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University of Groningen Exploring the mechanisms underlying the phenotype of MCAD deficiency with Systems Medicine Martines, Anne-Claire

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(1)University of Groningen. Exploring the mechanisms underlying the phenotype of MCAD deficiency with Systems Medicine Martines, Anne-Claire. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.. Document Version Publisher's PDF, also known as Version of record. Publication date: 2019 Link to publication in University of Groningen/UMCG research database. Citation for published version (APA): Martines, A-C. (2019). Exploring the mechanisms underlying the phenotype of MCAD deficiency with Systems Medicine: from computational model to mice to man. Rijksuniversiteit Groningen.. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.. Download date: 28-06-2021.

(2) Chapter 2 The promiscuous enzyme medium-chain 3-keto-acyl-CoA thiolase triggers a vicious cycle in fatty-acid beta-oxidation. Anne-Claire M.F. Martines1,2, Karen van Eunen1,2, Dirk-Jan Reijngoud1,2, and Barbara M. Bakker1,2,*. 1 Laboratory. of Pediatrics, University of Groningen, University Medical Center Groningen, The Netherlands 2 Systems Biology Centre for Energy Metabolism and Ageing, University of Groningen, University Medical Center Groningen, The Netherlands. PLoS Comput Biol. 2017;13(4):1–22.

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(4) The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation. Abstract ‹–‘ Š‘†”‹ƒŽ ˆƒ––›Ǧƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘ ȋ Ȍ ’Žƒ›• ƒ ‡–”ƒŽ ”‘Ž‡ ‹ ƒƒŽ‹ƒ ‡‡”‰› ‡–ƒ„‘Ž‹•Ǥ—Ž–‹’Ž‡•‡˜‡”‡†‹•‡ƒ•‡•ƒ”‡ƒ••‘ ‹ƒ–‡†™‹–Š†‡ˆ‡ –•‹–Š‹•’ƒ–Š™ƒ›Ǥ

(5) –•‹‡–‹  •–”— –—”‡ ‹• Šƒ”ƒ –‡”‹œ‡† „› ƒ ‘’Ž‡š ™‹”‹‰ ‘ˆ ™Š‹ Š –Š‡ ˆ— –‹‘ƒŽ ‹’Ž‹ ƒ–‹‘• Šƒ˜‡ Šƒ”†Ž› „‡‡ ‡š’Ž‘”‡†Ǥ ‡’‡–‹–‹˜‡ › Ž‡• ‘ˆ ”‡˜‡”•‹„Ž‡ ”‡ƒ –‹‘•ǡ ‡ƒ Š › Ž‡ •Š‘”–‡‹‰ –Š‡ ˆƒ––›ƒ ‹†„›–™‘ ƒ”„‘ƒ–‘•ǡ‡˜‘‡ ‘’‡–‹–‹‘„‡–™‡‡‹–‡”‡†‹ƒ–‡•‘ˆ†‹ˆˆ‡”‡– Šƒ‹ Ž‡‰–Š•ˆ‘”ƒ ‘‘•‡–‘ˆǮ’”‘‹• —‘—•ǯ‡œ›‡•ȋ‡œ›‡•™‹–Šƒ –‹˜‹–›–‘™ƒ”†•—Ž–‹’Ž‡ •—„•–”ƒ–‡•ȌǤ

(6) ‘—”˜ƒŽ‹†ƒ–‡†‹‡–‹ ‘†‡Ž‘ˆ–Š‡’ƒ–Š™ƒ›ǡ•—„•–”ƒ–‡‘˜‡”Ž‘ƒ† ƒ—•‡•ƒ•–‡‡’ ƒ† †‡–”‹‡–ƒŽ ˆŽ—š †‡ Ž‹‡Ǥ ‡”‡ǡ ™‡ —”ƒ˜‡Ž –Š‡ —†‡”Ž›‹‰ ‡ Šƒ‹• ƒ† –Š‡ ”‘Ž‡ ‘ˆ ‡œ›‡’”‘‹• —‹–›‹‹–Ǥ ‘’ƒ”‹•‘ ‘ˆ ƒŽ–‡”ƒ–‹˜‡ ‘†‡Ž ˜‡”•‹‘• ‡Ž— ‹†ƒ–‡† –Š‡ ”‘Ž‡ ‘ˆ ’”‘‹• —‹–› ‘ˆ ‹†‹˜‹†—ƒŽ ‡œ›‡•Ǥ ”‘‹• —‹–› ‘ˆ –Š‡ Žƒ•– ‡œ›‡ ‘ˆ –Š‡ ’ƒ–Š™ƒ›ǡ ‡†‹—Ǧ Šƒ‹ ‡–‘ƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ȋȌǡ™ƒ•„‘–Š‡ ‡••ƒ”›ƒ†•—ˆˆ‹ ‹‡––‘‡Ž‹ ‹––Š‡ˆŽ—š†‡ Ž‹‡Ǥ—„•‡“—‡–Ž›ǡ ‡–ƒ„‘Ž‹ ‘–”‘ŽƒŽ›•‹•”‡˜‡ƒŽ‡†–Šƒ–Šƒ†‹•—ˆˆ‹ ‹‡– ƒ’ƒ ‹–›–‘ ‘’‡™‹–ŠŠ‹‰Š •—„•–”ƒ–‡ ‹ˆŽ—šǤ ‡š–ǡ ™‡ “—ƒ–‹ˆ‹‡† –Š‡ ‹–‡”ƒŽ ‡–ƒ„‘Ž‹  ”‡‰—Žƒ–‹‘ǡ ”‡˜‡ƒŽ‹‰ ƒ ˜‹ ‹‘—• › Ž‡ƒ”‘—†Ǥ’‘•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†ǡǯ•‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡••–ƒ”–‡†–‘ ƒ ——Žƒ–‡Ǥ Š‡ —ˆƒ˜‘—”ƒ„Ž‡ ‡“—‹Ž‹„”‹— ‘•–ƒ– ‘ˆ –Š‡ ’”‡ ‡†‹‰ ‡œ›‡ǡ ‡†‹—Ȁ•Š‘”–Ǧ Šƒ‹ Š›†”‘š›ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ǡ ™‘”‡† ƒ• ƒ ƒ’Ž‹ˆ‹‡”ǡ Ž‡ƒ†‹‰ –‘ ƒ ——Žƒ–‹‘‘ˆ—’•–”‡ƒ‘‡•–‡”•ǡ‹ Ž—†‹‰ƒ ›ŽǦ‘‡•–‡”•ǤŠ‡•‡ƒ ›ŽǦ‘‡•–‡”•ƒ”‡ ƒ––Š‡•ƒ‡–‹‡’”‘†— –•‘ˆƒ†‹Š‹„‹–‡†‹–•ƒŽ”‡ƒ†›Ž‘™ƒ –‹˜‹–›ˆ—”–Š‡”Ǥ ‹ƒŽŽ›ǡ –Š‡ƒ ——Žƒ–‹‘‘ˆ‘‡•–‡”•Ž‡†–‘ƒ•‡“—‡•–”ƒ–‹‘‘ˆˆ”‡‡‘Ǥ‘„‡‹‰ƒ ‘ˆƒ –‘”ˆ‘” ǡ ‹–• •‡“—‡•–”ƒ–‹‘ Ž‹‹–‡† –Š‡  ƒ –‹˜‹–› ‡˜‡ ˆ—”–Š‡”ǡ –Š—• ‘’Ž‡–‹‰ –Š‡ ˜‹ ‹‘—• › Ž‡Ǥ‹ ‡‘‹•ƒŽ•‘ƒ•—„•–”ƒ–‡ˆ‘”†‹•–ƒ–‡œ›‡•ǡ‹–‡ˆˆ‹ ‹‡–Ž› ‘—‹ ƒ–‡† –Š‡Ǯ–”ƒˆˆ‹ Œƒǯƒ––‘–Š‡‡–‹”‡’ƒ–Š™ƒ›Ǥ Š‹•‘˜‡Ž‡ Šƒ‹•’”‘˜‹†‡•ƒ„ƒ•‹•–‘‡š’Ž‘”‡–Š‡”‘Ž‡‘ˆ ‹†‹•‡ƒ•‡ƒ†‡Ž— ‹†ƒ–‡ •‹‹Žƒ”’”‹ ‹’Ž‡•‹‘–Š‡”’ƒ–Š™ƒ›•‘ˆŽ‹’‹†‡–ƒ„‘Ž‹•Ǥ   . Ͷͳ. 2.

(7) Chapter 2. Introduction Š‡ ‹–‘ Š‘†”‹ƒŽ ˆƒ––›Ǧƒ ‹† ‘š‹†ƒ–‹‘ ȋ Ȍ ‹ –Š‡ Ž‹˜‡” ‹• ‘ˆ ‡› ‹’‘”–ƒ ‡ ˆ‘” –Š‡ ’”‘†— –‹‘ ‘ˆ  ƒ† ‡–‘‡ „‘†‹‡• †—”‹‰ ˆƒ•–‹‰Ǥ

(8) – ƒŽŽ‘™• ‰Ž— ‘•‡ –‘ „‡ •’ƒ”‡† ˆ‘” —–‹Ž‹œƒ–‹‘ ‹ –Š‡ ’‡”‹’Š‡”›ǡ ƒ •—’’Ž› –Š‡ ‡‡”‰› ˆ‘” †‡ ‘˜‘ ‰Ž— ‘•‡ ’”‘†— –‹‘ǡ ƒ† ’”‘˜‹†‡• ’”‡ —”•‘”• ˆ‘” ‡–‘‡Ǧ„‘†› •›–Š‡•‹•Ǥ Š—•ǡ   ‹• ‹–”‹ ƒ–‡Ž› ‘‡ –‡† ™‹–Š ‘–Š‡” ’ƒ–Š™ƒ›• ‘ˆ ‡‡”‰› ‡–ƒ„‘Ž‹•Ǥ ‘” ƒŽ‘•– ƒŽŽ ‡œ›‡• ‘ˆ –Š‡ ’ƒ–Š™ƒ›ǡ ‹„‘” †‡ˆ‹ ‹‡ ‹‡• ƒ••‘ ‹ƒ–‡† ™‹–Š Š—ƒ †‹•‡ƒ•‡ Šƒ˜‡ „‡‡ ‹†‡–‹ˆ‹‡† ȏͳǡʹȐǤ ŽŽ –Š‡•‡ †‹•‘”†‡”• •Šƒ”‡ –Š‡ ”‹• ‘ˆ ǮŠ›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒǯǡ ƒ Ž‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰ ‘†‹–‹‘ ™‹–Š Ž‘™ „Ž‘‘† Ž‡˜‡Ž•‘ˆ‰Ž— ‘•‡ƒ†‡–‘‡„‘†‹‡•ȏͳǡʹȐǤŠ‡•‡˜‡”‹–›‘ˆ–Š‡†‹•‡ƒ•‡•›’–‘•†‡’‡†•‘ ™Š‹ Š‡œ›‡‹•‹••‹‰ǡ™Š‡–Š‡”–Š‡”‡‹•”‡•‹†—ƒŽƒ –‹˜‹–›ǡ„—––Š‡”‡‹•ƒŽ•‘ƒ—‡š’Žƒ‹‡† ˜ƒ”‹ƒ„‹Ž‹–›„‡–™‡‡‹†‹˜‹†—ƒŽ•Ǥ ‘”‡šƒ’Ž‡ǡ•‘‡ Š‹Ž†”‡™‹–ŠƒŽ‘••Ǧ‘ˆǦˆ— –‹‘—–ƒ–‹‘ ‹ –Š‡ ‡œ›‡ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌ ’”‡•‡– ™‹–Š ƒ Ž‹ˆ‡Ǧ –Š”‡ƒ–‡‹‰ †”‘’ ‹ „Ž‘‘† ‰Ž— ‘•‡ǡ ™Š‹Ž‡ ‘–Š‡”• ™‹–Š –Š‡ •ƒ‡ —–ƒ–‹‘ ‡˜‡” •Š‘™ ƒ› ’Š‡‘–›’‡ ȏ͵Ȃ͸ȐǤ ‘”‡‘˜‡”ǡ Ǧ†‡ˆ‹ ‹‡– Š‹Ž†”‡ †‘ ‘š‹†‹œ‡ ‡†‹—Ǧ Šƒ‹ ˆƒ––› ƒ ‹†• ‘”ƒŽŽ› —†‡” ‘Ǧ ŠƒŽŽ‡‰‹‰ ‘†‹–‹‘• ȏ͹ȐǤ

