Characterization of the inactivation of rat fatty acid synthase by C75: inhibition of partial reactions and protection by substrates.
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 K(I) (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 degrees C. We also provide experimental evidence that C75 inactivates the beta-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 beta-ketoacyl synthase. In contrast with the overall reaction, the beta-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 beta-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.
Rendina
Alan R
AR
Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA. alan.rendina@bms.com
Cheng
Dong
D
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000494:pharmacology; D000105:Acetyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008316:Malonyl Coenzyme A / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013696:Temperature
C406734:4-methylene-2-octyl-5-oxofuran-3-carboxylic acid; D008316:Malonyl Coenzyme A; D009249:NADP; D000105:Acetyl Coenzyme A; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D064429:Fatty Acid Synthases; D015107:4-Butyrolactone; D004229:Dithiothreitol
Cancer Res. 2000 Jan 15;60(2):213-8
10667561
Eur J Pharmacol. 2005 Mar 21;511(1):31-41
15777777
Science. 2000 Jun 30;288(5475):2379-81
10875926
Biochem Biophys Res Commun. 2001 Nov 16;288(5):1200-6
11700039
Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):1921-5
11854492
Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9498-502
12060712
Prog Lipid Res. 2003 Jul;42(4):289-317
12689621
Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12624-9
14532332
J Biol Chem. 2004 Jan 30;279(5):3817-27
14615481
Nature. 2004 Apr 1;428(6982):569-74
15058305
J Biol Chem. 2004 May 7;279(19):19970-6
15028725
J Biol Chem. 1968 Oct 25;243(20):5471-8
4302787
J Biol Chem. 1970 Sep 25;245(18):4732-44
5456146
Arch Biochem Biophys. 1974 Jun;162(2):412-20
4152155
Methods Enzymol. 1975;35:37-44
1121291
J Biol Chem. 1981 May 25;256(10):5128-33
6112225
Arch Biochem Biophys. 1981 Jul;209(2):613-9
7294812
J Biochem. 1982 Jul;92(1):7-12
6749834
J Biochem. 1989 May;105(5):751-5
2666407
Biochemistry. 1989 May 30;28(11):4523-30
2669958
J Biol Chem. 1993 Oct 25;268(30):22508-13
8226759
Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8695-9
7567999
Biochemistry. 1996 Aug 13;35(32):10569-75
8756713
Biochemistry. 1999 Sep 7;38(36):11643-50
10512619
J Biol Chem. 1962 Oct;237:3245-9
14033211
Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3450-4
10716717