Characterization of the Interthiol Acyltransferase Reaction Catalyzed by the β-Ketoacyl Synthase Domain of the Animal Fatty Acid Synthase†

Biochemistry ◽  
1997 ◽  
Vol 36 (51) ◽  
pp. 16338-16344 ◽  
Author(s):  
Andrzej Witkowski ◽  
Anil K. Joshi ◽  
Stuart Smith
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Ketoacyl Synthase

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.

Characterization of fatty acid synthase in cell lines derived from experimental mammary tumors

1998 ◽  
Vol 1392 (1) ◽  
pp. 85-100 ◽  
Author(s):  
Randolph A Hennigar ◽  
Mildred Pochet ◽  
Dirk A Hunt ◽  
Aron E Lukacher ◽  
Virginia J Venema ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Cell Lines ◽  
Fatty Acid Synthase ◽  
Mammary Tumors

scholarly journals Purification and Biochemical Characterization of theMycobacterium tuberculosisβ-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

2001 ◽  
Vol 276 (50) ◽  
pp. 47029-47037 ◽  
Author(s):  
Merrill L. Schaeffer ◽  
Gautam Agnihotri ◽  
Craig Volker ◽  
Howard Kallender ◽  
Patrick J. Brennan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Mycolic Acids ◽  
E Coli ◽  
Long Chain

Mycolic acids are vital components of theMycobacterium tuberculosiscell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performsde novofatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two β-ketoacyl-ACP synthase (KAS) enzymes of theM. tuberculosisFASII system. KasA and KasB were expressed inE. coliand purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use ofM. tuberculosisAcpM, suggesting that structural differences between AcpM andE. coliACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

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