(9) – •‡‡• –Š‡”‡ˆ‘”‡ –Šƒ– ‘– –Š‡ ‘š‹†ƒ–‹‘ ƒ’ƒ ‹–›ǡ „—– –Š‡ ƒ’ƒ„‹Ž‹–› –‘ —•‡ –Š‹• ƒ’ƒ ‹–› —†‡” ƒŽŽ ‹” —•–ƒ ‡•ǡ Š‡ ‡ –Š‡ ”‘„—•–‡••‘ˆ–Š‡ ’ƒ–Š™ƒ›ǡ‹• ‘’”‘‹•‡†‹–Š‡•‡’ƒ–‹‡–•Ǥ ‡ ‡–Ž›ǡ ™‡ ‘•–”— –‡† ƒ† ‡š’‡”‹‡–ƒŽŽ› ˜ƒŽ‹†ƒ–‡† ƒ †›ƒ‹  ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž ‘ˆ  ’ƒ–Š™ƒ›‹”ƒ–Ž‹˜‡”ȏͺȐǤ‘†‡Ž•‹—Žƒ–‹‘••Š‘™‡†–Šƒ––Š‡’ƒ–Š™ƒ›‹•‹–”‹•‹ ƒŽŽ› •‡•‹–‹˜‡–‘„‡ ‘‹‰‘˜‡”Ž‘ƒ†‡†™‹–Š‹–••—„•–”ƒ–‡’ƒŽ‹–‘›ŽǦ‘ǡƒ•‹š–‡‡Ǧ ƒ”„‘ȋͳ͸Ȍǡ •ƒ–—”ƒ–‡† ƒ ›ŽǦ‘ †‡”‹˜ƒ–‹˜‡ ‘ˆ –Š‡ †‹‡–ƒ”› ˆƒ––› ƒ ‹† ’ƒŽ‹–‹  ƒ ‹†Ǥ – Š‹‰Š •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•–Š‡   ˆŽ—š†‡ Ž‹‡†ǡ ‹–‡”‡†‹ƒ–‡ ƒ ›ŽǦ‘ ‡•–‡”• ƒ ——Žƒ–‡† ƒ†–Š‡ ƒ ›Ž ƒ””‹‡” ‘‡œ›‡  ȋ‘Ȍ ™ƒ• †‡’Ž‡–‡†Ǥ —„•‡“—‡– •‹—Žƒ–‹‘• ‘ˆ   ‹ Ǧ †‡ˆ‹ ‹‡– ‘—•‡ Ž‹˜‡” ȏͻȐǡ „ƒ•‡† ‘ †‡–ƒ‹Ž‡† ‡š’‡”‹‡–ƒŽ Šƒ”ƒ –‡”‹œƒ–‹‘ ‘ˆ –Š‡•‡ ‹ ‡ǡ •Š‘™‡† –Šƒ– –Š‡ ‡œ›‡ †‡ˆ‹ ‹‡ › ”‡†— ‡† –Š‡ –‘Ž‡”ƒ–‡† •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘ ƒ† ƒ‰‰”ƒ˜ƒ–‡†–Š‡”‹•‘ˆ‘˜‡”Ž‘ƒ†ȏͻȐǤ‡Š›’‘–Š‡•‹œ‡–Šƒ–‹’ƒ–‹‡–•™‹–Šƒ †‹•‘”†‡”ƒ •‹‹Žƒ” ’Š‡‘‡‘ ƒ› „‡ ‹†— ‡† †—”‹‰ ˆƒ•–‹‰ǡ †—‡ –‘ –Š‡ ”‡ ”—‹–‡– ‘ˆ ˆƒ––› ƒ ‹†• ˆ”‘ ƒ†‹’‘•‡ •–‘”‡•ǡ ’‘••‹„Ž› ‹ ‘„‹ƒ–‹‘ ™‹–Š ƒ ‹†‡–ƒŽ ’‡”–—”„ƒ–‹‘• ‘ˆ ‘–Š‡” ‘Ǧ †‡’‡†‡–’”‘ ‡••‡•Ǥ‡Žƒ–‡†–‘–Š‹•ǡ‹–Šƒ•„‡‡’”‘’‘•‡†–Šƒ–ƒ‹„ƒŽƒ ‡„‡–™‡‡ˆƒ––› ƒ ‹† —’–ƒ‡ ‹–‘ –Š‡ ‹–‘ Š‘†”‹ƒ ƒ†  ǡ ƒ ——Žƒ–‹‘ ‘ˆ   ‹–‡”‡†‹ƒ–‡•ǡ ƒ† ‹ ‘’Ž‡–‡ Ž‹‡ƒ––Š‡„ƒ•‹•‘ˆ‹•—Ž‹”‡•‹•–ƒ ‡‹–Š‡—• Ž‡ȏͳͲȐǤ‘‰‡–‘”‡‹•‹‰Š– ‹–‘–Š‡‡ Šƒ‹••—†‡”Ž›‹‰–Š‡•‡†‹ˆˆ‡”‡–†‹•‡ƒ•‡•ǡ‹–‹•‹’‘”–ƒ––‘—†‡”•–ƒ†Š‘™ –Š‡ ‹„ƒŽƒ ‡ ƒ”‹•‡•ǡ ƒ† –Š‡ ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž ‰‹˜‡• —• •‘‡ ˆ‹”•– Š‹–•ǡ ƒ• ‘—–Ž‹‡† „‡Ž‘™Ǥ •’‡ ‹ƒŽˆ‡ƒ–—”‡‘ˆ–Š‡ „‹‘ Š‡‹•–”›‹•–Š‡’”‘‹• —‹–›‘ˆ‹–•‡œ›‡•ǡ‡ƒ‹‰–Šƒ– ‡ƒ Š ‡œ›‡ ƒ–ƒŽ›œ‡• –Š‡ ‘˜‡”•‹‘ ‘ˆ ˆƒ––›Ǧƒ ‹† †‡”‹˜ƒ–‹˜‡• ‘ˆ ƒ ”ƒ‰‡ ‘ˆ Šƒ‹ Ž‡‰–Š• ȏʹǡͺȐǤš‹†ƒ–‹‘‘ˆƒŽ‘‰Ǧ Šƒ‹ˆƒ––›ƒ ‹†”‡“—‹”‡•—’–ƒ‡‹–‘–Š‡‹–‘ Š‘†”‹ƒŽƒ–”‹š„› –Š‡ƒ ›ŽǦ ƒ”‹–‹‡•Š—––Ž‡ƒ†—Ž–‹’Ž‡”‘—†•‘ˆ ȋ ‹‰ͳȌǤ

(10) ‡ƒ Š”‘—†ǡ–Š‡ƒ ›ŽǦ‘ ‡•–‡”‹••Š‘”–‡‡†„›–™‘ ƒ”„‘ƒ–‘•ƒ†‘‡ƒ ‡–›ŽǦ‘‘Ž‡ —Ž‡‹•’”‘†— ‡†Ǥƒ Š › Ž‡ ‘•‹•–• ‘ˆ ˆ‘—” ‘•‡ —–‹˜‡ ”‡ƒ –‹‘• ƒ–ƒŽ›œ‡† „› ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋǤǤ ͳǤ͵ǤͻͻǤ͵ ƒ†ǤǤͳǤ͵ǤͺǤͳȌǡ‡‘›ŽǦ‘Š›†”ƒ–ƒ•‡‘” ”‘–‘ƒ•‡ȋǡǤǤͶǤʹǤͳǤ͹Ͷƒ†ǤǤͶǤʹǤͳǤͳͷͲȌǡ Š›†”‘š›ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋǤǤͳǤͳǤͳǤʹͳͳƒ†ǤǤͳǤͳǤͳǤ͵ͷȌƒ†‡–‘ƒ ›ŽǦ‘–Š‹‘Žƒ•‡ ȋǤǤ ʹǤ͵ǤͳǤͳ͸ȌȌǤ ‘—” ‹•‘‡œ›‡• ȋ–Š‡ ‡”›Ǧ‘‰Ǧǡ ‘‰Ǧǡ ‡†‹—Ǧǡ ƒ† Š‘”–ǦŠƒ‹  ›ŽǦ ‘‡Š›†”‘‰‡ƒ•‡•ǡǡǡƒ†Ȍ ƒ–ƒŽ›œ‡–Š‡ˆ‹”•–”‡ƒ –‹‘‘ˆ–Š‡ › Ž‡ǡ ƒ ‡’–‹‰ƒ”ƒ‰‡‘ˆƒ ›ŽǦ‘•—„•–”ƒ–‡•‘ˆ†‹ˆˆ‡”‡–ǡ‘˜‡”Žƒ’’‹‰ Šƒ‹Ž‡‰–Š•Ǥ Ͷʹ.

(11) The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation. Malonyl-CoA Palmitoyl-CoA Palmitoyl-carnitine carnitine CoASH carnitine. 2. CPT1 CACT. C16. CPT2. C4-C16. Mitochondrion. C4-C16. carnitine. CoASH carnitine. Palmitoyl-carnitine Acyl-CoA. MCKAT. Acetyl-CoA. SCAD. MCAD. LCAD. VLCAD. C4-C16. CoASH. C4-C6. C4-C12. C8-C16. C12-C16. Enoyl-CoA. Ketoacyl-CoA. MSCHAD C4-C16. NADH + H+ C4-ketoacyl-CoA NAD+. CROT C4-C16. FAD FADH2. Acetyl-CoA NADH + H+ C4-ketoacyl-CoA NAD+ CoASH. MTP C8-C16. Hydroxyacyl-CoA.  Fig 1. Schematic representation of the modelled mFAO pathway in rat. ‡–ƒ„‘Ž‹–‡•‹„Ž—‡ƒ”‡ˆ‹š‡† ’ƒ”ƒ‡–‡”•ǡ™Š‹Ž‡‘–Š‡”‡–ƒ„‘Ž‹–‡•ƒ”‡ˆ”‡‡˜ƒ”‹ƒ„Ž‡•ǤŠ‡•—‘ˆ˜ƒ”‹ƒ„Ž‡‘‡•–‡”•ƒ†ˆ”‡‡‘‹•ƒ ‘•‡”˜‡† ‘‹‡–›Ǥ ”‡‡ǣ ‡œ›‡• ’ƒ”–‹ ‹’ƒ–‹‰ ‹ –Š‡ ƒ”‹–‹‡ › Ž‡Ǣ ’—”’Ž‡ǣ ‡œ›‡• ’ƒ”–‹ ‹’ƒ–‹‰ ‹ –Š‡•Š‘”–Ǧ Šƒ‹„”ƒ ŠǢ‰”‡›ǣ‡œ›‡•’ƒ”–‹ ‹’ƒ–‹‰‹–Š‡‡†‹—Ǧƒ†Ž‘‰Ǧ Šƒ‹„”ƒ ŠǤŽŽ’”‘ ‡••‡• ƒ”‡”‡˜‡”•‹„Ž‡ƒ†–Š‡•‹œ‡‘ˆ–Š‡ƒ””‘™Š‡ƒ†•‹†‹ ƒ–‡•–Š‡‡–†‹”‡ –‹‘‘ˆ–Š‡ˆŽ—šǤ Š‹•‘†‡Ž‹•‡šƒ –Ž› –Š‡•ƒ‡ƒ•’—„Ž‹•Š‡†„‡ˆ‘”‡ȏͺȐǡ™‹–Š‘—–‘†‹ˆ‹ ƒ–‹‘•Ǥ.  ‹–‘ Š‘†”‹ƒŽ ”‹ˆ— –‹‘ƒŽ ”‘–‡‹ ȋȌ –Š‡ ƒ–ƒŽ›œ‡• –Š‡ ‡š– –Š”‡‡ ”‡ƒ –‹‘• ˆ‘” •—„•–”ƒ–‡•‘ˆͺ ƒ”„‘ƒ–‘•‘”Ž‘‰‡”ǡ™Š‹Ž‡•Š‘”–‡”•—„•–”ƒ–‡•ȋ͸ƒ†ͶȌƒ”‡‡š Ž—•‹˜‡Ž› ‘˜‡”–‡† „› ƒ †‹•–‹ – ǡ ‡†‹—Ȁ•Š‘”–Ǧ Šƒ‹ Š›†”‘š›ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȀ Ȍǡ ƒ† ‡†‹—Ǧ Šƒ‹ ‡–‘ƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ ȋȌǤ ‹ ‡ ‡ƒ Š ‡œ›‡ ‹• ’”‘‹• —‘—• ƒ† ƒ–ƒŽ›œ‡• ”‡ƒ –‹‘• ‹ —Ž–‹’Ž‡ ”‘—†•ǡ –Š‡ †‹ˆˆ‡”‡– •—„•–”ƒ–‡• ƒ† ”‡•—Ž–‹‰’”‘†— –• ‘’‡–‡ˆ‘”„‹†‹‰–‘–Š‡ƒ –‹˜‡•‹–‡Ǥ

