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P Dimroth

Publications and source records attributed to P Dimroth.

At least 127 records · Page 7Linked to original sources
PubMed1983

Purification and characterization of a new sodium-transport decarboxylase. Methylmalonyl-CoA decarboxylase from Veillonella alcalescens.

Upon resolution of the particulate cell fraction of Veillonella alcalescens by gel chromatography, membranes and ribosomes were clearly resolved. Methylmalonyl-CoA decarboxylase was bound to the membranes and not to ribosomes as reported earlier. Membrane vesicles containing methylmalonyl-CoA decarboxylase were prepared by disrupting V. alcalescens cells with a French pressure chamber. About 64% of the decarboxylase was oriented in these vesicles with the substrate binding site facing to the outside. The vesicles performed a rapid accumulation of Na+ ions in response to the decarboxylation of methylmalonyl-CoA. Decarboxylation and transport were highly uncoupled. The efficiency of the transport was considerably increased if methylmalonyl-CoA decarboxylation was retarded by using a low temperature or by slowly generating the substrate enzymically from propionyl-CoA. Under optimized conditions Na+ was concentrated inside the inverted vesicles eight-times higher than in the incubation medium. Methylmalonyl-CoA decarboxylase was solubilized from the membranes with Triton X-100 and purified about 20-fold by affinity chromatography on monomeric avidin-Sepharose columns. The decarboxylase was specifically activated by Na+ ions (apparent Km approximately equal to 0.6 mM). Whereas (S)-methylmalonyl-CoA was the superior substrate (apparent Km approximately equal to 7 microM), malonyl-CoA was also decarboxylated (apparent Km approximately equal to 35 microM). The decarboxylation of methylmalonyl-CoA yielded CO2 and not HCO-3 as the primary reaction product. Analysis of the purified enzyme by dodecylsulfate gel electrophoresis indicated the presence of four different polypeptides alpha, beta, gamma, delta with Mr 60 000, 33 000, 18 5000 and 14 000. The latter of these polypeptides was clearly visible only after silver staining but not after staining with Coomassie brilliant blue. A low molecular weight polypeptide with similar staining properties was also found in oxaloacetate decarboxylase. Methylmalonyl-CoA decarboxylase contained about 1 mol covalently bound biotin per 125 500 g protein which was localized exclusively in the gamma-subunit. This subunit therefore represents the biotin carboxyl carrier protein of methylmalonyl-CoA decarboxylase. A new very sensitive method for the detection of biotin-containing proteins is described.

Biological Transport↗
PubMed1982

The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes.

The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.

Bacterial Proteins↗
PubMed1981

Reconstitution of sodium transport from purified oxaloacetate decarboxylase and phospholipid vesicles.

The Na+-pumping oxaloacetate decarboxylase, isolated from Klebsiella aerogenes membranes, was incorporated into the membranes of lecithin liposomes. Reconstitution of Na+ transport in this artificial system was achieved by the detergent dilution method with octylglucoside as the detergent. An optimum Na+ transport activity upon dilution was obtained in the presence of about 2.7% octylglucoside. With these reconstituted vesicles, a steady state internal Na+ concentration was established by oxaloacetate decarboxylation which was about 30 times higher than that of the external medium. The presence of low concentrations of the Na+-carrying ionophores nigericin or trinactin almost completely abolished the oxaloacetate-dependent Na+ transport, whereas the uncoupler, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, was without effect. The results are in accord with the function of oxaloacetate decarboxylase as a primary active Na+ pump converting the energy of the decarboxylation reaction into a Na+ gradient.

Biological Transport, Active↗
PubMed1981

Characterization of a membrane-bound biotin-containing enzyme: oxaloacetate decarboxylase from Klebsiella aerogenes.

Oxaloacetate decarboxylase from Klebsiella aerogenes is firmly bound to the cytoplasmic membrane, from which it can be solubilized with nonionic detergents. The solubilized enzyme behaved like the membrane-bound enzyme with respect to its inhibition by avidin and to the requirement of sodium ions for catalytic activity. The decarboxylase was purified 4.5-fold over the solubilized membrane extract by conventional means. Dodecyl-sulfate disc-gel electrophoretic analysis indicated that the enzyme consists of polypeptides of a single size. The molecular weight of these polypeptides is 68000. Radioactive biotin was incorporated specifically into these polypeptide chains upon growth of the bacteria in the presence of the radioactive vitamin. Biotin as the prosthetic group of oxaloacetate decarboxylase is now firmly established. The enzyme in the absence of detergent occurs in a highly aggregated form which elutes in the exclusion volume of a Biogel A 1.5 m column. The reported inhibition of oxaloacetate decarboxylase by citrate could not be repeated. On the other hand oxalate, 2-oxomalonate and glyoxylate proved to be very potent inhibitors of the decarboxylase. The stereochemical course of the oxaloacetate decarboxylation reaction was determined starting from stereospecifically labelled malates, which by malate dehydrogenase and oxaloacetate decarboxylase were converted to chiral pyruvates. The chirality of these pyruvates was analysed via their conversion to acetates and malates by determining the extent of tritium retention upon incubation of the latter with fumarase. It was found that oxaloacetate decarboxylation occurs stereospecifically with retention of configuration.

