Limited proteolysis of pig liver CoA synthase: evidence for subunit identity

Worrall, Dawn; Lambert, Sarah F.; Tubbs, Philip K.

doi:10.1016/0014-5793(85)81258-8

Cited by 8 publications

(5 citation statements)

References 9 publications

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“…Sizeexclusion chromatography on FPLC Superdex 200 at the final stage of purification revealed that, in the presence of 150 mM NaCl, pig-liver PPAT\DPCK eluted as a peak with molecular mass of 62 kDa (Figure 1a). This indicates that the protein behaves as a monomer, and not a homodimer, as originally suggested [13]. N-terminal sequence analysis of the purified protein revealed a blocked N-terminus, and internal sequencing was performed following either limited trypsin proteolysis or cyanogen bromide cleavage (Figure 1b).…”

Section: Resultssupporting

confidence: 59%

“…Proteolytic nicking of the pig enzyme by trypsin to yield 40 kDa and 22 kDa fragments has been reported, which does not effect either activity or the proposed tertiary structure [13]. The sequence analysis of the resulting 40 kDa band resulting from SDS\PAGE (the 22 kDa band N-terminal was blocked) shows that this nicking occurs after position Arg")", suggesting that an exposed region exists between the N-terminal region and the PPAT domain.…”

Section: Substrate Ppat (Reverse Reaction)mentioning

confidence: 98%

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Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and dephospho-CoA kinase bifunctional enzyme (CoA synthase)

Aghajanian

Worrall

2002

Biochemical Journal

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The final two enzymes in the CoA biosynthetic pathway, phosphopantetheine adenylyltransferase (PPAT; EC 2.7.7.3) and dephospho-CoA kinase (DPCK; EC 2.7.1.24), are separate proteins in prokaryotes, but exist as a bifunctional enzyme in pig liver. In the present study we have obtained sequence information from purified pig-liver enzyme, and identified the corresponding cDNA in a number of species. The human gene localizes to chromosome 17q12-21 and contains regions with sequence similarity to the monofunctional Escherichia coli DPCK and PPAT. The recombinant 564-amino-acid human protein confirmed the associated transferase and kinase activities, and gave similar kinetic properties to the wild-type pig enzyme.

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Section: Resultssupporting

confidence: 59%

Section: Substrate Ppat (Reverse Reaction)mentioning

confidence: 98%

Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and dephospho-CoA kinase bifunctional enzyme (CoA synthase)

Aghajanian

Worrall

2002

Biochemical Journal

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“…The human and porcine CoA synthases have been shown to possess three domains, an N-terminal domain that helps target the bifunctional enzyme to the mitochondrial outer membrane and mediates protein-protein interactions, a middle CoaD domain and a C-terminal CoaE domain [10]. Interestingly, it has been shown that proteolytic cleavage of the human enzyme does not affect enzyme activity or the proposed tertiary structure [11]. Also, the N-terminal domain of the CoA synthase is found only in higher eukaryotes and has been shown to effect regulation of the CoaD and CoaE activities by phospholipids [12].…”

Section: Introductionmentioning

confidence: 99%

The Role of UPF0157 in the Folding of M. tuberculosis Dephosphocoenzyme A Kinase and the Regulation of the Latter by CTP

2009

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BackgroundTargeting the biosynthetic pathway of Coenzyme A (CoA) for drug development will compromise multiple cellular functions of the tubercular pathogen simultaneously. Structural divergence in the organization of the penultimate and final enzymes of CoA biosynthesis in the host and pathogen and the differences in their regulation mark out the final enzyme, dephosphocoenzyme A kinase (CoaE) as a potential drug target.Methodology/Principal FindingsWe report here a complete biochemical and biophysical characterization of the M. tuberculosis CoaE, an enzyme essential for the pathogen's survival, elucidating for the first time the interactions of a dephosphocoenzyme A kinase with its substrates, dephosphocoenzyme A and ATP; its product, CoA and an intrinsic yet novel inhibitor, CTP, which helps modulate the enzyme's kinetic capabilities providing interesting insights into the regulation of CoaE activity. We show that the mycobacterial enzyme is almost 21 times more catalytically proficient than its counterparts in other prokaryotes. ITC measurements illustrate that the enzyme follows an ordered mechanism of substrate addition with DCoA as the leading substrate and ATP following in tow. Kinetic and ITC experiments demonstrate that though CTP binds strongly to the enzyme, it is unable to participate in DCoA phosphorylation. We report that CTP actually inhibits the enzyme by decreasing its Vmax. Not surprisingly, a structural homology search for the modeled mycobacterial CoaE picks up cytidylmonophosphate kinases, deoxycytidine kinases, and cytidylate kinases as close homologs. Docking of DCoA and CTP to CoaE shows that both ligands bind at the same site, their interactions being stabilized by 26 and 28 hydrogen bonds respectively. We have also assigned a role for the universal Unknown Protein Family 0157 (UPF0157) domain in the mycobacterial CoaE in the proper folding of the full length enzyme.Conclusions/SignificanceIn view of the evidence presented, it is imperative to assign a greater role to the last enzyme of Coenzyme A biosynthesis in metabolite flow regulation through this critical biosynthetic pathway.

