scholarly journals Limited proteolysis and proton n.m.r. spectroscopy of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli

1981 ◽  
Vol 199 (3) ◽  
pp. 733-740 ◽  
Author(s):  
R N Perham ◽  
G C K Roberts
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Proton Nuclear Magnetic Resonance ◽  
Resonance Spectroscopy ◽  
Multienzyme Complex ◽  
Oxoglutarate Dehydrogenase ◽  
Polypeptide Chains ◽  
Dehydrogenase Complex

The 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was treated with trypsin at pH 7.0 at 0 degrees C. Loss of the overall catalytic activity was accompanied by rapid cleavage of the lipoate succinyltransferase polypeptide chains, this apparent Mr falling from 50 000 to 36 000 as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A slower shortening of the 2-oxoglutarate decarboxylase chains was also observed, whereas the lipoamide dehydrogenase chains were unaffected. The inactive trypsin-treated enzyme had lost the lipoic acid-containing regions of the lipoate succinyltransferase polypeptide chains, yet remained a highly assembled structure, as judged by gel filtration and electron microscopy. The lipoic acid-containing regions are therefore likely to be physically exposed in the complex, protruding from the structural core formed by the lipoate succinyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy of the 2-oxoglutarate dehydrogenase complex revealed the existence of substantial regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after removal of the lipoic acid-containing regions by treatment of the complex with trypsin. By analogy with the comparably mobile regions of the pyruvate dehydrogenase complex of E. coli, it is likely that the highly mobile regions of polypeptide chain in the 2-oxoglutarate complex are in the lipoate succinyltransferase component and encompass the lipoyl-lysine residues. It is clear, however, that the mobility of this polypeptide chain is not restricted to the immediate vicinity of these residues.

scholarly journals Amino acid sequence around lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1980 ◽  
Vol 187 (3) ◽  
pp. 905-908 ◽  
Author(s):  
G Hale ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Multienzyme Complex ◽  
Dehydrogenase Complex

Amino-acid sequences around two lipoic acid residues in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli were investigated. A single amino acid sequence of 13 residues was found. A repeated amino acid sequence in the lipoate acetyltransferase chain might explain this result.

scholarly journals Evidence for two lipoic acid residues per lipoate acetyltransferase chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1976 ◽  
Vol 159 (3) ◽  
pp. 677-682 ◽  
Author(s):  
M J Danson ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Acid Content ◽  
Polypeptide Chain ◽  
Multienzyme Complex ◽  
Covalently Bound

The reaction of two maleimides, N-ethylmaleimide and bis-(N-maleimidomethyl) ether, with the pyruvate dehydrogenase multienzyme complex of Escherichia coli in the presence of the substrate, pyruvate, was examined. In both cases, the reaction was demonstrated to be almost exclusively with the lipoate acetyltransferase component, and evidence is presented to show that the most likely sites of reaction are the lipoic acid residues covalently bound to this component. With both reagents the stoicheiometry of the reaction was measured: 2 mol of reagent reacted with each polypeptide chain of lipoate acetyltransferase, implying that each chain bears two functionally active lipolic acid residues. This observation can be reconciled with previous determinations of the lipoic acid content of the complex by allowing for the variability of the subunit polypeptide-chain ratio that can be demonstrated for this multimeric enzyme.

scholarly journals Polypeptide-chain stoicheiometry and lipoic acid content of the pyruvate dehydrogenase complex of Escherichia coli

1979 ◽  
Vol 177 (1) ◽  
pp. 129-136 ◽  
Author(s):  
G Hale ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Acid Content ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pyruvate Decarboxylase ◽  
Molar Ratios

The pyruvate dehydrogenase multienzyme complex was isolated from Escherichia coli grown in the presence of [35S]sulphate. The three component enzymes were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the molar ratios of the three polypeptide chains were determined by measurement of the radioactivity in each band. The chain ratio of lipoamide dehydrogenase to lipoate acetyltransferase approached unity, but there was a molar excess of chains of the pyruvate decarboxylase component. The 35S-labelled complex was also used in a new determination of the total lipoic acid content. It was found that each polypeptide chain of the lipoate acetyltransferase component appears to bear at least three lipoyl groups.

scholarly journals Lipoylation of the E2 components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli

