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H G Nimmo

Publications and source records attributed to H G Nimmo.

At least 37 records · Page 2Linked to original sources
PubMed1989

Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase.

Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.

Amino Acid Sequence↗
PubMed1989

Evidence for an arginine residue at the coenzyme-binding site of Escherichia coli isocitrate dehydrogenase.

The arginine-specific reagent phenylglyoxal inactivated the active, dephosphorylated, form of Escherichia coli isocitrate dehydrogenase rapidly in a pseudo-first-order process. Both NADP+ and NADPH protected the enzyme against inactivation. Phenylglyoxal appeared to react with one arginine residue per subunit, and the extent of the reaction was proportional to the extent of the inactivation. In contrast, the phosphorylated form of isocitrate dehydrogenase did not react detectably with phenylglyoxal. The data indicate that the coenzyme-binding site of isocitrate dehydrogenase contains a reactive arginine residue that is protected by phosphorylation, and are consistent with the hypothesis that phosphorylation of the enzyme occurs close to or at its active site.

Arginine↗
PubMed1988

Photoaffinity labelling shows that Escherichia coli isocitrate dehydrogenase kinase/phosphatase contains a single ATP-binding site.

Ultraviolet irradiation of E.coli isocitrate dehydrogenase kinase/phosphatase in the presence of 8-azidoATP resulted in parallel losses of its kinase and phosphatase activities, and in covalent attachment of the reagent to the protein at a single site. ATP and ADP protected the two activities to similar extents. The data suggest that the activation of the phosphatase by adenine nucleotides results from binding of the nucleotides to the active site of the kinase.

Adenosine Diphosphate↗
PubMed1988

Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308.

Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.

Anions↗
PubMed1987

Specific antibodies and the selective inhibitor ICI 118233 demonstrate that the hormonally stimulated 'dense-vesicle' and peripheral-plasma-membrane cyclic AMP phosphodiesterases display distinct tissue distributions in the rat.

Polyclonal-antibody preparations DV1 and PM1, raised against purified preparations of rat liver insulin-stimulated 'dense-vesicle' and peripheral-plasma-membrane cyclic AMP phosphodiesterases, were used to analyse rat liver homogenates by Western-blotting techniques. The antibody DV1 identified only the 63 kDa native subunit of the 'dense-vesicle' enzyme, and the antibody PM1 only the 52 kDa subunit of the plasma-membrane enzyme. These antibodies also detected the subunits of these two enzymes in homogenates of kidney, heart and white adipose tissue from rat. Quantitative immunoblotting demonstrated that the amount of these enzymes (by wt.) varied in these different tissues, as did the expression of these two enzymes, relative to each other, by a factor of as much as 7-fold. The ratio of the dense-vesicle enzyme to the peripheral-plasma-membrane enzyme was lowest in liver and kidney and highest in heart and white adipose tissue. ICI 118233 was shown to inhibit selectively the 'dense-vesicle' cyclic AMP phosphodiesterase in liver. It did this in a competitive fashion, with a Ki value of 3.5 microM. Inhibition of tissue-homogenate cyclic AMP phosphodiesterase activity by ICI 118233 was used as an index of the contribution to activity by the 'dense-vesicle' enzyme. By this method, a tissue distribution of the 'dense-vesicle' enzyme was obtained which was similar to that found by using the immunoblotting technique. The differential expression of isoenzymes of cyclic AMP phosphodiesterase activity in various tissues might reflect a functional adaptation, and may provide the basis for the different physiological actions of compounds which act as selective inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Molecular cloning and over-expression of the glyoxylate bypass operon from Escherichia coli ML308.

A recombinant plasmid carrying an 11 kb restriction-endonuclease-ClaI fragment of genomic DNA from Escherichia coli ML308 was constructed. This plasmid complements an aceA mutation. The plasmid encodes the structural genes of the glyoxylate bypass operon, namely malate synthase A (aceB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK), as judged by overexpression of enzyme activities and transcription/translation experiments in vitro. Subcloning confirmed that expression of the aceK gene is essential for growth on acetate.

Cloning, Molecular↗
PubMed1987

Regulation of the enzymes at the branchpoint between the citric acid cycle and the glyoxylate bypass in Escherichia coli.