(12) –Š‡ ‘•–”— –‹‘‘ˆ–Š‡‘†‡ŽȏͺȐǡ–Š‡–‘–ƒŽ ‘ ‡–”ƒ–‹‘‘ˆ‘Šƒ•„‡‡ ‘•‹†‡”‡†ƒ•ƒ ‘•‡”˜‡†‘‹‡–›ǡ‹Ǥ‡Ǥˆ”‡‡‘ƒ†‘‡•–‡”•ƒ”‡‘Ž›‹–‡” ‘˜‡”–‡†ƒ†‘•›–Š‡•‹•‹• ‘—–•‹†‡ –Š‡ • ‘’‡ ‘ˆ –Š‡ ‘†‡ŽǤ ŽŽ ”‡ƒ –‹‘• ™‡”‡ –”‡ƒ–‡† ƒ• ”‡˜‡”•‹„Ž‡ ƒ† ’”‘†— – •‡•‹–‹˜‡Ǥƒ‹‰‹–‘ƒ ‘—––Š‡’”‘‹• —‹–›‘ˆ–Š‡‡œ›‡•ǡ”‡˜‡”•‹„Ž‡‹ Šƒ‡Ž‹•Ǧ‡–‡ ‡“—ƒ–‹‘•™‡”‡‘†‹ˆ‹‡†–‘‹ Ž—†‡ƒŽŽ‘‡•–‡”•–Šƒ–ƒ‡œ›‡ ƒ’”‘†— ‡‘” ‘•—‡ ƒ• ‘’‡–‹–‹˜‡ ‹Š‹„‹–‘”•ǡ ™‹–Š –Š‡‹” ƒˆˆ‹‹–› ‘•–ƒ– Km •‡”˜‹‰ ƒ• ‘’‡–‹–‹˜‡ ‹Š‹„‹–‹‘ ‘•–ƒ–Ǥ  ‡šƒ’Ž‡ ‘ˆ •— Š ƒ ”ƒ–‡ ‡“—ƒ–‹‘ǡ ˆ‘” –Š‡ ‘˜‡”•‹‘ ‘ˆ ͳ͸Ǧƒ ›ŽǦ‘ „› –Š‡ ‡œ›‡ǡ‹•ǣ ሾಷಲವሿ ሾ಴భలషೌ೎೤೗ష಴೚ಲሿ ሾ಴భలష೐೙೚೤೗ష಴೚ಲሿ ሾಷಲವಹమሿ ή ି ή ಼೘ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ಼೘ೇಽ಴ಲವǡಷಲವ ಼೘ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ಼೘ ೇಽ಴ಲವǡಷಲವή‫ܭ‬. ௦௙ೇಽ಴ಲವǡ಴భల ή௏೘ೌೣǡೇಽ಴ಲವ ήቌ. ቍ. ݁‫ݍ‬ǡܸ‫ܦܣܥܮ‬. ‫ݒ‬௏௅஼஺஽ǡ஼ଵ଺ ൌ. ሾಷಲವሿ ሾ಴భలషೌ೎೤೗ష಴೚ಲሿ ሾ಴భలష೐೙೚೤೗ష಴೚ಲሿ ሾ಴೙షೌ೎೤೗ష಴೚ಲሿ ሾ಴೙ష೐೙೚೤೗ష಴೚ಲሿ ሾಷಲವಹమ ሿ ‫ۊ‬ήቆଵା ା ା σ೙సభర ା ା ቇ ೙సభమ಼೘ ಼೘ೇಽ಴ಲವǡಷಲವ ಼೘ೇಽ಴ಲವǡಷಲವಹ ಼೘ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ಼೘ೇಽ಴ಲವǡ಴భలష೐೙೚೤೗ష಴೚ಲ ೇಽ಴ಲವǡ಴೙షೌ೎೤೗ష಴೚ಲ ಼೘ೇಽ಴ಲವǡ಴೙ష೐೙೚೤೗ష಴೚ಲ ᇣᇧᇧᇧᇧᇧᇧᇧᇧᇤᇧᇧᇧᇧᇧᇧᇧᇧᇥ ᇣᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇤᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇥ మ ೔೙೓೔್೔೟೔೚೙್೤ೝ೐ೌ೎೟೔೚೙೛ೝ೚೏ೠ೎೟ ೔೙೓೔್೔೟೔೚೙್೤ೞೠ್ೞ೟ೝೌ೟೐ೞೌ೙೏೛ೝ೚೏ೠ೎೟ೞ೚೑೚೟೓೐ೝ೎೓ೌ೔೙೗೐೙೒೟೓ೞ‫ی‬. ‫ۇ‬ଵା ‫ۉ‬. ȋͳȌ

(13)  –Š‹• ‡“—ƒ–‹‘ǡ –Š‡ ‡–ƒ„‘Ž‹–‡• ‹ –Š‡ ‰”‡› „‘š ƒ”‡ –Š‡ •—„•–”ƒ–‡• ƒ† ’”‘†— –• ‘ˆ ‘’‡–‹‰”‡ƒ –‹‘•ƒ†ƒ –ƒ•‹Š‹„‹–‘”•Ǥ

(14) ˆ–Š‹•’”‘‹• —‹–›™ƒ•”‡‘˜‡†in silico„›–ƒ‹‰ ‘—–ƒŽŽ ‘’‡–‹–‹˜‡‹Š‹„‹–‹‘–‡”•‡š ‡’–†‹”‡ –’”‘†— –‹Š‹„‹–‹‘ȋ‹Ǥ‡ǤƒŽŽ–‡”•‹–Š‡‰”‡› „‘šȌǡ –Š‡ ˆŽ—š ‡šŠ‹„‹–‡† ‘”ƒŽ •ƒ–—”ƒ–‹‘ „‡Šƒ˜‹‘—”Ǥ Š‹• •—‰‰‡•–• –Šƒ– –Š‡ ‡œ›‡. Ͷ͵.

(15) Chapter 2 ’”‘‹• —‹–›ƒ†–Š‡”‡•—Ž–‹‰‡–ƒ„‘Ž‹  ‘’‡–‹–‹‘ ƒ—•‡–Š‡ˆŽ—š†‡ Ž‹‡ƒ–Š‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘• ȏͺǡͻȐǤ How ‘’‡–‹–‹‘ ‡†‹ƒ–‡• –Š‹• ‡‡”‰‡– ƒ† ’‘–‡–‹ƒŽŽ› †‡Ž‡–‡”‹‘—• ˆŽ—š†‡ Ž‹‡’Š‡‘‡‘Šƒ•ǡŠ‘™‡˜‡”ǡ‘–„‡‡•–—†‹‡†›‡–Ǥ‘„‡ƒ„Ž‡–‘‡š’Ž‘”‡‹–•”‘Ž‡‹ †‹•‡ƒ•‡ ƒ† ‰‡– ƒ „‡––‡” ‹•‹‰Š– ‹–‘ –Š‡ ‘”ƒŽ ˆ— –‹‘‹‰ ‘ˆ –Š‡   ’ƒ–Š™ƒ›ǡ ‹– ‹• ‹’‘”–ƒ– –‘ —”ƒ˜‡Ž –Š‡ ‘’Ž‡–‡ •‡“—‡ ‡ ‘ˆ ‡˜‡–•Ǥ Š‹• ™‹ŽŽ „‡ ”— ‹ƒŽ –‘ —†‡”•–ƒ† Š‘™ ƒ Š‡ƒŽ–Š›   ‹•’”‘–‡ –‡† ƒ‰ƒ‹•– –Š‹• ’Š‡‘‡‘ ‘ –Š‡ ‘‡ Šƒ† ƒ† Š‘™ –Š‡ ”‹•ˆ‘”’ƒ–‹‡–• ƒ„‡‹‹‹œ‡†‘–Š‡‘–Š‡”Šƒ†Ǥ Š‡ ƒ‹ ‘ˆ –Š‹• •–—†› ‹• –‘ †‹••‡ – –Š‡ ‡ Šƒ‹• ˜‹ƒ ™Š‹ Š ‡œ›‡ ’”‘‹• —‹–› ƒ† –Š‡ ”‡•—Ž–‹‰ ‡–ƒ„‘Ž‹  ‘’‡–‹–‹‘ ‹†— ‡• ˆŽ—š †‡ Ž‹‡ ƒ– Š‹‰Š •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•Ǥ ’‡ ‹ˆ‹ ƒŽŽ›ǡ™‡™‹ŽŽ†‹••‡ –ȋ‹Ȍ–Š‡”‘Ž‡‘ˆ’”‘‹• —‹–›‘ˆ‡ƒ Š‹†‹˜‹†—ƒŽ‡œ›‡ƒ†ȋ‹‹Ȍ–Š‡ ”‘Ž‡‘ˆ‡ƒ Š‘ˆ–Š‡‡–ƒ„‘Ž‹–‡•‹–Š‡‹–‡”ƒŽ”‡‰—Žƒ–‹‘‘ˆ–Š‡’ƒ–Š™ƒ›Ǥ‘–Š‹•‡†™‡—•‡ ‘’—–ƒ–‹‘ƒŽ ‘†‡ŽŽ‹‰ ƒ† ‡–ƒ„‘Ž‹  ‘–”‘Ž ƒƒŽ›•‹• ȏͳͳȐǡ ƒ† “—ƒ–‹ˆ› –Š‡ ”‡‰—Žƒ–‘”› •–”‡‰–Š‘ˆ–Š‡‡–ƒ„‘Ž‹–‡•„›ƒ’”‡˜‹‘—•Ž›’—„Ž‹•Š‡†‡–Š‘†ȏͳʹȐǤ‡™‹ŽŽ”‡˜‡ƒŽƒ˜‹ ‹‘—• › Ž‡ –Šƒ– •–ƒ”–• ƒ– –Š‡ ˜‡”› ‡† ‘ˆ –Š‡ ’ƒ–Š™ƒ› ƒ”‘—†  ƒ† ‹• ƒ’Ž‹ˆ‹‡† ƒ† –”ƒ•‹––‡† –Š”‘—‰Š‘—– –Š‡ ‡–‹”‡ ’ƒ–Š™ƒ›Ǥ ‡ ™‹ŽŽ †‹• —•• Š‘™ ƒ–—”ƒŽ ”‡‰—Žƒ–‹‘ ƒ† ’”‘’‡” ‡„‡††‹‰ ‹ •—””‘—†‹‰ ’ƒ–Š™ƒ›• ƒ› ’”‡˜‡– –Š‹• ’Š‡‘‡‘ ‹ Š‡ƒŽ–Š› ‹†‹˜‹†—ƒŽ•ƒ†Š‘™‹–ƒ›’‘–‡–‹ƒŽŽ›„‡ƒ––‡—ƒ–‡†‘”ƒ‰‰”ƒ˜ƒ–‡†‹†‹•‡ƒ•‡Ǥ . Results Description of the phenotype

(16)  ‘—” ’”‡˜‹‘—• •–—†‹‡• ™‡ •Š‘™‡† –Šƒ– –Š‡ ˆŽ—š †‡ Ž‹‡ ƒ– Š‹‰Š ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•ȋȏ’ƒŽ‹–‘›ŽǦ‘ȐȌȋ ‹‰ʹȌ™ƒ•†—‡–‘–Š‡†‡’Ž‡–‹‘‘ˆ–Š‡ˆ”‡‡‘’‘‘Žǡ ™Š‹ Š”‡•—Ž–‡†ˆ”‘–Š‡ƒ ——Žƒ–‹‘‘ˆ‘‡•–‡”•ȋ ‹‰ʹȌǤ B 2.0. 6000. 1.6. 5000. free CoA. [Metabolite] (ȝM). Juptake (μmol.min-1.gProtein-1). A. 1.2. 0.8 0.4. 4000. Intermediate CoA esters. 3000. C6/C4 CoA esters. 2000. C4-CoA esters. 1000. C6-CoA esters. 0. 0.0 0. 0. 250. D. 8. vmtpC8 vmtpC10 vmtpC12 vmtpC14 vmtpC16. 7 6 5 4 3. 2 1. 8. J (μmol.min-1.gProtein-1). J (μmol.min-1.gProtein-1). C. 50 100 150 200 [Palmitoyl-CoA]CYT (ȝM). 50 100 150 200 250 [Palmitoyl-CoA]CYT (ȝM). vmckatC4 vmckatC6 vmckatC8 vmckatC10 vmckatC12 vmckatC14 vmckatC16. 7 6 5 4 3 2 1 0. 0. 0. 50 100 150 200 250 [Palmitoyl-CoA]CYT ȝ0

(17). 0. 50 100 150 200 [Palmitoyl-CoA]CYT ȝ0

(18). 250. . Fig 2. Simulated steady-state fluxes and concentrations in the mFAO model. (A) Š‡ ‡ˆˆ‡ – ‘ˆ ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ ȋȏ’ƒŽ‹–‘›ŽǦ‘ȐȌ ‘ –Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ ˆŽ—šǤ Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ —’–ƒ‡ ˆŽ—š‘ˆ ’ƒŽ‹–‘›ŽǦ‘ȋ —’–ƒ‡Ȍ‹•’Ž‘––‡†ȋ ƒŽ —Žƒ–‡†ƒ•–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—š ‘ˆ ’ƒŽ‹–‘›ŽǦ ƒ”‹–‹‡. ͶͶ.