1991 ◽  
Vol 277 (1) ◽  
pp. 153-158 ◽  
Author(s):  
L C Packman ◽  
B Green ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Catalytic Mechanism ◽  
Chemical Reduction ◽  
Pyruvate Dehydrogenase Complex ◽  
Exchange Chromatography ◽  
Lysine Residue ◽  
Multienzyme Complex ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The number of functional lipoyl groups in the dihydrolipoyl acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex from Escherichia coli has been re-assessed by means of a combination of protein-chemical and mass-spectrometric techniques. (1) After the complex had been treated with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, the lipoyl domains were excised from the complex, treated with NaBH4 and re-exposed to N-ethyl[2,3-14C]maleimide. All the chemically reactive lipoyl groups in the native complex were found to be catalytically active. (2) Proteolytic digests of the separated lipoyl domains were examined for the presence of the lipoylation-site peptide, GDKASME, with and without the lipoyl group in N6-linkage to the lysine residue. Only the lipoylated form of the peptide was detected, suggesting that all three lipoyl domains are fully substituted at this site. (3) The behaviour of each lipoyl domain was examined on ion-exchange chromatography in response to alkylation with 4-vinylpyridine after either chemical reduction of the lipoyl group with dithiothreitol or reductive acetylation by the pyruvate dehydrogenase complex in the presence of pyruvate. All three domains exhibited a quantitative shift in retention time, confirming that each domain was fully substituted by an enzymically reactive lipoyl group. (4) When subjected to electrospray mass spectrometry, each domain gave a mass consistent with a fully lipoylated domain, and no aberrant substitution of the target lysine residue was detected. The same result was obtained for the lipoyl domain from the E. coli 2-oxoglutarate dehydrogenase complex. (5) Previous widespread attempts to assess the number of functional lipoyl groups in the pyruvate dehydrogenase multienzyme complex, which have led to the view that a maximum of two lipoyl groups per E2 chain may be involved in the catalytic mechanism, are in error.

scholarly journals Detection of bacterial lipoic acid. A modified gas-chromatographic-mass-spectrometric procedure

1989 ◽  
Vol 258 (3) ◽  
pp. 749-754 ◽  
Author(s):  
K J Pratt ◽  
C Carles ◽  
T J Carne ◽  
M J Danson ◽  
K J Stevenson
Keyword(s):  
Escherichia Coli ◽  
Mass Spectrum ◽  
Lipoic Acid ◽  
Mass Spectrometric ◽  
Halobacterium Halobium ◽  
Multienzyme Complex ◽  
Whole Cells ◽  
Oxoglutarate Dehydrogenase ◽  
Covalent Chromatography ◽  
Chromatographic Mass

The detection of bacterial lipoic acid by a modified g.c.-m.s. procedure is reported. Cells were hydrolysed in HCl to release protein-bound lipoic acid, which, after extraction into benzene, was reduced with NaBH4. The dihydrolipic acid so generated was then isolated by covalent chromatography on dithiolspecific p-aminophenylarsenoxide-agarose and, after elution by 2,3-dimercaptopropane-1-sulphonic acid and extraction into benzene, was allowed to O2-oxidize to the disulphide form. The isolated lipoic acid was allowed to react with diazomethane, and the methyl ester so produced was detected by g.c.-m.s. Analysis of the mass spectrum showed the characteristic molecular ion and seven fragmentation ions, which, along with the identification of those ions retaining the two sulphur atoms, allows the definitive detection of lipoic acid. The methodology has been successfully tested with authentic lipoic acid, the 2-oxoglutarate dehydrogenase multienzyme complex and with whole cells of Escherichia coli. In addition, it has been used to search for and identify lipoic acid in the archaebacterium Halobacterium halobium. The significance of this discovery and the possible roles of the cofactor in H. halobium are discussed.

scholarly journals Lipoic acid residues in a take-over mechanism for the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1981 ◽  
Vol 199 (3) ◽  
pp. 513-520 ◽  
Author(s):  
J N Berman ◽  
G X Chen ◽  
G Hale ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Decarboxylase ◽  
Enzymic Activity ◽  
Enzyme Mechanism ◽  
Enzyme Complex ◽  
Complex Activity ◽  
Dehydrogenase Complex

The pyruvate dehydrogenase complex of Escherichia coli contains two lipoic acid residues per dihydrolipoamide acetyltransferase chain, and these are known to engage in the part-reactions of the enzyme. The enzyme complex was treated with trypsin at pH 7.0, and a partly proteolysed complex was obtained that had lost almost 60% of its lipoic acid residues although it retained 80% of its pyruvate dehydrogenase-complex activity. When this complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, the rate of modification of the remaining S-acetyldihydrolipoic acid residues was approximately equal to the accompanying rate of loss of enzymic activity. This is in contrast with the native pyruvate dehydrogenase complex, where under the same conditions modification proceeds appreciably faster than the loss of enzymic activity. The native pyruvate dehydrogenase complex was also treated with lipoamidase prepared from Streptococcus faecalis. The release of lipoic acid from the complex followed zero-order kinetics for most of the reaction, whereas the accompanying loss of pyruvate dehydrogenase-complex activity lagged substantially behind. These results eliminate a model for the enzyme mechanism in which specifically one of the two lipoic acid residues on each dihydrolipoamide acetyltransferase chain is essential for the reaction. They are consistent with a model in which the dihydrolipoamide acetyltransferase component contains more lipoic acid residues than are required to serve the pyruvate decarboxylase subunits under conditions of saturating substrates, enabling the function of an excised or inactivated lipoic acid residue to be taken over by another one. Unusual structural properties of the enzyme complex might permit this novel feature of the enzyme mechanism.

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