Binding and dissociation of the pyruvate dehydrogenase complex of <i>Azotobacter vinelandii</i> on thiol—Sepharose

Graaf-Hess, A.C. de; Kok, A. de

doi:10.1016/0014-5793(82)80112-9

Cited by 25 publications

(9 citation statements)

References 17 publications

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“…Lipoamide dehydrogenase was resolved from the pyruvate dehydrogenase complex according to standard procedures [7]. Both enzymes were used as 0.1-1/zM solutions in 50 mM potassium phosphate, pH 7.0, containing 0.5 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time‐resolved flavin fluorescence

Kok

Visser

1987

FEBS Letters

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Subnanosecond-resolved fluorescence measurements of the FAD bound in glutathione reductase and lipoamide dehydrogenase revealed characteristic differences in dynamic properties of both enzymes, which are considered to have common structural features. The flavin fluorescence in glutathione reductase is quenched mainly via a dynamic mechanism, in agreement with enhanced flexibility of the flavin as inferred from rapid depolarization of the fluorescence.

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Section: Methodsmentioning

confidence: 99%

Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time‐resolved flavin fluorescence

Kok

Visser

1987

FEBS Letters

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“…The El and E3 components were eluted as described before [17]. After washing with standard buffer without PhMeS02F the E2 component, still bound on the matrix, was digested by incubating with standard buffer containing 10 pg/ml trypsin at 4°C for 1 h. After washing with standard buffer containing 0.2 mM PhMeSOzF the catalytic domain fragment was eluted with standard buffer containing 3 M KBr.…”

Section: Isolation Of the Main Fragments Obtained By Lirnitedproteolymentioning

confidence: 99%

“…After ethanolamine -Sepharose chromatography the enzyme was concentrated by precipitation with 10% poly(ethyleneglyco1) 6000 instead of ultracentrifugation. The complex was resolved into its components by covalent chromatography on thiolSepharose 4B as described before [17] with the following modifications. Dissociation of El was performed at pH 9.4 instead of pH 8.8.…”

Section: Isolation Of the Complex And Resolution Into Its Componentsmentioning

confidence: 99%

The domain structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Hanemaaijer

Kok²,

Jollès

et al. 1987

Eur J Biochem

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Limited proteolysis with trypsin has been used to study the domain structure of the dihydrolipoyltransacetylase (E2) component of the pyruvate dehydrogenase complex of Azotobacter vinelundii. Two stable end products were obtained and identified as the N-terminal lipoyl domain and the C-terminal catalytic domain. By performing proteolysis of E2, which was covalently attached via its lipoyl groups to an activated thiol-Sepharose matrix, a separation was obtained between the catalytic domain and the covalently attached lipoyl domain. The latter was removed from the column after reduction of the S-S bond and purified by ultrafiltration. The lipoyl domain is monomeric with a mass of 32.6 kDa. It is an elongated structure with flf0 = 1.62. Circulair dichroic studies indicates little secondary structure. The catalytic domain is polymeric with s20.w = 17 S and mass = 530 kDa. It is a compact structure withflh = 1.24 and shows 40% of the secondary structure of E2. The cubic structure of the native E2 is retained by this fragment as observed by electron microscopy. Ultracentrifugation in 6 M guanidine hydrochloride in the presence of 2 mM dithiothreitol yields a mass of 15.8 kDa. An N-terminal sequence of 36 amino acids is homologous with residues 370-406 of Escherichiu coli E2. The catalytic domain possesses the catalytic site, but in contrast to the E. coli subunit binding domain the pyruvate dehydrogenase (El) and lipoamide dehydrogenase (E3) binding sites are lost during proteolysis. From comparison with the E. coli E2 sequence a model is presented in which the several functions, such as lipoyl domain, the E3 binding site, the catalytic site, the Ez/E2 interaction sites, and the El binding site, are indicated.The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate resulting in the production of acetyl-coenzyme A and NADH. In gram-negative bacteria three enzymes take part in this reaction: pyruvate dehydrogenase (El), lipoylacetyltransferase (E2) and lipoamide dehydrogenase (E3). El and E3 are non-covalently bound as dimers to a core of E2 [l]. The structure of PDC from Azotobucter vinelundii is based on a tetrameric core of E2, to which three El dimers and one E3 dimer are bound [2]. This quaternary structure differs considerably from that of the Escherichiu coli PDC which is based on a core of 24 E2 chains arranged in a cubic structure with 432 symmetry [3]. To this core twelve El dimers and six E3 dimers are bound in the catalytically optimum stoichiometry [4]. Upon removal of the peripheral components, the A . vinelundii E2 associates to a multimer. Although its appearance in the electron microscope is similar to that of E. coli E2, hydrodynamic studies indicate differences in quaternary structure [5, 61. Experiments with hybrid E. coli/A. vinelandii PDC complexes Correspondence f a A.