(19) The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation –Š”‘—‰Šǡ‹Ǥ‡Ǥ–Š‡—’–ƒ‡‘ˆ’ƒŽ‹–‘›ŽǦ ƒ”‹–‹‡‹–‘–Š‡‹–‘ Š‘†”‹ƒȌǡ‹ ‘–”ƒ•––‘˜ƒ—‡et al. ʹͲͳ͵ ȏͺȐ ‹ ™Š‹ Š –Š‡   ˆŽ—š ™ƒ• ’Ž‘––‡†Ǥ Š‡ –™‘ ƒ”‡ —‹“—‡Ž› Ž‹‡† ™‹–Š –Š‡ Žƒ––‡” „‡‹‰ ͹Ǧˆ‘Ž† Š‹‰Š‡”Ǥ (B) Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ ‘ ‡–”ƒ–‹‘• ‘ˆ ˆ”‡‡ ‘ ȋ‹†‡–‹ ƒŽ –‘ †ƒ–ƒ ˆ”‘ ȏͺȐȌǡ –Š‡ •— ‘ˆ ƒŽŽ ‹–‡”‡†‹ƒ–‡ ‘ ‡•–‡”•ǡ –Š‡ •— ‘ˆ ƒŽŽ ͶǦ ƒ† ͸ ‘ ‡•–‡”• ƒ† –Š‡ •—„•‡– ‘ˆ ͶǦ ƒ† ͸ ‘ ‡•–‡”• ȋ‡™ ”‡•—Ž–•ȌǤ (C-D) ‹•–”‹„—–‹‘ ‘ˆ •–‡ƒ†›Ǧ•–ƒ–‡ ˆŽ—š‡• ȋ Ȍ ‘ˆ †‹ˆˆ‡”‡– Šƒ‹ǦŽ‡‰–Š •—„•–”ƒ–‡• –Š”‘—‰Š ƒ†ǡ”‡•’‡ –‹˜‡Ž›ǤŽ›–Š‡ Šƒ‹Ž‡‰–Š•™Š‹ Šƒ”‡ ‘˜‡”–‡†„›–Š‡•‡‡œ›‡•ȋ ˆǤ ‹‰ͳȌƒ”‡ ‹ Ž—†‡†Ǥ. ‡ˆ‹”•– ƒŽ —Žƒ–‡†™Š‹ Š‘‡•–‡”•ƒ ——Žƒ–‡†ǡƒ†–Š‡•‡–—”‡†‘—––‘„‡’”‡†‘‹ƒ–Ž› –Š‡ ‘ ‡•–‡”• ‘ˆ Šƒ‹ Ž‡‰–Š• Ͷ ƒ† ͸ ȋ ‹‰ ʹȌǤ ”‘ ‡‘›ŽǦ‘ ‘™ƒ”† –Š‡   ’ƒ–Š™ƒ› ‘•‹•–• ‘ˆ –™‘ „”ƒ Š‡•ǣ –Š‡  „”ƒ Š ȋ‰”‡› ‹ ‹‰ ͳȌǡ ™Š‹ Š ‡š Ž—•‹˜‡Ž› ‘˜‡”–• –Š‡ Ž‘‰‡” Šƒ‹ Ž‡‰–Š• ‘ˆ ͺ ƒ† Š‹‰Š‡”ǡ ƒ† –Š‡ „”‘ƒ† •’‡ ‹ˆ‹ ‹–› Ǧ Ȁ Ǧ „”ƒ Š ȋ’—”’Ž‡ ‹ ‹‰ ͳȌǡ ™Š‹ Š ‘˜‡”• –Š‡ ‘’Ž‡–‡ ”ƒ‰‡ ‘ˆ Šƒ‹ Ž‡‰–Š•ˆ”‘Ͷ–Š”‘—‰Šͳ͸ǤŠ—•ǡ–Š‡ͶǦƒ†͸‘‡•–‡”• ƒ‘Ž›„‡‘š‹†‹œ‡†˜‹ƒ–Š‡ ǦȀ Ǧ „”ƒ Šǡ ™Š‹Ž‡ Ž‘‰‡” Šƒ‹ Ž‡‰–Š• ƒ „‡ ‘š‹†‹œ‡† ˜‹ƒ „‘–Š „”ƒ Š‡•Ǥ ‘™‡˜‡”ǡ –Š‡ ƒ –—ƒŽ ˆŽ—š ‘ˆ ͺ ƒ† Ž‘‰‡” ‡‘›ŽǦ‘ ‡•–‡”• ™‡– ƒŽ‘•– ‡š Ž—•‹˜‡Ž› ˜‹ƒ –Š‡  ”‘—–‡ ȋ ‹‰ ʹȌǤ Ž› Ͷ ƒ† ͸ ‘ ‡•–‡”•ǡ ™Š‹ Š Šƒ† ‘ ‘–Š‡” ‘’–‹‘ǡ–‘‘–Š‡ǦȀ Ǧ„”ƒ Šȋ ‹‰ʹȌǤŠ‡”‡ˆ‘”‡ǡ™‡”‡ˆ‡”–‘–Š‡Žƒ––‡” ƒ• –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ ŠǤ Š‡ ˆƒ – –Šƒ– ’”‡ ‹•‡Ž› –Š‡ ‡•–‡”• ‹ –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Š ƒ ——Žƒ–‡†™ƒ•ƒˆ‹”•–‹†‹ ƒ–‹‘ˆ‘”ƒ„‘––Ž‡‡ ‹–Š‹•„”ƒ ŠǤ. The flux decline originates at the promiscuous MCKAT enzyme ‘ ‰ƒ‹ ‘”‡ ‹•‹‰Š– ‹–‘ –Š‡ ‡ Šƒ‹• ˜‹ƒ ™Š‹ Š ‡œ›‡ ’”‘‹• —‹–› ‹†— ‡• –Š‡ ˆŽ—š †‡ Ž‹‡ǡ ™‡ ˆ‹”•– †‹••‡ –‡† –Š‡ ”‘Ž‡ ‘ˆ ’”‘‹• —‹–› ‘ˆ ‡ƒ Š ‹†‹˜‹†—ƒŽ ‡œ›‡Ǥ ‘ –Š‹• ‡†ǡ ‡œ›‡ ’”‘‹• —‹–› ™ƒ• ”‡‘˜‡† ‘’—–ƒ–‹‘ƒŽŽ› Ȃ ‡‹–Š‡” ˆ”‘ ƒŽŽ ‡œ›‡• •‹—Ž–ƒ‡‘—•Ž› ‘” ˆ”‘ ‡ƒ Š ‡œ›‡ ‹†‹˜‹†—ƒŽŽ› Ȃ „› ’ƒ”–‹–‹‘‹‰ ƒ ‡œ›‡ ’‘‘Ž –‘ †‹•–‹ –ˆ”ƒ –‹‘•†‡†‹ ƒ–‡†–‘ƒ•‹‰Ž‡ Šƒ‹Ž‡‰–Šȋ•‡‡‡–Š‘†•ȌǤ

(20) –Š‡‘Ǧ’”‘‹• —‘—• ”ƒ–‡ ‡“—ƒ–‹‘ǡ –Š‡ ‡œ›‡ ™ƒ• •–‹ŽŽ ‹Š‹„‹–‡† „› ‹–• ‘™ ’”‘†— –ǡ „—– ‘– „› ’”‘†— –• ƒ† •—„•–”ƒ–‡• ‘ˆ ‘–Š‡” Šƒ‹ Ž‡‰–Š•Ǥ – –Š‡ •ƒ‡ –‹‡ –Š‡ –‘–ƒŽ ƒ–ƒŽ›–‹  ƒ’ƒ ‹–› ȋVmaxȌ ™ƒ• ’ƒ”–‹–‹‘‡† ‘˜‡” –Š‡ †‹ˆˆ‡”‡– ”‡ƒ –‹‘• ƒ–ƒŽ›œ‡† „› ‡ƒ Š ‡œ›‡ ȋ•‡‡ ‡–Š‘†• ˆ‘” ƒ ƒ–Š‡ƒ–‹ ƒŽ †‡• ”‹’–‹‘ȌǤ Š‡ ˆŽ—š †‡ Ž‹‡ ‹ –Š‡ ’—„Ž‹•Š‡† ‘†‡Ž ™‹–Š ˆ—ŽŽ ’”‘‹• —‹–› ™ƒ• ’Ž‘––‡† ƒ• ƒ ”‡ˆ‡”‡ ‡ ȋ ‹‰ ͵ȌǤ

(21) ˆ ’”‘‹• —‹–› ™ƒ• ”‡‘˜‡† ˆ”‘ ƒŽŽ ‡œ›‡• •‹—Ž–ƒ‡‘—•Ž›ǡ–Š‡ˆŽ—š†‹†‘–†‡ Ž‹‡ƒ›‘”‡ǡ„—–ƒ’’”‘ƒ Š‡†ƒƒš‹—ƒ–•ƒ–—”ƒ–‹‰ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ ȋ ‹‰ ͵ȌǤ

(22)  ‘–”ƒ•–ǡ ”‡‘˜‹‰ –Š‡ ’”‘‹• —‹–› ˆ”‘ ƒ› ‡œ›‡ ‹†‹˜‹†—ƒŽŽ› †‹† ‘– ’”‡˜‡– ˆŽ—š †‡ Ž‹‡ ȋˆ‹‰—”‡  ‹ ͳ ‹‰Ȍǡ ‡š ‡’– ™Š‡ ’”‘‹• —‹–›™ƒ•”‡‘˜‡†ˆ”‘–Š‡”‡ƒ –‹‘ȋ ‹‰͵ȌǤ‘˜‡”•‡Ž›ǡ™Š‡’”‘‹• —‹–› ™ƒ•”‡‘˜‡†ˆ”‘ƒŽŽ‡œ›‡•exceptǡ–Š‡ˆŽ—š†”‘’’‡†‡˜‡‘”‡•–‡‡’Ž›–Šƒ‹ –Š‡‘”‹‰‹ƒŽ‘†‡Ž™‹–Šˆ—ŽŽ’”‘‹• —‹–›ȋ ‹‰͵ȌǤŠ‡•‡”‡•—Ž–••Š‘™–Šƒ–Ȃ—†‡”–Š‡‰‹˜‡ ‘†‹–‹‘•Ǧ’”‘‹• —‹–›‘ˆ‹•„‘–Š‡ ‡••ƒ”›ƒ†•—ˆˆ‹ ‹‡–ˆ‘”–Š‡ˆŽ—š†‡ Ž‹‡ƒ–ƒ ”‹–‹ ƒŽ ‘ ‡–”ƒ–‹‘ ‘ˆ ƒ„‘—– ͷͲ ρ ‘ˆ ’ƒŽ‹–‘›ŽǦ‘Ǥ ˆ ‘–‡ǡ ™Š‡ –Š‡ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘™ƒ•‹ ”‡ƒ•‡†–‘•—’”ƒ’Š›•‹‘Ž‘‰‹ ƒŽ ‘ ‡–”ƒ–‹‘•ƒ„‘˜‡ʹͷͲρǡ‡˜‡–Š‡ ‘†‡ŽŽƒ ‹‰’”‘‹• —‹–›‡šŠ‹„‹–‡†ƒˆŽ—š†‡ Ž‹‡ȋˆ‹‰—”‡‹ͳ ‹‰ȌǤ

(23) –Š‡‘†‡Ž ™‹–Š’”‘‹• —‹–›‹‘Ž›ǡ–Š‡ˆŽ—š†”‘’’‡†ƒ–ƒŠ‹‰Š‡”’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ –Šƒ ‹ –Š‡ ‘†‡Ž ™‹–Š ˆ—ŽŽ ’”‘‹• —‹–› ȋ ‹‰ ͵ȌǤ ‘‰‡–Š‡”ǡ –Š‹• †‡‘•–”ƒ–‡• –Šƒ– –Š‡ ’”‘‹• —‹–› ‘ˆ –Š‡ ‘–Š‡” ‡œ›‡• ‘—Ž† ‹ ’”‹ ‹’Ž‡ ‘–”‹„—–‡ –‘ ˆŽ—š †‡ Ž‹‡ǡ „—– —†‡” ’Š›•‹‘Ž‘‰‹ ƒŽ ‘†‹–‹‘•–Š‡’”‘‹• —‹–›‘ˆ‹•„›ˆƒ”–Š‡‘•–‹’‘”–ƒ–Ǥ Ͷͷ. 2.