During growth of Escherichia coli on acetate, the glyoxylate bypass supplies the precursors needed for biosynthesis. The glyoxylate bypass enzyme isocitrate lyase competes with the citric acid cycle enzyme isocitrate dehydrogenase for the available isocitrate. We have studied the control of metabolic flux at this branchpoint by examining the regulatory properties of the enzymes concerned. Isocitrate dehydrogenase is controlled by reversible phosphorylation catalysed by a bifunctional kinase/phosphatase whose activities are regulated by isocitrate, biosynthetic precursors and adenine nucleotides. The flux through isocitrate lyase is mainly controlled by the intracellular concentration of isocitrate. The phosphorylation system responds to the availability of energy and precursors and maintains isocitrate at a concentration high enough to sustain the flux through the glyoxylate bypass necessary for biosynthesis.

Citric Acid Cycle↗
PubMed1986

Purification of the phosphorylated night form and dephosphorylated day form of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi.

Phosphoenolpyruvate carboxylase of Bryophyllum fedtschenkoi was shown to exist in two forms: a night form, which is phosphorylated and has low sensitivity to inhibition by malate, and a day form, which is dephosphorylated and 10 times more sensitive to malate. The day and night forms of the enzyme were purified retaining their distinct malate sensitivities and phosphorylation states. The purified enzymes contained a major protein (subunit Mr 112,000) and a minor protein (subunit Mr 123,000). The two polypeptides appeared to have closely related amino acid sequences and were present in a similar ratio in extracts that had been prepared rapidly. The phosphate present in the night form of the enzyme was covalently bound to serine. It was not a catalytic intermediate. Alkaline phosphatase removed the phosphate group in vitro and increased the malate sensitivity of the enzyme to that observed for the day form. Both the day and night forms of the enzyme were probably tetramers, and their apparent Mr was lowered by the presence of malate, but was unaffected by Mg2+ ions, EDTA, a rise in pH or a 10-fold change in enzyme concentration. The rapid loss of malate sensitivity, observed in extracts of leaves prepared during the day and at night, was shown to be due to proteolysis of the enzyme. It was slowed in the presence of malate and by phosphorylation of the enzyme.

Carboxy-Lyases↗
PubMed1986

Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate.

The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant.

Chromatography, High Pressure Liquid↗
PubMed1986

Pyruvate metabolism and the phosphorylation state of isocitrate dehydrogenase in Escherichia coli.

During growth of Escherichia coli on acetate, isocitrate dehydrogenase (ICDH) is partially inactivated by phosphorylation and is thus rendered rate-limiting in the Krebs cycle so that the intracellular concentration of isocitrate rises which, in turn, permits an increased flux of carbon through the anaplerotic sequence of the glyoxylate bypass. A large number of metabolites stimulate ICDH phosphatase and inhibit ICDH kinase in the wild-type (E. coli ML308) and thus regulate the utilization of isocitrate by the two competing enzymes, ICDH and isocitrate lyase. Addition of pyruvate to acetate grown cultures triggers a rapid dephosphorylation and threefold activation of ICDH, both in the wild-type (ML308) and in mutants lacking pyruvate dehydrogenase (ML308/Pdh-), PEP synthase (ML308/Pps-) or both enzymes (ML308/Pdh-Pps-). Pyruvate stimulates the growth on acetate of those strains with an active PEP synthase but inhibits the growth of those strains that lack this enzyme. When pyruvate is exhausted, ICDH is again inactivated and the growth rate reverts to that characteristic of growth on acetate. Because pyruvate stimulates dephosphorylation of ICDH in strains with differing capabilities for pyruvate metabolism, it seems likely that pyruvate itself is a sufficient signal to activate the dephosphorylation mechanism, but this does not discount the importance of other signals under other circumstances.

Acetates↗
PubMed1985

Effects of phosphorylation on the kinetic properties of rat liver ATP-citrate lyase.