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“…The complex was resolved into its component enzymes by the method of de Graaf and de Kok [17]. Protein concentrations were measured by the method of Lowry et al [18], using bovine serum albumin a s a standard.…”

Section: Pyruvate Dehydrogenase Complex Was Isolated Frommentioning

confidence: 99%

The size of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Association phenomena

et al. 1984

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Sedimentation analysis and light-scattering studies indicate that the aggregation state of the pyruvate dehydrogenase complex of Azotobucter vinelundii in 50 mM potassium phosphate (pH 7.0) can be described in terms of a monomer-dimer equilibrium with a dissociation constant of 6.8 pM. The apparent molecular mass of the monomeric particle is 750000 -850000 Da.The equilibrium is shifted to the monomeric species when pressure is applied on the system. Pressure-jump experiments yielded a relaxation time of about 70 ms. In the presence of 3 % poly(ethy1ene glycol) 6000 and 10 mM MgCI2, further association takes place to a system that can be described in terms of dimer-tetramer-octamer equilibria. Upon applying a pressure of 80 MPa to this system these equilibria are shifted to the dimeric state but some monomer formation cannot be excluded. Release of pressure shows that the relaxation time of the dimertetramer equilibrium is less than 5 s, that of tetramer-octamer equilibrium is of the order of minutes. The isolated E2 component has a molecular mass of 2000000k 100000 Da; and thus consists of about 30 E2 peptide chains. Electron micrographs are similar to those of the E2 component of the Escherichiu coli complex, which were interpreted as cubic structures with an octagonal symmetry. Upon addition of El to the pure E2 component, changes in the assembly occur and mixtures of large (E. coli-like, 22 -45 S) and small ( A . uinelundii-like, 11 -I8 S) subcomplexes are obtained. The two forms of the subcomplexes are in slow equilibrium (relaxation time 10 -30 min).It is proposed that the E2 tetramer of the intact pyruvate dehydrogenase complex of A . vinelundii is represented by the corner structures of the isolated E2 component.Pyruvate dehydrogenase complexes (PDC) catalyse the oxidative decarboxylation of pyruvate to yield acetyl-coenzyme A and NADH. Three enzymes participate in this reaction: pyruvate dehydrogenase (El), lipoate acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). These enzymes form large assemblies of non-covalently associated polypeptide chains [l 1. The well-studied PDC of Escherichiu coli has a sedimentation coefficient of 53 -60 S and estimations of its molecular mass range over 3.75-6.1 MDa [2 -51. PDCs from gram-positive bacteria and eukaryotes are even larger [6 -81. The PDC from Azotobucter vinelundii is the smallest complex known : its sedimentation coefficient was determined to be 19 S and from light-scattering measurements a molecular mass of l -l .2 MDa was calculated [9]. The A . vinelundii PDC preparations that were used in these experiments had a low specific activity (6 U/mg protein) and contained the so-called fourth component [9,10]. We have therefore repeated these measurements with the pure threecomponent, high-specific-activity preparations (1 5 -19 U/mg protein) that were obtained by a revised purification procedure [ll].Abbreviations. PDC, pyruvate dehydrogenase complex; PEG 6000, poly(ethy1ene glycol) 6000; SDS, sodium dodecyl sulfate.Enzymes. Pyruvate dehydrogenase...

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Binding and dissociation of the pyruvate dehydrogenase complex of Azotobacter vinelandii on thiol—Sepharose

Cited by 25 publications

References 17 publications

Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time‐resolved flavin fluorescence

Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time‐resolved flavin fluorescence

The domain structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

The size of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Association phenomena

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