(24) Chapter 2 B 4. 2.0 1.6. Published model. 1.2. No promiscuity at MCKAT. 0.8. Flux control coefficient (-). No promiscuity except at MCKAT. 0.4. No promiscuity. CPT1 2 MCAD. 0 SCAD -2 MCKAT -4. 0.0. 0. 50 100 150 200 250 [Palmitoyl-CoA]CYT ȝ0

(25). C. D. J. Rpuptake -2.5. sfcpt1C16 Vcpt1 CarCYT Kmcpt1CarCYT CoACYT Kmcpt1CoACYT C16AcylCoACYT Kmcpt1C16AcylCoACYT Keqcpt1 Keqcact Vfcact sfcactC16 [NAD+]/[NADH] Vmckat Keqmschad. 0.0. 50 100 150 200 250 [Palmitoyl-CoA]CYT (ȝM). E. J. -2.5. 2.5. Rpuptake 0.0. 2.5. Keqmschad Vmckat [NAD+]/[NADH] CoACYT Keqcrot sfmckatC4 sfcpt1C16 Vcpt1 KmmckatC4ketoacylCoA Kmcpt1CarCYT CarCYT Kmcpt1CoACYT C16AcylCoACYT KmmckatC4acylCoA KmmckatCoAMAT. Juptake. 0. Reaction-partitioned R[NAD+ ]/[NADH]. Juptake (μmol.min-1.gProtein-1). A. 3. vmschadC4 vmschadC6. 2. vmtpC8 vmtpC10. 1. vmtpC12 vmtpC14. 0. vmtpC16. -1 5. 15. 25 53.37 60. 250. [Palmitoyl-CoA]CYT (μM). . Fig 3. The role of the short-chain branch. (A) –‡ƒ†›Ǧ•–ƒ–‡ ˆŽ—š ˜‡”•—• ȏ’ƒŽ‹–‘›ŽǦ‘Ȑ ‹ –Š‡ ’—„Ž‹•Š‡†‘†‡Žȋ„Žƒ Ž‹‡Ȍǡƒ‘†‡Ž™‹–Š‘—–ƒ›‡œ›ƒ–‹ ’”‘‹• —‹–›ȋ„Žƒ †ƒ•Š‡†Ž‹‡ǡ”‡’”‘†— ‹‰ ”‡•—Ž–•ˆ”‘ȏͺȐƒ•ƒ”‡ˆ‡”‡ ‡Ȍǡƒ‘†‡Ž™‹–Š‘—–’”‘‹• —‹–›‘ˆȋ‰”‡‡Ž‹‡Ȍǡƒ†ˆ‹ƒŽŽ›ƒ‘†‡Ž ™‹–Š‘Ž›’”‘‹• —‹–›‘ˆǡ„—–‘–‘ˆƒ›‘ˆ–Š‡‘–Š‡”‡œ›‡•ȋ’—”’Ž‡Ž‹‡ȌǤ (B)Š‡ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–• ‹ –Š‡ ‘”‹‰‹ƒŽ ‘†‡Ž ƒ• ƒ ˆ— –‹‘ ‘ˆ ȏ’ƒŽ‹–‘›ŽǦ‘ȐǤ Ž› ‡œ›‡• ™‹–Š ƒ •—„•–ƒ–‹ƒŽ ‘–”‹„—–‹‘ƒ”‡‹ Ž—†‡†Ǣˆ‘”ƒ ‘’Ž‡–‡•‡–‘ˆˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•ǡ•‡‡ ˆ‹‰—”‡‹ʹ ‹‰Ǥ(C-D)‘’ ͳͷˆŽ—š”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•™‹–Š–Š‡Š‹‰Š‡•–ƒ„•‘Ž—–‡˜ƒŽ—‡•ƒ–ʹͷɊȋȌƒ†͸ͲɊȋȌ‘ˆȏ’ƒŽ‹–‘›ŽǦ ‘ȐǤŠ‡’ƒ”ƒ‡–‡”•p ˆ‘”™Š‹ Š–Š‡ˆŽ—š”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–™ƒ• ƒŽ —Žƒ–‡†‹•‹†‹ ƒ–‡†‘–Š‡Ǧƒš‹•Ǥ ƒ” ‰”‡› „ƒ”• ”‡’”‡•‡– ͳǦ”‡Žƒ–‡† ’ƒ”ƒ‡–‡”•ǡ ’‹ „ƒ”• ”‡’”‡•‡– Ȁ Ǧ”‡Žƒ–‡† ’ƒ”ƒ‡–‡”•ǡ ’—”’Ž‡ „ƒ”• ”‡’”‡•‡– Ǧ”‡Žƒ–‡† ’ƒ”ƒ‡–‡”• ƒ† –Š‡ Ž‹‰Š– „Ž—‡ „ƒ” ”‡’”‡•‡–• ƒ ”‘–‘ƒ•‡Ǧ”‡Žƒ–‡† ୎୳୮୲ୟ୩ୣ. ’ƒ”ƒ‡–‡”Ǥ(E) Ž—š”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–‘ˆȏΪȐȀȏ Ȑȋ ሾ୒୅ୈశሿȀሾ୒୅ୈୌሿ Ȍ’ƒ”–‹–‹‘‡†‹ ‘–”‹„—–‹‘• ௃. ‘ˆ‹†‹˜‹†—ƒŽΪǦ†‡’‡†‡–”‡ƒ –‹‘•ƒ–ͷ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•ƒ ‘”†‹‰–‘ ܴሾே஺஽ శ ሿȀሾே஺஽ுሿ ൌ ௩. ୎୳୮୲ୟ୩ୣ. ೔ σ௜ ‫ܥ‬௜௃ ή ߝሾே஺஽ శ ሿȀሾே஺஽ுሿ . (cf. Eq. 4). Š‡ ‘–”‹„—–‹‘•‘ˆȀ  ”‡ƒ –‹‘•ͺǦͳ͸–‘  ሾ୒୅ୈశ ሿȀሾ୒୅ୈୌሿ ™‡”‡. ‡‰Ž‹‰‹„Ž‡ƒ†–Š‡”‡ˆ‘”‡‘–•Š‘™‹–Š‡Ž‡‰‡†Ǥ. . MCKAT becomes the most important flux-controlling enzyme when the flux declines ƒŽ‹–‘›ŽǦ‘‹•–Š‡•—„•–”ƒ–‡‘ˆͳǡ–Š‡ˆ‹”•–‡œ›‡‘ˆ–Š‡–”ƒ•’‘”–•Š—––Ž‡ǡ ‘•‹•–‹‰ ‘ˆͳǡǡƒ†ʹȋ ‹‰ͳȌǤŠ‡†‡ Ž‹‡‘ˆˆŽ—šƒ•ƒˆ— –‹‘‘ˆ’ƒŽ‹–‘›ŽǦ‘•—‰‰‡•–• –Šƒ–ͳƒ –‹˜‹–›Šƒ•ƒ‡‰ƒ–‹˜‡ ‘–”‘Ž‘˜‡”–Š‡’ƒ–Š™ƒ›ˆŽ—šǤ‘‹˜‡•–‹‰ƒ–‡Š‘™–Š‡ˆŽ—š ‹• ‘–”‘ŽŽ‡†—†‡”–Š‡•‡ ‘†‹–‹‘•ǡ–Š‡ ‘–”‘Ž‡š‡”–‡†„›‡ƒ Š‡œ›‡‘˜‡”–Š‡ˆŽ—š™ƒ• “—ƒ–‹ˆ‹‡†Ǥ

(26)  ‡–ƒ„‘Ž‹  ‘–”‘Ž ƒŽ›•‹• ȏͳͳǡͳ͵ȂͳͷȐǡ –Š‡ ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ‫ܥ‬௜௃  ‘ˆ ƒ ‡œ›‡i‘˜‡”–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—šJ ‹•†‡ˆ‹‡†ƒ•ǣ ‫ܥ‬௜௃ ൌ. ௗ௟௡௃Ȁௗ௣ డ௟௡௩೔Τడ௣. ȋʹȌ. ‹™Š‹ Šp‹•ƒ’ƒ”ƒ‡–‡”–Šƒ–‘Ž›ƒˆˆ‡ –•–Š‡”ƒ–‡v‘ˆ‡œ›‡iǤˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ‡“—ƒŽ –‘ ͳ ‡ƒ•–Šƒ– –Š‡ †‹”‡ – ‡ˆˆ‡ – ‘ˆ p ȋ‡Ǥ‰Ǥ –Š‡ ‡œ›‡ ‘ ‡–”ƒ–‹‘ ‘” VmaxȌ ‘ –Š‡ ‡œ›‡‹•ˆ—ŽŽ›–”ƒ•Žƒ–‡†–‘–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—šǤ

(27) ˆ–Š‡ƒ„•‘Ž—–‡ ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•‘ˆ–Š‡ Ͷ͸.

(28) The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation ‘–Š‡” ‡œ›‡• ƒ”‡ Ž‘•‡ –‘ œ‡”‘ǡ –Š‹• ‹†‹ ƒ–‡• –Šƒ– –Š‡ ‡œ›‡ ‹• ‘’Ž‡–‡Ž› ”ƒ–‡Ǧ ‘–”‘ŽŽ‹‰Ǥ  Ž‘™ ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ‹†‹ ƒ–‡• –Šƒ– –Š‡ ’”‹ƒ”› ‡ˆˆ‡ – ‘ˆ p ‹• ‘—–‡”ƒ –‡† „› ƒ†ƒ’–ƒ–‹‘• ‘ˆ ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘•ǡ •— Š –Šƒ– –Š‡ ˆ‹ƒŽ ‡ˆˆ‡ – ‘ –Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ ˆŽ—š ‹• •ƒŽŽǤ Š‡ ”‡•—Ž–• ƒ”‡ •Š‘™ ‹ ‹‰ ͵Ǥ – Ž‘™ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘• ͳ ˆ—ŽŽ› ‘–”‘ŽŽ‡† –Š‡ ˆŽ—šǡ ƒ• ™ƒ• ƒ’’ƒ”‡– ˆ”‘ ‹–• ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ‡“—ƒŽ –‘ ͳǡ ™Š‹Ž‡ ƒŽŽ ‘–Š‡” ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–• ™‡”‡ ‡‰Ž‹‰‹„Ž‡ ȋ ‹‰ ͵ ƒ† ʹȌǤ–Š‹‰Š‡”’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•ǡŠ‘™‡˜‡”ǡ–Š‡ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–‘ˆͳ „‡ ƒ‡ •–”‘‰Ž› ‡‰ƒ–‹˜‡ǡ ‹†‡‡† ‹†‹ ƒ–‹‰ –Šƒ– ƒ •–‹—Žƒ–‹‘ ‘ˆ ‹–• ƒ–ƒŽ›–‹  ƒ’ƒ ‹–› ”‡†— ‡† –Š‡ ˆŽ—š •–‡‡’Ž›Ǥ  ‘”†‹‰ –‘ –Š‡ •—ƒ–‹‘ –Š‡‘”‡ ȏͳͳǡͳͶȐ –Š‡ •— ‘ˆ ƒŽŽ ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•‹•‡“—ƒŽ–‘ͳǡ‹’Ž›‹‰–Šƒ––Š‡‡‰ƒ–‹˜‡ ‘–”‘Ž‡š‡”–‡†„›ͳ•Š‘—Ž† „‡ ‘’‡•ƒ–‡†„›Š‹‰Š’‘•‹–‹˜‡ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•‘ˆ‘‡‘”‘”‡‘–Š‡”‡œ›‡•ǤŠ‡ ƒŒ‘”•Šƒ”‡‘ˆ–Š‹•’‘•‹–‹˜‡ˆŽ—š ‘–”‘Ž™ƒ•ƒ†‘’–‡†„›ǡƒ†ƒ‹‘”’ƒ”–„› ƒ†  ȋ ‹‰ ͵ ƒ† ʹȌǤ ‹••‡ –‹‰ –Š‡ ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ‘ˆ  ‹ –Šƒ– ‘ˆ ‹†‹˜‹†—ƒŽǡ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹  ”‡ƒ –‹‘• •Š‘™‡† –Šƒ– ‘Ž› ‹–• Ͷ ƒ† ͸ ”‡ƒ –‹‘• ‘–”‹„—–‡† •—„•–ƒ–‹ƒŽŽ› ȋˆ‹‰—”‡  ‹ ʹ ‹‰Ȍǡ ƒ‰ƒ‹ †‡‘•–”ƒ–‹‰ ƒ ’‹˜‘–ƒŽ ”‘Ž‡ ‘ˆ –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Š ‘ˆ –Š‡ ’ƒ–Š™ƒ›Ǥ –Š‡” ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–• ™‡”‡ •ƒŽŽ —†‡” ƒŽŽ ‘†‹–‹‘•ȋˆ‹‰—”‡‹ʹ ‹‰ȌǤ . The accumulation of intermediates is amplified by the short-chain branch ‡ –Š‡ ™‘†‡”‡† ™Š‡–Š‡” ‘–Š‡” ”‡ƒ –‹‘•ǡ „‡•‹†‡• ǡ ͳǡ ǡ ƒ†  ‘†—Žƒ–‡† –Š‡ ’Š‡‘–›’‡Ǥ Š‡”‡ˆ‘”‡ǡ ™‡ “—ƒ–‹ˆ‹‡† –Š‡ ˆŽ—š ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–• ˆ‘” ƒŽŽ ’ƒ”ƒ‡–‡”•p‹–Š‡‘†‡Žƒ––™‘ ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•ǣ„‡Ž‘™–Š‡ ”‹–‹ ƒŽ ‘ ‡–”ƒ–‹‘ˆ‘”ˆŽ—š†‡ Ž‹‡ȋʹͷɊȌƒ†„‡›‘†–Š‹• ”‹–‹ ƒŽ ‘ ‡–”ƒ–‹‘™Š‡–Š‡ˆŽ—š ‹•„‡Ž‘™‹–•ƒš‹— ƒ’ƒ ‹–›ȋ͸ͲɊȌǤŠ‡ˆŽ—š”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–ܴ௣௃ ȏͳͷȐ“—ƒ–‹ˆ‹‡•–Š‡ ”‡Žƒ–‹˜‡‡ˆˆ‡ –‘ˆƒ•ƒŽŽ Šƒ‰‡‘ˆƒ’ƒ”ƒ‡–‡”p‘–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—šƒ ‘”†‹‰–‘ǣ ܴ௣௃ ൌ  ݈݀݊‫ܬ‬Τ݈݀݊‫݌‬ ȋ͵Ȍ ‹‰ ͵ ƒ† ͵ •Š‘™ –Š‡ –‘’ ͳͷ ’ƒ”ƒ‡–‡”• ™‹–Š –Š‡ Š‹‰Š‡•– ƒ„•‘Ž—–‡ ˆŽ—š ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•ƒ–ʹͷɊƒ†͸ͲɊ‘ˆ’ƒŽ‹–‘›ŽǦ‘ǡ”‡•’‡ –‹˜‡Ž›Ǥ

