scholarly journals Characterization of the inactivation of rat fatty acid synthase by C75: inhibition of partial reactions and protection by substrates

2005 ◽  
Vol 388 (3) ◽  
pp. 895-903 ◽  
Author(s):  
Alan R. RENDINA ◽  
Dong CHENG
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Room Temperature ◽  
Fatty Acid Synthase ◽  
Enzyme Inhibitor ◽  
Acetyl Coa ◽  
Enoyl Reductase ◽  
Malonyl Coa ◽  
Ketoacyl Synthase

C75, a synthetic inhibitor of FAS (fatty acid synthase), has both anti-tumour and anti-obesity properties. In this study we provide a detailed kinetic characterization of the mechanism of in vitro inhibition of rat liver FAS. At room temperature, C75 is a competitive irreversible inhibitor of the overall reaction with regard to all three substrates, i.e. acetyl-CoA, malonyl-CoA and NADPH, exhibiting pseudo-first-order kinetics of the complexing type, i.e. a weak non-covalent enzyme–inhibitor complex is formed before irreversible enzyme modification. C75 is a relatively inefficient inactivator of FAS, with a maximal rate of inactivation of 1 min−1 and an extrapolated KI (dissociation constant for the initial complex) of approx. 16 mM. The apparent second-order rate constants calculated from these values are 0.06 mM−1·min−1 at room temperature and 0.21 mM−1·min−1 at 37 °C. We also provide experimental evidence that C75 inactivates the β-ketoacyl synthase (3-oxoacyl synthase) partial activity of FAS. Unexpectedly, C75 also inactivates the enoyl reductase and thioesterase partial activities of FAS with about the same rates as for inactivation of the β-ketoacyl synthase. In contrast with the overall reaction, the β-ketoacyl synthase activity and the enoyl reductase activity, substrates do not protect the thioesterase activity of rat liver FAS from inactivation by C75. These results differentiate inactivation by C75 from that by cerulenin, which only inactivates the β-ketoacyl synthase activity of FAS, by forming an adduct with an active-site cysteine. Interference by dithiothreitol and protection by the substrates, acetyl-CoA, malonyl-CoA and NADPH, further distinguish the mechanism of C75-mediated inactivation from that of cerulenin. The most likely explanation for the multiple effects observed with C75 on rat liver FAS and its partial reactions is that there are multiple sites of interaction between C75 and FAS.

scholarly journals Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase

1997 ◽  
Vol 327 (1) ◽  
pp. 267-273 ◽  
Author(s):  
P. Grattan ROUGHAN
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Acetyl Coa ◽  
Minimal Processing ◽  
Malonyl Coa ◽  
Substrate Channelling ◽  
Isolated Chloroplasts

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10–20 μM, assuming a stromal volume of 66 μl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 μM. Malonyl-CoA concentrations were also very low (< 0.1–3.0 μM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 μM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA–acyl-carrier-protein transacylase reactions at the concentrations of metabolites measured here are < 0.1–4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.

scholarly journals Tsc13p Is Required for Fatty Acid Elongation and Localizes to a Novel Structure at the Nuclear-Vacuolar Interface inSaccharomyces cerevisiae

2001 ◽  
Vol 21 (1) ◽  
pp. 109-125 ◽  
Author(s):  
Sepp D. Kohlwein ◽  
Sandra Eder ◽  
Chan-Seok Oh ◽  
Charles E. Martin ◽  
Ken Gable ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Calcium Sensitivity ◽  
Long Chain Fatty Acid ◽  
Enoyl Reductase ◽  
Novel Structure ◽  
Malonyl Coa ◽  
Chain Lengths ◽  
Long Chain

