Palmitoyl-coenzyme A synthetase. Isolation of an enzyme-bound intermediate

Bar‐Tana, Jacob; Rose, G.; Shapiro, B.

doi:10.1042/bj1350411

Cited by 24 publications

(8 citation statements)

References 13 publications

(12 reference statements)

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“…4 and 5), meaning that the fatty acyl-AMP intermediate is unlikely to be released from the enzyme. This is consistent with extensive experimentation that has been unable to isolate such intermediates (23,24). In contrast to LC-FACS, SC-FACS is suggested to adopt two different closed conformations for the two steps of the reaction (31,32), whereas the catalytic Bi Uni Uni Bi PingPong mechanism should be the same in all FACSs (22,(25)(26)(27).…”

Section: Resultssupporting

confidence: 67%

“…In contrast to LC-FACS, SC-FACS is suggested to adopt two different closed conformations for the two steps of the reaction (31,32), whereas the catalytic Bi Uni Uni Bi PingPong mechanism should be the same in all FACSs (22,(25)(26)(27). If it is the case, the conformational change via the open form between the two distinct C-terminal forms in the two-step catalysis should take place in SC-or MC-FACSs, because these FACSs were shown to release and utilize the acetyl-AMP or butyryl-AMP as the two-step reaction intermediate, respectively, but not in LC-FACS (23,24,26,27).…”

Section: Resultsmentioning

confidence: 99%

“…However, to date the fatty acyl-AMP intermediate has not been isolated nor utilized experimentally as substrate for the second step (Reaction 3) or as product for the first step (Reaction 2) in reverse catalysis (23,24) in contrast to the acetyl-AMP or butyryl-AMP for SC-or MC-FACSs (25)(26)(27).…”

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confidence: 99%

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Structural Basis of the Substrate-specific Two-step Catalysis of Long Chain Fatty Acyl-CoA Synthetase Dimer

Hisanaga

Ago²,

Nakagawa³

et al. 2004

Journal of Biological Chemistry

188

262

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Section: Resultssupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

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Structural Basis of the Substrate-specific Two-step Catalysis of Long Chain Fatty Acyl-CoA Synthetase Dimer

Hisanaga

Ago²,

Nakagawa³

et al. 2004

Journal of Biological Chemistry

188

262

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“…Furthermore, the surface charge of the complex is affected by the hydrophilic or hydrophobic nature of the individual proteins that make up the complexes, especially with membrane proteins where lipids also may be present. The dimeric form of ACSL has been reported as the active form in rat and in S. cerevisiae (24,25), and VDAC has been described as a dimer or tetramer (26,27). On the basis of these findings, we estimated the possible composition of the band at 280 kDa as consisting of a CPT1a monomer, an ACSL dimer, and a VDAC dimer that yield a sum of 296 kDa.…”

Section: Proteinmentioning

confidence: 99%

Mitochondrial Carnitine Palmitoyltransferase 1a (CPT1a) Is Part of an Outer Membrane Fatty Acid Transfer Complex

Lee

Kerner

Hoppel

2011

Journal of Biological Chemistry

215

159

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CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM.Long chain fatty acids, an important energy source in mitochondria, are oxidized in the matrix via ␤-oxidation. Long chain fatty acids are activated on the cytosolic side of the mitochondrial outer membrane (MOM) 2 by long chain acyl-CoA synthetase (ACSL) (1). To be metabolized, activated fatty acids, as well as other substrates, ions, and nucleotides, cross the MOM through the voltage-dependent anion channel (VDAC), also called mitochondrial porin.Historically, the MOM was considered to be a sieve through which small compounds passed unimpeded. That view is no longer tenable. In Trypanosoma brucei, which expresses only one VDAC, knock-out of VDAC leads to mitochondria that no longer oxidize substrates (2). However, with disruption of the outer membrane, the mitoplasts are fully capable of oxidizing substrates. These studies highlight the essential role of VDAC in mitochondrial substrate oxidation in transportation of small anion substrates through the MOM.We studied PA22, a 22-kDa polyanion VDAC inhibitor (3), in mitochondrial substrate oxidation and observed that ADPstimulated glutamate, succinate, (ϩrotenone), and palmitoylcarnitine (ϩmalate) oxidation rates were unaffected. However, oxidation of palmitate (ϩATP, Mg 2ϩ , CoA, carnitine, and malate) and palmitoyl-CoA (ϩcarnitine and malate) was inhibited at 1-2 nmol of PA22/mg of mitochondrial protein. These data indicate that PA22 is selective for the first two steps in fatty acid oxidation without affecting other substrates. Additionally, we showed that PA22 interacts with VDAC by demonstrating that binding of hexokinase to rat liver mitochondria was inhibited by PA22 (3). VDAC was initially identified as the hexokinase-binding protein (4).PA10, a 10-kDa polyanion VDAC inhibitor (5), decreases ADP-stimulated oxidation of glutamate, malate, and succinat...

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“…The ACL family catalyzes the formation of a fatty acyl-CoA from a fatty acid substrate, ATP, and CoA in a Mg 2+ -dependent two-step reaction (Bar-Tana et al, 1973a, 1973bBlack et al, 1997) (Fig. 3).…”

Section: Enzyme Kinetics Of the Al Domainmentioning

confidence: 99%

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Fritzler

Zhu

2007

International Journal for Parasitology

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The apicomplexan Cryptosporidium parvum possesses a unique 1,500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesizing a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [ 3 H] palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h = 1.46, K 50 = 0.751 μM, V max = 2.236 μmol mg −1 min −1 ) and CoA (h = 0.704, K 50 = 5.627 μM, V max = 0.557 μmol mg −1 min −1 ), and biphasic kinetics towards adenosine 5′-triphosphate (K m1 = 3.149 μM, V max1 = 373.3 nmol mg −1 min −1 , K m2 = 121.0 μM, and V max2 = 563.7 nmol mg −1 min −1 ). The AL domain is Mg 2+ -dependent and its activity could be inhibited by triacsin C (IC 50 = 6.64 μM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.

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Palmitoyl-coenzyme A synthetase. Isolation of an enzyme-bound intermediate

Cited by 24 publications

References 13 publications

Structural Basis of the Substrate-specific Two-step Catalysis of Long Chain Fatty Acyl-CoA Synthetase Dimer

Structural Basis of the Substrate-specific Two-step Catalysis of Long Chain Fatty Acyl-CoA Synthetase Dimer

Mitochondrial Carnitine Palmitoyltransferase 1a (CPT1a) Is Part of an Outer Membrane Fatty Acid Transfer Complex

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

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