(29) ƒ‰”‡‡‡–™‹–Š–Š‡Š‹‰Š ˆŽ—š ‘–”‘Ž‘ˆͳǡ–Š‡‡‹‰Š–Š‹‰Š‡•–”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•ƒ–ʹͷρ ‘’”‹•‡†‡š Ž—•‹˜‡Ž› ’ƒ”ƒ‡–‡”• –Šƒ– ƒˆˆ‡ – –Š‡ ”ƒ–‡ ‘ˆ ͳǡ ‹ Ž—†‹‰ ‹–• ȋˆ‹š‡†Ȍ •—„•–”ƒ–‡Ǧ ƒ† ’”‘†— – ‘ ‡–”ƒ–‹‘• ȋƒ”ǡ ‘ǡ ͳ͸ ›Ž‘Ȍǡ ƒ–ƒŽ›–‹  ƒ’ƒ ‹–› ȋ•ˆͳǡͳ͸ ƒ†  ’–ͳȌǡ ƒ† ƒˆˆ‹‹–› ‘•–ƒ–• ȋǡͳǡƒ”ǡ ǡͳǡ‘ǡ ǡͳǡͳ͸ ›Ž‘Ȍ ȋ ‹‰ ͵Ȍǡ ƒ† –Š‡ ‡š– ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–• ™‡”‡ ƒŽ”‡ƒ†› –‘‘ Ž‘™ –‘ ˜‹•—ƒŽ‹œ‡Ǥ – ͸Ͳ ρǡ Š‘™‡˜‡”ǡ ƒ–‹ ‹’ƒ–‡† ’ƒ”ƒ‡–‡”•”‡Žƒ–‹‰–‘ƒ’’‡ƒ”‡†ȋ‹Ǥ‡ǤͶǡ•ˆǡͶǡǡ ƒ–ǡͶǦ‡–‘ƒ ›ŽǦ‘ǡǡ ƒ–ǡͶǦ Ϊ ƒ ›ŽǦ‘ƒ†ǡ‘ǡǡƒ••Š‘™„›–Š‡’—”’Ž‡„ƒ”•ȌǤ

(30) ƒ††‹–‹‘ǡ–Š‡ȏ ȐȀȏ Ȑ”ƒ–‹‘‹ –Š‡ ‹–‘ Š‘†”‹ƒŽ ƒ–”‹š ƒ† –Š‡ ‡“—‹Ž‹„”‹— ‘•–ƒ– ‘ˆ Ȁ  ȋ•Š‘™ „› –Š‡ ’‹ „ƒ”•Ȍ ƒ†  ȋ•Š‘™ „› –Š‡ Ž‹‰Š– „Ž—‡ „ƒ”Ȍ ™‡”‡ ‹ –Š‡ –‘’ Ͷ ‘ˆ Š‹‰Š‡•– ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•ȋ ‹‰͵ȌǤ ‹ ‡ Ϊ ‹• ƒ •—„•–”ƒ–‡ ‘ˆ Ȁ ǡ ‹– •‡‡• Ž‹‡Ž› –Šƒ– –Š‡ Š‹‰Š ˆŽ—š ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–• ‘ˆ ȏΪȐȀȏ Ȑ ƒ† –Š‡ ‡“—‹Ž‹„”‹— ‘•–ƒ– ƒ”‡ —–—ƒŽŽ› ”‡Žƒ–‡†Ǥ ‡ –Š‡”‡ˆ‘”‡ •‡– ‘—– –‘ ‡Ž— ‹†ƒ–‡ –Š‡ ‡ Šƒ‹• ˜‹ƒ ™Š‹ Š –Š‡› ƒˆˆ‡ –‡† –Š‡ ˆŽ—šǤ Š‡ ”‘Ž‡ ‘ˆ Ϊ ‹• ‘’Ž‡šǡ •‹ ‡‹– ‹•ƒŽ•‘ ƒ •—„•–”ƒ–‡ ˆ‘” –Š‡  ”‡ƒ –‹‘ƒ†„‘–Š Ȁ  ƒ†  ƒ–ƒŽ›œ‡”‡ƒ –‹‘•‘ˆ•—„•–”ƒ–‡•™‹–Šƒ”ƒ‰‡‘ˆˆƒ––›Ǧƒ ‹† Šƒ‹Ž‡‰–Š•ǤŠ‡”‡ˆ‘”‡ǡ™‡. Ͷ͹. 2.

(31) Chapter 2 †‹••‡ –‡† –Š‡ ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡– ‘ˆ ȏΪȐȀȏ Ȑ ‹–‘ ‘–”‹„—–‹‘• ‘ˆ ƒŽŽ –Š‡ †‹ˆˆ‡”‡– ”‡ƒ –‹‘•–Šƒ–ƒ”‡‹ˆŽ—‡ ‡†„›–Š‹•”ƒ–‹‘ǡ„ƒ•‡†‘–Š‡ ‘„‹‡†”‡•’‘•‡–Š‡‘”‡ȏͳͷȐǣ ௃. ௩. ೔ ܴሾே஺஽శሿȀሾே஺஽ுሿ ൌ  σ௜ ‫ܥ‬௜௃ ή ߝሾே஺஽ శ ሿȀሾே஺஽ுሿ. ȋͶȌ. ௜ ‹™Š‹ Š–Š‡•—ƒ–‹‘‹•‘˜‡”ƒŽŽ”‡ƒ –‹‘•iǡƒ†ߝே஺஽ାȀே஺஽ு †‡‘–‡•–Š‡‡Žƒ•–‹ ‹–›ȋ‹Ǥ‡Ǥ–Š‡. †‹”‡ –•‡•‹–‹˜‹–›Ȍ‘ˆ–Š‡”ƒ–‡‘ˆ”‡ƒ –‹‘i–‘™ƒ”†•–Š‡ΪǦ ”ƒ–‹‘ǡƒ ‘”†‹‰–‘ǣ ௩. ೔ ା ߝሾே஺஽ శ ሿΤሾே஺஽ுሿ ൌ  ߲݈݊‫ݒ‬௜ Τ߲Žሺሾܰ‫ ܦܣ‬ሿΤሾܰ‫ܪܦܣ‬ሿሻ. ௃ Š‡†‹••‡ –‹‘‘ˆ“ǤͶ‹‹–•”‡ƒ –‹‘Ǧ•’‡ ‹ˆ‹ –‡”•‫ܥ‬௜. ή. ȋͷȌ. ௩೔ ߝሾே஺஽ శ ሿȀሾே஺஽ுሿ ȋ ‹‰͵Ȍ”‡˜‡ƒŽ‡†–™‘. Žƒ”‰‡ƒ†’‘•‹–‹˜‡–‡”•ǡ”‡’”‡•‡–‹‰–Š‡Ͷƒ†͸”‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„›Ȁ ǤŠ‡ –‡”•”‡’”‡•‡–‹‰™‡”‡•ƒŽŽƒ†‡‰ƒ–‹˜‡Ǥ‡ ‘ Ž—†‡ˆ”‘–Š‹•–Šƒ––Š‡ ’‘•‹–‹˜‡ ‡ˆˆ‡ – ‘ˆ ȏΪȐȀȏ Ȑ ‘ –Š‡ ˆŽ—š ƒ „‡ ƒ––”‹„—–‡† ‡š Ž—•‹˜‡Ž› –‘ –Š‡ •Š‘”–Ǧ Šƒ‹ ”‡ƒ –‹‘•ȋͶƒ†͸Ȍ‘ˆȀ ǡ™Š‹Ž‡‡˜‡‡š‡”–‡†ƒ•ƒŽŽ‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘Ǥ Š‡ Ȁ  ”‡ƒ –‹‘ ‹• Ž‘•‡ –‘ –Š‡”‘†›ƒ‹  ‡“—‹Ž‹„”‹— ™Š‡ –Š‡ ’ƒ–Š™ƒ› ‹• ƒ– •–‡ƒ†›•–ƒ–‡ȋ ˆǤ–Š‡•‹‹Žƒ”•Šƒ’‡•‘ˆȀ •—„•–”ƒ–‡•ƒ†’”‘†— –•‹ˆ‹‰—”‡ƒ†‹ ͵ ‹‰ǡƒ•‘’’‘•‡†–‘–Š‘•‡‘ˆ•—„•–”ƒ–‡•ƒ†’”‘†— –•‹ˆ‹‰—”‡ƒ†‹͵ ‹‰ȌǤ ‹ ”‡ƒ•‡‘ˆ–Š‡ȏΪȐȀȏ Ȑ”ƒ–‹‘•Š‹ˆ–•–Š‡‡“—‹Ž‹„”‹—‘ˆ–Š‡”‡ƒ –‹‘‹–Š‡ˆ‘”™ƒ”† †‹”‡ –‹‘ǡ‡š’Žƒ‹‹‰™Š›‹–Šƒ•–Š‡•ƒ‡‡ˆˆ‡ –ƒ•ƒ Šƒ‰‡‘ˆ–Š‡‡“—‹Ž‹„”‹— ‘•–ƒ–ǤŠ‡ ”‡ƒ•‘ –Šƒ– –Š‡ ‡“—‹Ž‹„”‹— ‘•–ƒ–•ƒ”‡ •‘‹’‘”–ƒ– ˆ‘” –Š‡ ˆŽ—š ‹•–Šƒ– –Š‡ ‡“—‹Ž‹„”‹— ‘•–ƒ–‘ˆȀ ‹•‡š–”‡‡Ž›—ˆƒ˜‘—”ƒ„Ž‡ƒ–ʹǤͳ͹ȉͳͲǦͶȋ†‹‡•‹‘Ž‡••ǡ ˆǤ ‹‰ͳȌ‹–Š‡ ˆ‘”™ƒ”††‹”‡ –‹‘Ǥ•ƒŽŽƒ ——Žƒ–‹‘‘ˆͶǦƒ†͸‡–‘ƒ ›ŽǦ‘ǡ–Š‡†‹”‡ –•—„•–”ƒ–‡•‘ˆ ǡ ™‹ŽŽ –Š‡”‡ˆ‘”‡ ‡–”ƒ‹ –Š‡ Š›†”‘š›ƒ ›ŽǦ‘ •—„•–”ƒ–‡• ‘ˆ Ȁ  –‘ ƒ ——Žƒ–‡ •‡˜‡”ƒŽ ‘”†‡”• ‘ˆ ƒ‰‹–—†‡ ‘”‡ǡ –Š‡ ‡šƒ – ”ƒ–‹‘ †‡’‡†‹‰ ‘ –Š‡ ȏΪȐȀȏ Ȑ ”ƒ–‹‘ ȋ ‹‰—”‡‹͵ ‹‰ȌǤŠ‡‡“—‹Ž‹„”‹— ‘•–ƒ–‘ˆ‹•͵Ǥͳ͵ǡ–Š—•‹†— ‹‰ ƒ‡‘›ŽǦ‘ ƒ ——Žƒ–‹‘‘ˆƒŽ‘•––Š‡•ƒ‡‘”†‡”‘ˆƒ‰‹–—†‡ȋ ‹‰—”‡‹͵ ‹‰ȌǤŠ—•ǡȀ  ƒ†  ƒ’Ž‹ˆ› –Š‡ ƒ ——Žƒ–‹‘ ‘ˆ ‘ ‡•–‡”• ‡‘”‘—•Ž›ǡ –Š‡”‡„› †”ƒ‹‹‰ –Š‡ ˆ”‡‡ ‘’‘‘ŽǤ ‹ƒŽŽ›ǡ ‹ ‹‰ ͵ ‘Ž› ’ƒ”ƒ‡–‡”•”‡Žƒ–‡† –‘ –Š‡ Ͷ”‡ƒ –‹‘ ƒ’’‡ƒ” ȋ‹Ǥ‡Ǥ •ˆǡͶǡ ǡ ƒ–ǡͶǦ‡–‘ƒ ›ŽǦ‘Ȍǡ „—– Šƒ”†Ž› ƒ› ’ƒ”ƒ‡–‡”• ”‡Žƒ–‡† –‘ –Š‡ ͸ ”‡ƒ –‹‘ǡ ‡š ‡’– ˆ‘” –Š‡ ƒˆˆ‹‹–› –‘™ƒ”† ‹–• ’”‘†— – ͶǦƒ ›ŽǦ‘  ȋ‹Ǥ‡Ǥ ǡ ƒ–ǡͶǦƒ ›ŽǦ‘Ȍǡ  •‹ ‡ –Š‡ Žƒ––‡” Šƒ˜‡ Ž‘™‡” ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•Ǥ Š‹• ƒ› „‡ ƒ––”‹„—–‡† –‘ †‹ˆˆ‡”‡ ‡• ‹ –Š‡ ƒ–ƒŽ›–‹  ‘•–ƒ–• „‡–™‡‡ –Š‡ Ͷ ƒ† ͸ ”‡ƒ –‹‘•ǡ ‘–ƒ„Ž› ȋ‹Ȍ –Š‡ ͶǦ•’‡ ‹ˆ‹   ƒ–ƒŽ›–‹  ƒ’ƒ ‹–› ȋ•ˆͶǡ ƒ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹  ‘†‹ˆ›‹‰ ˆƒ –‘” ˆ‘” ƒšȌ „‡‹‰ ʹǦˆ‘Ž† Ž‘™‡” –Šƒ ‹–•͸Ǧ •’‡ ‹ˆ‹  ‘—–‡”’ƒ”– ȋ•ˆ͸Ȍ ȋͲǤͶͻ ˜‡”•—• ͳȌ ƒ† ȋ‹‹Ȍ –Š‡ ƒŽ‘•– ʹǦˆ‘Ž† Ž‘™‡” ƒˆˆ‹‹–› ‘ˆ ˆ‘”‹–•ͶǦ‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡–Šƒˆ‘”‹–•͸Ǧ‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡ǡ“—ƒ–‹ˆ‹‡† „›ƒʹǦˆ‘Ž†Š‹‰Š‡” ‘””‡•’‘†‹‰‹ Šƒ‡Ž‹•Ǧ‡–‡ ‘•–ƒ–ȋȌȋͳʹǤͶ˜‡”•—•͸Ǥ͹ɊȌǤ . Internal regulation reveals a vicious cycle around MCKAT ‘ —†‡”•–ƒ† –Š‡ •‡“—‡ ‡ ‘ˆ ‡˜‡–• Ž‡ƒ†‹‰ –‘ ˆŽ—š †‡ Ž‹‡ ƒ† –Š‡ ’‘–‡–‹ƒŽ ”‘Ž‡ ‘ˆ ‘’‡–‹–‹˜‡‹Š‹„‹–‹‘–Š‡”‡‹ǡ™‡‹•’‡ –‡†–‹‡ ‘—”•‡•‘ˆ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘•ƒ† ‡œ›‡ ”ƒ–‡•Ǥ –ƒ”–‹‰ ˆ”‘ ƒ •–‡ƒ†› •–ƒ–‡ ƒ– ͲǤͳ ρǡ –Š‡ ‘ ‡–”ƒ–‹‘ ‘ˆ –Š‡ •—„•–”ƒ–‡ ’ƒŽ‹–‘›ŽǦ‘ ™ƒ• ‹ ”‡ƒ•‡† ƒ– –‹‡ ’‘‹– œ‡”‘ –‘ ʹͷ ‘” ͸Ͳ ρ ƒ† –Š‡ ”‡•’‘•‡ ‘ˆ –Š‡ ’ƒ–Š™ƒ› ™ƒ• ˆ‘ŽŽ‘™‡† —–‹Ž –Š‡ •›•–‡ ”‡Žƒš‡† ‹–‘ ƒ ‡™ •–‡ƒ†› •–ƒ–‡Ǥ – „‘–Š •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘• ‡–ƒ„‘Ž‹–‡• ƒ ——Žƒ–‡† ‘” †‡ Ž‹‡† ‰”ƒ†—ƒŽŽ›ǡ ™‹–Š Šƒ”†Ž› ƒ› †‡–‡ –ƒ„Ž‡. Ͷͺ.