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“…The bi-functional complex ("CoA synthase") was purified from pig liver (10) and shown to exist in solution as a homodimer with subunits of 57 kDa. Limited proteolysis of the latter revealed that the subunits are identical and that each subunit contains both PPAT and dPCoA kinase (11). Although similar bi-functional complexes have been partially purified from other mammalian sources (12,13), the PPAT of bakers' yeast (14) was identified as part of a much larger (375-400 kDa) CoA-synthesizing protein complex, which contained six different enzyme activities involved in the synthesis and metabolism of CoA.…”

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confidence: 99%

Purification and Characterization of Phosphopantetheine Adenylyltransferase from Escherichia coli

Geerlof

Lewendon

Shaw³

1999

Journal of Biological Chemistry

111

112

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Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4-phosphopantetheine yielding 3-dephospho-CoA and pyrophosphate. Wild-type PPAT from Escherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamed coaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.Coenzyme A (CoA) 1 is an essential cofactor in numerous biosynthetic, degradative, and energy-yielding metabolic pathways and is involved in the control of several key reactions in intermediary metabolism (1). CoA also donates the 4Ј-phosphopantetheinyl cofactor to the acyl carrier protein of the fatty acid synthase complex (2).The synthesis of CoA occurs in five steps which, utilize pantothenate (vitamin B 5 ), cysteine, and ATP (for review, see Ref.3). In all systems studied, the rate of CoA biosynthesis appears to be regulated by feedback inhibition of the first enzyme of the pathway, pantothenate kinase (4 -7). In vitro studies of pantothenate kinase from Escherichia coli showed that (a) CoA and, to a lesser extent, its acyl thioesters are competitive inhibitors with respect to ATP and (b) the K i values are within the physiological range of intracellular CoA concentrations (6). Studies of the intermediates in CoA biosynthesis have shown that both pantothenate and 4Ј-phosphopantetheine can accumulate in the cell (8). Hence, in addition to control of CoA synthesis on the level of pantothenate kinase, further modulation of flux through the pathway could occur at phosphopantetheine adenylyltransferase (PPAT), which catalyzes the penultimate step in the pathway (Fig. 1), the reversible adenylation of 4Ј-phosphopantetheine to form 3Ј-dephospho-CoA (dPCoA) and pyrophosphate (PP i ). Regulation at this step may control the reutilization of 4Ј-phosphopantetheine arising either from the turnover of the 4Ј-phosphopantetheinyl cofactor of the acyl carrier protein (8) or the cleavage of CoA by a phosphodiesterase (9).Despite the above arguments for a role in the regulation of CoA biosynthesis, PPAT has not been the subject of a detailed study. Enzymes with PPAT activity have been purified from a number of different organisms. In mammals PPAT has been shown to be part of a complex that also includes dPCoA kinase, the effector of the fina...

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Limited proteolysis of pig liver CoA synthase: evidence for subunit identity

Cited by 8 publications

References 9 publications

Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and dephospho-CoA kinase bifunctional enzyme (CoA synthase)

Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and dephospho-CoA kinase bifunctional enzyme (CoA synthase)

The Role of UPF0157 in the Folding of M. tuberculosis Dephosphocoenzyme A Kinase and the Regulation of the Latter by CTP

Purification and Characterization of Phosphopantetheine Adenylyltransferase from Escherichia coli

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