ABSTRACT The TSC13/YDL015c gene was identified in a screen for suppressors of the calcium sensitivity of csg2Δ mutants that are defective in sphingolipid synthesis. The fatty acid moiety of sphingolipids in Saccharomyces cerevisiae is a very long chain fatty acid (VLCFA) that is synthesized by a microsomal enzyme system that lengthens the palmitate produced by cytosolic fatty acid synthase by two carbon units in each cycle of elongation. TheTSC13 gene encodes a protein required for elongation, possibly the enoyl reductase that catalyzes the last step in each cycle of elongation. The tsc13 mutant accumulates high levels of long-chain bases as well as ceramides that harbor fatty acids with chain lengths shorter than 26 carbons. These phenotypes are exacerbated by the deletion of either the ELO2 or ELO3gene, both of which have previously been shown to be required for VLCFA synthesis. Compromising the synthesis of malonyl coenzyme A (malonyl-CoA) by inactivating acetyl-CoA carboxylase in atsc13 mutant is lethal, further supporting a role of Tsc13p in VLCFA synthesis. Tsc13p coimmunoprecipitates with Elo2p and Elo3p, suggesting that the elongating proteins are organized in a complex. Tsc13p localizes to the endoplasmic reticulum and is highly enriched in a novel structure marking nuclear-vacuolar junctions.

Mammalian fatty acid synthetase. III. Characterization of human liver synthetase products and kinetics of methylmalonyl-CoA inhibition

1976 ◽  
Vol 54 (11) ◽  
pp. 923-926 ◽  
Author(s):  
Daniel A. K. Roncari ◽  
Esther Y. W. Mack
Keyword(s):  
Fatty Acid ◽  
Human Liver ◽  
Fatty Acid Synthetase ◽  
Acid Synthesis ◽  
Acetyl Coa ◽  
Malonyl Coa ◽  
Direct Explanation ◽  
Vitro Model

When propionyl-CoA was substituted for either acetyl-CoA or butyryl-CoA in the presence of [14C]malonyl-CoA and NADPH, the pure human liver fatty acid synthetase complex synthesized only straight-chain, saturated, 15- and 17-carbon radioactive fatty acids. At optimal concentrations, propionyl-CoA was a better primer of fatty acid synthesis than acetyl-CoA. Methylmalonyl-CoA inhibited the synthetase competitively with respect to malonyl-CoA. The Ki was calculated to be 8.4 μM. These findings provide an in vitro model and offer a direct explanation at the molecular level for some of the abnormal manifestations observed in diseases characterized by increased cellular concentrations of propionyl-CoA and methylmalonyl-CoA.

scholarly journals Characterization of rat liver malonyl-CoA decarboxylase and the study of its role in regulating fatty acid metabolism

2000 ◽  
Vol 350 (2) ◽  
pp. 599 ◽  
Author(s):  
Jason R.B. DYCK ◽  
Luc G. BERTHIAUME ◽  
Panakkezhum D. THOMAS ◽  
Paul F. KANTOR ◽  
Amy J. BARR ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Fatty Acid Metabolism ◽  
Acid Metabolism ◽  
Malonyl Coa

scholarly journals Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase

2021 ◽  
Author(s):  
Satyam Khanal ◽  
Roberto Javier Brea Fernandez ◽  
Michael Burkart ◽  
Neal Devaraj
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Type I ◽  
Phospholipid Membrane ◽  
Acetyl Coa ◽  
Chemical Ligation ◽  
Malonyl Coa ◽  
Synthetic Cell ◽  
Membrane Bound

The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

scholarly journals Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase

2019 ◽  
Author(s):  
Jennifer W. Lou ◽  
Kali R. Iyer ◽  
S. M. Naimul Hasan ◽  
Leah E. Cowen ◽  
Mohammad T. Mazhab-Jafari
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Protein Translocation ◽  
Acyl Carrier Protein ◽  
Reaction Chamber ◽  
Type I ◽  
Acyl Carrier Proteins ◽  
Enoyl Reductase ◽  
Ketoacyl Synthase

ABSTRACTDuring fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

scholarly journals Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jennifer W. Lou ◽  
Kali R. Iyer ◽  
S. M. Naimul Hasan ◽  
Leah E. Cowen ◽  
Mohammad T. Mazhab-Jafari
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Protein Translocation ◽  
Acyl Carrier Protein ◽  
Reaction Chamber ◽  
Type I ◽  
Acyl Carrier Proteins ◽  
Enoyl Reductase ◽  
Ketoacyl Synthase

Abstract During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

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