(32) The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation ‘˜‡”Ǧ‘”—†‡”•Š‘‘–•ȋ ‹‰—”‡‹Ͷ ‹‰ȌǤ–ƒŽ‘™ ‘ ‡–”ƒ–‹‘‘ˆ’ƒŽ‹–‘›ŽǦ‘ȋʹͷρȌ –Š‡ ”ƒ–‡• ‘ˆ ƒŽŽ ‡œ›‡• ƒŽ•‘ ‹ ”‡ƒ•‡† ‰”ƒ†—ƒŽŽ› ‹ –‹‡ ȋ ‹‰—”‡ ‹ ͷ ‹‰ȌǤ – ͸Ͳ ρǡ Š‘™‡˜‡”ǡ –Š‡ ’ƒ––‡” ™ƒ• ‘•’‹ —‘—•Ž› †‹ˆˆ‡”‡–ǡ ƒ• ‡š‡’Ž‹ˆ‹‡† „› ‡œ›‡ ”ƒ–‡• ƒ”‘—† –Š‡Ͷ‡–ƒ„‘Ž‹–‡•ȋ ‹‰ͶȌǤ A. B vMCKATC6 vMCADC4 + vSCADC4 vMCKATC4. 1.4 1.3 1.2 1.1. 0.01. vmckatC4 ĬX j (μmol.miní2.mgProteiní). v (μmol.min-1.gProtein-1). 1.5. C16-Ketoacyl-CoA C14-Ketoacyl-CoA C12-Ketoacyl-CoA C10-Ketoacyl-CoA C8-Ketoacyl-CoA C6-Ketoacyl-CoA C4-Ketoacyl-CoA C16-Acyl-CoA C14-Acyl-CoA C12-Acyl-CoA C10-Acyl-CoA C8-Acyl-CoA C6-Acyl-CoA C4-Acyl-CoA Free CoA. 0. -0.01. 0.0 0. 100 200 time (min). 300. 0. 100. 200 time (min). 300. . Fig 4. Time course of key short-chain reaction rates and regulation of the MCKAT-C4 reaction. (A) ‹‡ ‘—”•‡ ‘ˆ –Š‡ ”‡ƒ –‹‘ ”ƒ–‡• ‘ˆ  ˆ‘” ‹–• ͸ ƒ† Ͷ •—„•–”ƒ–‡• ȋ˜͸ ƒ† ˜Ͷǡ ”‡•’‡ –‹˜‡Ž›Ȍ ƒ† ‘ˆ –Š‡ •—‡† ƒ –‹˜‹–‹‡• ‘ˆ  ƒ†  ‘ –Š‡‹” Ͷ •—„•–”ƒ–‡ ȋ˜Ͷ Ϊ ˜ͶȌƒˆ–‡”ƒ•—††‡—’•Š‹ˆ–‘ˆȏ’ƒŽ‹–‘›ŽǦ‘Ȑˆ”‘ͲǤͳ–‘͸ͲɊǤ(B) ‹‡ ‘—”•‡‘ˆ”‡‰—Žƒ–‘”› ‡–ƒ„‘Ž‹–‡ ‘–”‹„—–‹‘•–‘˜Ͷƒˆ–‡”ƒ•—††‡ȏ’ƒŽ‹–‘›ŽǦ‘Ȑ‹ ”‡ƒ•‡ˆ”‘ͲǤͳ–‘͸ͲɊǤ .  Š‡”ƒ–‡‘ˆ‘‹–•ͶǦ‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡Žƒ‰‰‡†•—„•–ƒ–‹ƒŽŽ›„‡Š‹†–Š‡”ƒ–‡ƒ– ™Š‹ Š –Š‡ ͶǦƒ ›ŽǦ‘ ‡–‡”‡† –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Š ˜‹ƒ  ƒ† Ǥ ‘–Š ”ƒ–‡• ‹ ”‡ƒ•‡† ‹ –‹‡ ƒ• –Š‡‹” •—„•–”ƒ–‡ ’‘‘Ž• ˆ‹ŽŽ‡†ǡ —–‹Ž ƒ– –‹‡ ’‘‹– ʹͺ ‹ǡ ™Š‡ –Š‡› •–ƒ”–‡†–‘†‡ Ž‹‡ǤŠ‡•ƒ‡Šƒ’’‡‡†ƒ––Š‡Ž‡˜‡Ž‘ˆ–Š‡͸”‡ƒ –‹‘•‘ˆȀƒ† ǡƒŽ„‡‹––‘ƒŽ‡••‡”‡š–‡–ȋ ‹‰—”‡‹͸ ‹‰ȌǤŠ‹••—‰‰‡•–‡†–Šƒ–‘‡‘”‘”‡‘ˆ–Š‡ ‡–ƒ„‘Ž‹–‡•‹Š‹„‹–‡†–Š‡•‡‡œ›‡•‘”„‡ ƒ‡Ž‹‹–‹‰Ǥ ‡ ™‘†‡”‡† ™Š‹ Š ‡–ƒ„‘Ž‹–‡• ƒ—•‡† –Š‡ ‡œ›‡ ”ƒ–‡• –‘ †‡ Ž‹‡Ǥ ‹ ‡ ‡ƒ Š ”‡ƒ –‹‘ ”ƒ–‡†‡’‡†•‘–Š‡ ‘ ‡–”ƒ–‹‘•‘ˆ—Ž–‹’Ž‡’”‹ƒ”›•—„•–”ƒ–‡•ƒ†’”‘†— –•ƒ•™‡ŽŽƒ• ‘’‡–‹‰‡–ƒ„‘Ž‹–‡•‘ˆ‘–Š‡” Šƒ‹Ž‡‰–Š•ǡ–Š‡ ‘–”‹„—–‹‘‘ˆ‡ƒ Š‹†‹˜‹†—ƒŽ‡–ƒ„‘Ž‹–‡ –‘–Š‡”‡‰—Žƒ–‹‘‘ˆ–Š‡”‡ƒ –‹‘”ƒ–‡•™ƒ•“—ƒ–‹ˆ‹‡†ǡƒ ‘”†‹‰–‘–Š‡‡–Š‘†‘ˆ”—‰‰‡ƒ ‡– ƒŽǤ ʹͲͲͷ ȏͳʹȐǤ Š‡ ‘ ‡’– „‡Š‹† –Š‹• ƒƒŽ›•‹• ‹• –Šƒ– ƒ Šƒ‰‡ ‹ ‡œ›‡ ”ƒ–‡ v ‹• –Š‡ ƒ–Š‡ƒ–‹ ƒŽ ’”‘†— – ‘ˆ ƒ Šƒ‰‡ ‹ ƒ ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘ Xj —Ž–‹’Ž‹‡† „› –Š‡ ‡Žƒ•–‹ ‹–› ‘ˆ –Š‡ ‡œ›‡ ”ƒ–‡ –‘ –Š‹• ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘ǡ ƒ† •—‡† ‘˜‡” ƒŽŽ ‡–ƒ„‘Ž‹–‡•j–Šƒ–ƒˆˆ‡ ––Š‡‡œ›‡ǣ ௗ௟௡௩ ሺ‫ݐ‬ሻ ௗ௧. ൌ σ௝. ௗ௟௡௑ೕ డ௟௡௩ ሺ‫ݐ‬ሻ ή ሺ‫ݐ‬ሻ ௗ௧ డ௟௡௑ೕ. ȣ௑௩ ೕ ሺ‫ݐ‬ሻ ‫ؠ‬. ™‹–Šǣ. ‫ ؠ‬σ௝ ȣ௑௩ ೕ ሺ‫ݐ‬ሻ. ȋ͸ƒȌ. ௗ௟௡௑ೕ డ௟௡௩ ሺ‫ݐ‬ሻ ή ௗ௧ ሺ‫ݐ‬ሻ డ௟௡௑ೕ. ȋ͸„Ȍ.

(33) –‡‰”ƒ–‹‘ ‘˜‡” –Š‡ ‡–‹”‡ ”‡Žƒšƒ–‹‘ –‹‡ ˆ”‘ ‘‡ •–‡ƒ†› •–ƒ–‡ –‘ ƒ‘–Š‡” ƒŽŽ‘™• –Š‡ 4ఫ  ‘ˆ ‡ƒ Š †‹••‡ –‹‘ ‘ˆ –Š‡ ‘˜‡”ƒŽŽ ”‡‰—Žƒ–‹‘ ‘ˆ –Š‡ ”ƒ–‡ ‹–‘ ƒ˜‡”ƒ‰‡ ‘–”‹„—–‹‘• തതത ଵ ௧ ௗ௟௡௩ ሺ߬ሻ݀߬ ‫׬‬ ௧ ଴ ௗ௧. ‹†‹˜‹†—ƒŽ‡–ƒ„‘Ž‹–‡Xjǣ ଵ ௧ σ௝ ‫׬‬଴ ȣ௑௩ ೕ ሺ߬ሻ݀߬ ௧. . . . ഥ ௑௩ ൌ σ௝ ȣ ೕ. . . . ଵ. ௧ డ௟௡௩. ൌ σ௝ ‫׬‬଴ ሺ߬ሻ ή ௧ డ௟௡௑. ௗ௟௡௑ೕ ௗ௧. ೕ. . . . ሺ߬ሻ݀߬ ൌ. . . ȋ͹Ȍ. Š‡ƒ˜‡”ƒ‰‡ƒ„•‘Ž—–‡ ‘–”‹„—–‹‘‘ˆ‡ƒ Š‡–ƒ„‘Ž‹–‡‹•‰‹˜‡„›ǣ. Ͷͻ. 2.

