Temperature-dependence of intramolecular coupling of active sites in pyruvate dehydrogenase multienzyme complexes

Packman, Leonard C.; Stanley, Christopher J.; Perham, Richard N.

doi:10.1042/bj2130331

Cited by 17 publications

(11 citation statements)

References 36 publications

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“…It should be noted that all the experiments described above were performed at 30 °C, appropriate to the laboratory but well below the physiological growth temperature (60 °C) at which the B. stearothermophilus PDH complex exhibits full catalytic activity and displays maximal active‐site coupling [39]. This will need to be taken into account in future work.…”

Section: Discussionmentioning

confidence: 99%

Self‐assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus

Domingo

Chauhan

Lessard³

et al. 1999

European Journal of Biochemistry

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The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was reconstituted in vitro from recombinant proteins derived from genes over-expressed in Escherichia coli. Titrations of the icosahedral (60-mer) dihydrolipoyl acetyltransferase (E2) core component with the pyruvate decarboxylase (E1, a 2 b 2 ) and dihydrolipoyl dehydrogenase (E3, a 2 ) peripheral components indicated a variable composition defined predominantly by tight and mutually exclusive binding of E1 and E3 with the peripheral subunit-binding domain of each E2 chain. However, both analysis of the polypeptide chain ratios in complexes generated from various mixtures of E1 and E3, and displacement of E1 or E3 from E1±E2 or E3±E2 subcomplexes by E3 or E1, respectively, showed that the multienzyme complex does not behave as a simple competitive binding system. This implies the existence of secondary interactions between the E1 and E3 subunits and E2 that only become apparent on assembly. Exact geometrical distribution of E1 and E3 is unlikely and the results are best explained by preferential arrangements of E1 and E3 on the surface of the E2 core, superimposed on their mutually exclusive binding to the peripheral subunit-binding domain of the E2 chain. Correlation of the subunit composition with the overall catalytic activity of the enzyme complex confirmed the lack of any requirement for precise stoichiometry or strict geometric arrangement of the three catalytic sites and emphasized the crucial importance of the flexibility associated with the lipoyl domains and intramolecular acetyl group transfer in the mechanism of active-site coupling.Keywords: multienzyme complex; pyruvate dehydrogenase complex; dihydrolipoyl acetyltransferase; selfassembly; competitive binding.The pyruvate dehydrogenase (PDH) multienzyme complex is a member of the family of 2-oxo acid dehydrogenase multienzyme complexes, all of which catalyse the oxidative decarboxylation of a 2-oxo acid and the reductive acylation of CoA, with concomitant reduction of NAD 1 and the release of CO 2 (reviewed in [1±3]). The complexes comprise multiple copies of three enzymes: a specific 2-oxo acid decarboxylase (E1); a specific dihydrolipoyl acyltransferase (E2); and dihydrolipoyl dehydrogenase (E3). In the PDH complex, E1 is pyruvate decarboxylase (EC 1.2.4.1), E2 is dihydrolipoyl acetyltransferase (EC 2.3.1.12) and E3 is dihydrolipoyl dehydrogenase (EC 1.8.1.4), which serves to reoxidize the dihydrolipoyl intermediate attached to E2 and exhibits no specificity towards the 2-oxo acid substrate.E2 forms the core of the multienzyme complex, to which E1 and E3 bind tightly but noncovalently. E1 may be dimeric (a 2 ) or heterotetrameric (a 2 b 2 ), depending on the source, and E3 is a dimer (a 2 ). The E2 polypeptide chain is composed of three types of domain, separated by long, flexible linker regions: one to three lipoyl domains (9 kDa) at the N-terminus (the number depending on the source of the complex); a small peripheral (E1 and/or E3) subunit-binding domain (PSBD, 4 k...

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Section: Discussionmentioning

confidence: 99%

Self‐assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus

Domingo

Chauhan

Lessard³

et al. 1999

European Journal of Biochemistry

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“…The effect of the antibodies on the intramolecular coupling of active sites [18,28,29] was therefore investigated in more detail. As the PDH complex was inhibited by the addition of increasing amounts of anti-PEP3 Fab fragments, the extent of acetylation of the E2p core with [2-14C]pyruvate in the absence of CoA fell in direct proportion to the loss of overall complex activity ( fig.3).…”

Section: Effect Of Antibody Binding On Catalysis and Active-site Coupmentioning

confidence: 99%

“…To each sample thiamin pyrophosphate (TPP) and MgCI2 were added to give final concentrations of 0.2 and 1.0 mM, respectively, and the mixture was incubated at 37°C for 2 rain. Sodium [2-14C]pyruvate (1-3 /~l of 23 mM, 13.1 mCi/mmol) was then added, and the extent of acetylation of the E2p core after 45 s measured as in [18]. A control experiment was performed by replacing the Fab fragments with equivalent amounts of non-specific rabbit IgG.…”

Section: Reductive Acetylation Of the Pdh Complex Andmentioning

confidence: 99%

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Antibodies against an inter‐domain segment of polypeptide chain inhibit active‐site coupling in the pyruvate dehydrogenase multienzyme complex

et al. 1989

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A synthetic peptide, AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKA, proved to be an efficient and specific immunogen in rabbits. The amino acid sequence of the peptide is identical to that of the inter-domain region (PEP3) linking the innermost of the three lipoyl domains to the dihydrolipoamide dehydrogenase-binding domain in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coll. Fab fragments from anti-PEP3 antibodies selectively inhibited active-site coupling in the complex without affecting the individual activities of the three component enzymes, highlighting the role of the inter-domain regions as flexible linkers in catalysis.

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“…El is a thiamin pyrophosphate-dependent 2-oxo acid deyhydrogenase, and E3 is an FAD-dependent lipoamide dehydrogenase. E2 is an acyltransferase that forms the central core of the complexes and uses lipoic acid, which is covalently bound to the epsilon amino group of lysine, as an essential cofactor (7). Antibodies to PDH-E2 are not found in patients with other chronic autoimmune liver diseases.…”

mentioning

confidence: 99%

The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase.

Water

Gershwin

Leung

et al. 1988

The Journal of Experimental Medicine

275

147

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Autoantibodies to mitochondrial antigens are characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC), but the precise antigenic determinants recognized by these antibodies have not been defined. Recently, our laboratory identified a 1,370-bp rat liver cDNA clone that coded for a polypeptide recognized specifically by sera from patients with PBC but not by sera from patients with other forms of liver disease. This recombinant protein was identified as the 74-kD M2 mitochondrial inner membrane autoantigen, now known to be dihydrolipoamide acetyltransferase. In the present study, we have identified a 603-bp fragment that codes for a polypeptide containing all of the autoreactivity of the original clone. In addition, based on hydrophobicity/hydrophilicity plots of the amino acid sequence of this polypeptide segment, several peptides were synthesized and tested for reactivity by an inhibition assay using sera from patients with PBC. One peptide, defined by the amino acids AEIETDKATIGFEVQEEGYL, absorbed serum reactivity to the protein product of the original clone. Of particular interest was the finding that this peptide contains the lipoic acid binding site KATIGF of the dihydrolipoamide acetyltransferase found in the inner mitochondrial membrane. Thus, it appears that for this autoantigen, the target of the autoantibodies corresponds to a functional site of the dihydrolipoamide acetyltransferase.

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Temperature-dependence of intramolecular coupling of active sites in pyruvate dehydrogenase multienzyme complexes

Cited by 17 publications

References 36 publications

Self‐assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus

Self‐assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus

Antibodies against an inter‐domain segment of polypeptide chain inhibit active‐site coupling in the pyruvate dehydrogenase multienzyme complex

The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase.

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