Biotin↗
PubMed1976

6-Methylsalicylic acid synthetase from Penicillium patulum. Some catalytic properties of the enzyme and its relation to fatty acid synthetase.

1. The specificity of 6-methylsalicylic acid synthetase with respect to the priming substrate was studied. If acetyl-CoA was replaced by propionyl-CoA 6-ethylsalicylic acid was synthesized. The rate of this synthesis was about 13% that of 6-methylsalicylic acid synthesis in presence of acetyl-CoA. 2. 6-Methylsalicylic acid synthetase contains an acetyl transferase activity as demonstrated by the transfer of acetyl residues from acetyl-CoA to pantetheine. This transferase also catalyses propionyl transfer but only with an about 13 times slower rate. 3. Analogous to fatty acid synthetase, treatment with iodoacetamide converts 6-methylsalicylic acid synthetase into a malonyl-CoA decarboxylase. Under certain conditons the iodoacetamide-treated enzyme could catalyse 6-methylsalicylic acid formation from malonyl CoA and NADPH without the external addition of acetyl CoA.

Acetyl Coenzyme A↗
PubMed1976

The prosthetic group of citrate-lyase acyl-carrier protein.

The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, beta-alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components were linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide. Incubation of the prosthetic-group-containing peptide with phosphodiesterase I yielded 4'-phosphopantetheine and adenylic acid. The 5'-AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5'-AMP. It was concluded from these results that the prosthetic group of citrate lyase acyl carrier protein consists of a substituted isomeric dephospho-CoA. This is bound to the protein by the 5'-phosphate group of adenylic acid. The 4'-phosphopantetheine residue is bound by a phosphodiester linkage to the 2' or 3' position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate lyase.

ATP Citrate (pro-S)-Lyase↗
PubMed1975

On the mechanism of action of isocitrate lyase.

1. The enzymes citrate lyase and isocitrate lyase catalyse similar reactions in the cleavage of citrate to acetate plus oxaloacetate and of isocitrate to succinate plus glyoxylate, respectively. 2. Nevertheless, the mechanism of action of each enzyme appears to be different from each other. Citrate lyase is an acyl carrier protein-containing enzyme complex whereas isocitrate lyase is not. The active form of citrate lyase is an acetyl-S-enzyme but that of isocitrate lyase is not a corresponding succinyl-S-enzyme. 3. In contrast to citrate lyase, the isocitrate enzyme is not inhibited by hydroxylamine nor does it acquire label if treated with appropriately labelled radioactive substrate. 4. Isotopic exchange experiments performed in H18-2O with isocitrate as a substrate produced no labelling in the product succinate. This was shown by mass-spectrometric analysis. 5. The conclusion drawn from these results is that no activation of succinate takes place on the enzyme through transient formation of succinic anhydride or a covalently-linked succinyl-enzyme, derived from this anhydride.

ATP Citrate (pro-S)-Lyase↗
PubMed1975

Isolation of subunits of citrate lyase and characterization of their function in the enzyme complex.

Citrate lyase [EC 4.1.3.6; citrate oxaloacetate-lyase (pro-3S-CH2-COO--leads to acetate)] from Klebsiella aerogenes has been dissociated with urea; the three different subunits, alpha-chain (molecular weight congruent to 54,000), beta-chain (molecular weight congruent to 32,000), and gamma-chain (acyl carrier protein; molecular weight congruent to 10,000), have been isolated in pure and catalytically active state. Recombination of the three subunits produced citrate lyase that was indistinguishable from the untreated enzyme. The alpha-chain in the presence of acetyl-S-acyl carrier protein catalyzed the formation of the corresponding citryl thioester with liberation of acetate, and the beta-chain catalyzed the cleavage of citryl-S-acyl carrier protein with liberation of oxaloacetate. A simple enzymic method for the preparation of citryl-S-acyl carrier protein is described.

Binding Sites↗