood storage conditions. For a given parameter combination there are in general three stationary points of the system, one of which is unstable. At a low ATP-need the ATP-level is relatively constant for variations in the rate constant of the ATP-consuming processes. Above a critical level of the energy consumption the system breaks down. An important role of the 2.3P2G-bypass of the erythrocytes is its action as an "energy buffer", wasting ATP in case of ATP-overproduction and producing ATP in case of underproduction. A parameter combination consistent with the data on the isolated enzymes was found which gives a good agreement of theoretical predictions with the measured metabolite concentrations. Under blood preservation conditions the difference of the rates of ATP-production and -consumption is the most important factor for a high ATP-level over long periods.

Adenosine Triphosphatases↗
PubMed1975

Location of gluconeogenesis from phosphoenolpyruvate in cotyledons of Cucurbita pepo.

1. The aim of this work was to discover the location of the enzymes that convert phosphoenolpyruvate to fructose 6-phosphate during gluconeogenesis in fatty seeds. Cotyledons of 5-day-old dark-grown seedlings of marrow (Cucurbita pepo) were used as experimental material. 2. Cotyledons were separated into palisade and mesophyll tissue. Extracts of the two tissues had comparable activities of gluconeogenic enzymes. 3. Extracts of cotyledons were fractionated by density gradient centrifugation to yeild mitochondria and glyoxysomes, and by gel filtration to yield proplastids. The isolated organelles retained their characteristic ultrastructure and appreciable amounts of marker enzymes. The proportions of the total activities of phosphoglyceromutase and fructose-1, 6-diphosphatase recovered in the mitochondrial and glyoxysomal preparations were insignificant. The same was true for the activities of phosphoglyceromutase and phosphopyruvate hydratase found in the proplastid preparations. 4. Extracts of a number of other gluconeigenic plant tissues were centrifuged at 2500 times g to yield particulate preparations. None of these preparations contained a significant proportion of the total activity of phosphoglyceromutase. 5. It is suggested that gluconeogenesis from phosphoenolpyruvate in plants occurs in the cytoplasm.

Cell Fractionation↗
PubMed1988

Rabbit M type phosphoglyceromutase: comparative effects of two thiol reagents antibody reaction and hybridization studies.

1. Treatment of purified rabbit phosphoglyceromutase (M type) with N-ethylmaleimide or with iodoacetamide produces the concurrent loss of phosphoglyceromutase activity with its collateral glycerate-2,3-P2 phosphatase activity. 2. Differences are observed in the protective effect of glycerate-2,3-P2 and of glycolate-2-P against N-ethylmaleimide and iodoacetamide treatments. 3. Specific chicken antibodies obtained by injection of the purified rabbit M type phosphoglyceromutase do not cross-react with the B type but neutralize both rabbit and human M type phosphoglyceromutase. 4. Purified rabbit M type phosphoglyceromutase can hybridize in vitro with the purified human B type or with purified human glycerate-2,3-P2 synthase. 5. Its ability to hybridize with glycerate-2,3-P2 synthase is unchanged after iodoacetamide treatment.

Animals↗