Homogeneous rat liver ATP-citrate lyase (EC 4.1.3.8) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. In agreement with other workers, the maximum level of phosphorylation that we observed was approx. 2 mol/mol of tetramer. Phosphorylated and non-phosphorylated forms of ATP-citrate lyase were prepared. Their kinetic properties were examined using an assay system in which the concentrations of Mg.ATP, magnesium.citrate and CoA were varied systematically at a constant concentration of Mg2+. The phosphorylated form had a two-fold higher Km for Mg.ATP than did the non-phosphorylated form, but no other kinetic differences between the two forms were detected. When ATP-citrate lyase was assayed at a concentration of Mg.ATP well below Km, it was found that phosphorylation of the enzyme correlated well with a decrease of approx. 50% in its activity. This is the first demonstration that phosphorylation can affect the activity of ATP-citrate lyase.

ATP Citrate (pro-S)-Lyase↗
PubMed1984

Purification and some kinetic properties of rat liver ATP citrate lyase.

A new purification procedure for rat liver ATP citrate lyase is described. The method reproducibly gives homogenous undegraded enzyme. Steady-state kinetic analysis of ATP citrate lyase was complicated by the presence of ADP, a product of the reaction, in solutions of ATP. The kinetic patterns observed were dependent on whether ADP was removed by the assay system. When assays were performed with a method in which ADP was removed, the results showed that the enzyme obeys a double-displacement mechanism with a phosphoenzyme intermediate. This resolves a controversy between the results of previous kinetic studies and those of isotope-exchange and enzyme-labelling experiments.

ATP Citrate (pro-S)-Lyase↗
PubMed1984

Studies of rat adipose-tissue microsomal glycerol phosphate acyltransferase.

Incubation of rat adipose-tissue microsomal fractions with iodoacetate caused an inactivation of glycerol phosphate acyltransferase that could be prevented by the presence of palmitoyl-CoA. A microsomal protein of subunit Mr 54 000 was found to react with radioactively labelled iodoacetate in the absence, but not in the presence, of palmitoyl-CoA. It is suggested that this protein is a component of glycerol phosphate acyltransferase. Incubation of rat adipose-tissue microsomal fractions with the catalytic subunit of cyclic AMP-dependent protein kinase, ATP and Mg2+ caused an inactivation of glycerol phosphate acyltransferase whose magnitude depended on the conditions used for assay of the acyltransferase. Rat adipose tissue microsomal proteins were phosphorylated by using protein kinase and [gamma-32P]ATP. One of the phosphorylated proteins was very similar, but not identical, in mobility to the Mr-54 000 protein labelled by iodoacetate. In contrast with a previous report [Sooranna & Saggerson (1976) FEBS Lett. 64, 36-39], no changes could be detected in the activity of glycerol phosphate acyltransferase in adipocytes treated with adrenaline. Adipocytes were labelled with [32P]Pi and treated with adrenaline, but no 32P was incorporated into the Mr-54000 protein labelled by iodoacetate. The results suggest that the activity of adipose-tissue microsomal glycerol phosphate acyltransferase is not directly controlled by phosphorylation.

Acyltransferases↗
PubMed1984

The phosphorylation of Escherichia coli isocitrate dehydrogenase in intact cells.

The isocitrate dehydrogenase of Escherichia coli ML308 can be reversibly activated by addition of pyruvate to cells growing on acetate [Bennett & Holms (1975) J. Gen. Microbiol. 87, 37-51]. By using cells pulse-labelled with [32P]Pi we showed that the activation and inactivation of the enzyme in these conditions correlate with its dephosphorylation and rephosphorylation respectively. Incubation of cell extracts prepared during an activation/inactivation cycle with purified isocitrate dehydrogenase phosphatase confirmed that the pyruvate-induced activation of the dehydrogenase goes essentially to completion. The results show that the reversible changes in the activity of the dehydrogenase in cells grown on acetate are solely due to phosphorylation/dephosphorylation. Inactive 32P-labelled isocitrate dehydrogenase was isolated from cells incubated with [32P]Pi in the presence of acetate. Both this material and purified enzyme phosphorylated in vitro were digested with chymotrypsin, and the phosphopeptides were isolated and analysed. Only one phosphopeptide was observed in each case; the results show that the residue phosphorylated in vivo is identical with that phosphorylated by purified isocitrate dehydrogenase kinase in vitro.

Amino Acids↗