(34) Chapter 2 ഥ ȁ௩௑ ൌ ଵ ‫׬‬௧ ቚȣ௑௩ ሺ߬ሻቚ ݀߬ ȁȣ ೕ ೕ ௧ ଴. ȋͺȌ. ‹‰ Ͷ •Š‘™• –Š‡ ‘–”‹„—–‹‘ȣ௑௩ ೕ ሺ‫ݐ‬ሻ ‘ˆ ƒŽŽ ‡–ƒ„‘Ž‹–‡• –Šƒ– ƒˆˆ‡ – –Š‡ ‘˜‡”•‹‘ ‘ˆ ͶǦ ‡–‘ƒ ›ŽǦ‘„›’Ž‘––‡†ƒ•ƒˆ— –‹‘‘ˆ–‹‡ˆ‘”ƒ–”ƒ•‹–‹‘ˆ”‘ͲǤͳ–‘͸Ͳρ‘ˆ ’ƒŽ‹–‘›ŽǦ‘ǡ ƒŽ —Žƒ–‡† ƒ ‘”†‹‰ –‘ “Ǥ ͸„Ǥ Š‡ ƒ„•‘Ž—–‡ ƒ”‡ƒ —†‡” ‡ƒ Š —”˜‡ ‹• ഥ ȁ௩ெ஼௄஺்ି஼ସ ƒ††‡‘–‡•–Š‡ƒ˜‡”ƒ‰‡ƒ„•‘Ž—–‡ ‘–”‹„—–‹‘‘ˆƒ•’‡ ‹ˆ‹ ‡–ƒ„‘Ž‹–‡j–‘–Š‡ ȁȣ ௑ೕ Šƒ‰‡‘ˆ–Š‡”ƒ–‡‘ˆ–Š‡Ͷ”‡ƒ –‹‘‘ˆ†—”‹‰–Š‡”‡Žƒšƒ–‹‘–‘–Š‡‡™•–‡ƒ†›•–ƒ–‡Ǥ  Žƒ”‰‡ ’‘•‹–‹˜‡ ‘–”‹„—–‹‘ ‹• ƒ†‡ „› –Š‡ •—„•–”ƒ–‡ ͶǦ‡–‘ƒ ›ŽǦ‘Ǥ ‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘•™‡”‡ƒ†‡„›ƒ ›ŽǦ‘‡•–‡”•‘ˆ˜ƒ”‹‘—• Šƒ‹Ž‡‰–Š•ǡ™Š‹ Šƒ”‡–Š‡’”‘†— –• ‘ˆƒŽŽ–Š‡‘–Š‡””‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„›Ǥ‘–ƒ„Ž›ƒŽ•‘–Š‡ˆ”‡‡‘ ‘ ‡–”ƒ–‹‘ƒ†‡ ƒ •—„•–ƒ–‹ƒŽ ‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘Ǥ ”‡‡ ‘ ‹• ƒ •—„•–”ƒ–‡ ‘ˆ  ƒ† –Š‡”‡ˆ‘”‡ ‹–• ‡Žƒ•–‹ ‹–› ‘‡ˆˆ‹ ‹‡–. డ௟௡௩ಾ಴಼ಲ೅ష಴ర డ୪୬ሾ஼௢஺ሿ. ‹•’‘•‹–‹˜‡ǡ„—–•‹ ‡–Š‡ ‘ ‡–”ƒ–‹‘†‡ ”‡ƒ•‡†™‹–Š–‹‡ǡ. –Š‡‘˜‡”ƒŽŽ ‘–”‹„—–‹‘„‡ ƒ‡‡‰ƒ–‹˜‡Ǥ

(35)  ‹‰ ͷǦ  –Š‡•‡ ƒ˜‡”ƒ‰‡ ‘–”‹„—–‹‘• ™‡”‡ ’Ž‘––‡† ƒ• ƒ ˆ— –‹‘ ‘ˆ –Š‡ •—„•–”ƒ–‡ ’ƒŽ‹–‘›ŽǦ‘ˆ‘”ƒ–”ƒ•‹–‹‘ˆ”‘ͲǤͳρ–‘–Š‡‹†‹ ƒ–‡† ‘ ‡–”ƒ–‹‘‘ˆ’ƒŽ‹–‘›ŽǦ‘Ǥ ‡ •–ƒ”–‡† ˆ”‘ –Š‡  ”‡ƒ –‹‘• ˆ‘” ‹–••—„•–”ƒ–‡• ͸ƒ†ͶǦ‡–‘ƒ ›ŽǦ‘ǡ ƒ– ™Š‹ Š –Š‡ˆŽ—š†‡ Ž‹‡ƒ’’‡ƒ”‡†–‘‘”‹‰‹ƒ–‡ȋ ‹‰ͷƒ†ȌǤ‹‡ˆ‘”–Š‡–”ƒ•‹–‹‘ˆ”‘ͲǤͳ–‘͸Ͳ ρǡ ƒŽ•‘ ƒ– ‘–Š‡” ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•–Š‡  ”‡ƒ –‹‘ ™ƒ• ‘•–Ž› ”‡‰—Žƒ–‡† „›–Š‡‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡‘ˆ–Š‡”‡ƒ –‹‘–Šƒ–‹•‹•’‡ –‡†ȋ‹Ǥ‡Ǥ–Š‡ͶǦ‡–‘ƒ ›ŽǦ‘ˆ‘” –Š‡ ͶǦ ”‡ƒ –‹‘ ƒ† –Š‡ ͸Ǧ‡–‘ƒ ›ŽǦ‘ ˆ‘” –Š‡ ͸Ǧ ”‡ƒ –‹‘Ȍǡ ‹–• ‘Ǧ •—„•–”ƒ–‡ ˆ”‡‡ ‘ǡ ƒ† ƒ ›ŽǦ‘ ‡•–‡”• ‘ˆ ˜ƒ”‹‘—• Šƒ‹ Ž‡‰–Š•ǡ ‹ Ž—†‹‰ –Š‡ ƒ ›ŽǦ‘ ’”‘†— –‘ˆ–Š‡”‡ƒ –‹‘‹–•‡ŽˆǤŠ‡‘Ž›‘˜‡”ƒŽŽ’‘•‹–‹˜‡ ‘–”‹„—–‹‘™ƒ•„›–Š‡‡–‘ƒ ›ŽǦ‘ •—„•–”ƒ–‡ǡ†—‡–‘–Š‡‹ ”‡ƒ•‡‘ˆ‹–• ‘ ‡–”ƒ–‹‘‹–‹‡ȋ ˆ ‹‰—”‡‹͹ȌǤŠ‡†‡ ”‡ƒ•‡‘ˆ –Š‡ ‘Ǧ•—„•–”ƒ–‡ ‘ ›‹‡Ž†‡† ƒ ‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘Ǥ Š‡ ”‘Ž‡ ‘ˆ –Š‡ ƒ ›ŽǦ‘ ‡•–‡”• ‹• ’ƒ”ƒ†‘š‹ ƒŽǤŠ‡›ƒ”‡’”‘†— –•‘ˆƒ†–Š‡”‡ˆ‘”‡‹Š‹„‹––Š‡‡œ›‡ǡƒ• ƒ„‡•‡‡ ˆ”‘–Š‡‡‰ƒ–‹˜‡”‡‰—Žƒ–‘”› ‘–”‹„—–‹‘‘ˆ–Š‡ƒ ›ŽǦ‘‡•–‡”•‹ ‹‰ͶǤ––Š‡•ƒ‡–‹‡ –Š‡͸Ǧƒ†ͶǦƒ ›ŽǦ‘‡•–‡”•ƒ”‡•—„•–”ƒ–‡•ˆ‘”–Š‡•Š‘”–Ǧ Šƒ‹„”ƒ Šƒ†ƒ”‡ ‘˜‡”–‡† –‘ ͸Ǧ ƒ† ͶǦ‡–‘ƒ ›Ž •—„•–”ƒ–‡• ˆ‘” Ǥ › ‘„‹‹‰ ƒŽŽ †ƒ–ƒǡ ™‡ †‡†— ‡† –Š‡ ˆ‘ŽŽ‘™‹‰‡ Šƒ‹•ȋ•—ƒ”‹œ‡†‹ ‹‰͸ȌǤ—’•Š‹ˆ–‘ˆ’ƒŽ‹–‘›ŽǦ‘ˆ‹ŽŽ•–Š‡ƒ ›ŽǦ‘ ’‘‘Ž•ǡ™Š‹ Šƒ”‡ ‘˜‡”–‡†‹–‘‡‘›ŽǦ‘‡•–‡”•ƒ†Žƒ”‰‡Ž›ˆ‘ŽŽ‘™–Š‡”‘—–‡†‘™–‘ ͸Ǧƒ ›ŽǦ‘Ǥ͸Ǧƒ†ͶǦƒ ›ŽǦ‘–Š‡ˆ‘ŽŽ‘™–Š‡•Š‘”–Ǧ Šƒ‹„”ƒ Š’”‘˜‹†‹‰•—„•–”ƒ–‡–‘ ǡ™Š‹ ŠŠƒ•‹•—ˆˆ‹ ‹‡– ƒ’ƒ ‹–›–‘‡–ƒ„‘Ž‹•‡–Š‡ƒ––Š‡•ƒ‡”ƒ–‡ǤŠ‹•Ž‡ƒ†•–‘ƒ ƒ ——Žƒ–‹‘ ‘ˆ ǯ• •—„•–”ƒ–‡• ͶǦ ƒ† ͸Ǧ‡–‘ƒ ›ŽǦ‘Ǥ • ™‡ Šƒ˜‡ •‡‡ǡ –Š‡ ƒ„•‘Ž—–‡ ‘ ‡–”ƒ–‹‘•‘ˆ–Š‡‡–‘ƒ ›ŽǦ‘‡•–‡”•ƒ”‡•ƒŽŽǡ„—––Š‡‹”‡ˆˆ‡ –•ƒ”‡ƒ’Ž‹ˆ‹‡† „›‡“—‹Ž‹„”ƒ–‹‰™‹–ŠŠ›†”‘š›ƒ ›ŽǦ‘‡•–‡”•ƒ†‡‘›ŽǦ‘‡•–‡”•Ǥ Š‡•‡‹Š‹„‹––Š‡ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡•ƒ†–Š—•–Š‡—’•–”‡ƒƒ ›ŽǦ‘‡•–‡”••–ƒ”––‘ ƒ ——Žƒ–‡Ǥ Š‡•‡ ƒ ›ŽǦ‘ ‡•–‡”• ƒ”‡ ƒ– –Š‡ •ƒ‡ –‹‡ ’”‘†— –•‘ˆ  ƒ†–Š‡”‡ˆ‘”‡ ‹Š‹„‹––Š‹•‡œ›‡ǤŠ‹•ƒ‰‰”ƒ˜ƒ–‡•–Š‡„‘––Ž‡‡ ƒ–Ǥ‘”‡‘˜‡”ǡ–Š‡ƒ ——Žƒ–‹‰ ‘‡•–‡”••‡“—‡•–‡”–Š‡ˆ”‡‡‘ƒ†–Š‡‘˜‡”ƒŽŽ”‡•—Ž–‹•ƒ†”ƒ•–‹ ”‡†— –‹‘‘ˆ–Š‡ˆ”‡‡‘ ’‘‘ŽǤ‹ ‡‘‹•ƒ ‘Ǧ•—„•–”ƒ–‡‘ˆǡ–Š‹•Ž‹‹–•–Š‡”‡ƒ –‹‘‡˜‡ˆ—”–Š‡”ƒ† –”ƒ’•–Š‡’ƒ–Š™ƒ›‹ƒ˜‹ ‹‘—• › Ž‡Ǥ

(36) –Š‡–‹‡ ‘—”•‡ȋ ‹‰ͶȌ–Š‡†‡ ”‡ƒ•‡‘ˆ–Š‡”ƒ–‡•‡–•‹™Š‡–Š‡‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘• ‘ˆ‘†‡ Ž‹‡ƒ†ƒ ›ŽǦ‘ƒ ——Žƒ–‹‘•—”’ƒ••–Š‡’‘•‹–‹˜‡ ‘–”‹„—–‹‘‘ˆ–Š‡‹ ”‡ƒ•‡† ‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡ȋ ‹‰ͶȌǤ‘•—’’‘”––Š‡‘–‹‘‘ˆ‹Š‹„‹–‹‘„›–Š‡ƒ ›ŽǦ‘. ͷͲ.

(37) The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation ‡•–‡”•ǡ–Š‡‘ —’ƒ ›‘ˆ™‹–Š’”‘†— –•ƒ†•—„•–”ƒ–‡•‘ˆ†‹ˆˆ‡”‡– Šƒ‹ǦŽ‡‰–Š•™ƒ• ƒŽ —Žƒ–‡†ȋ ‹‰—”‡‹ͺ ‹‰ȌǤ

(38) †‡‡†ǡ–Š‡†‘‹ƒ–‘ —’ƒ–•‘ˆ–Š‡ƒ –‹˜‡•‹–‡™‡”‡ͶǦ ƒ† ͸Ǧƒ ›ŽǦ‘ǡ –‘‰‡–Š‡” ‘ —’›‹‰ ͸͸ǤͶ Ψ ‘ˆ –Š‡ ƒ˜ƒ‹Žƒ„Ž‡ ƒ –‹˜‡ •‹–‡• ƒ– ͸Ͳ ρ ‘ˆ ’ƒŽ‹–‘›ŽǦ‘ȋ ‹‰—”‡‹ͺ ‹‰ȌǤ  A. j  ഥ |v σj |Ĭ Xj. ഥ |v |Ĭ X. B. vMCKATC6. 100% 75% 50%. C6-ketoacyl-CoA (substrate). 25% 0% 0. 50. 100. 150. 200. vMCKATC4. Free CoA C4-Acyl-CoA C6-Acyl-CoA C8-Acyl-CoA C10-Acyl-CoA C12-Acyl-CoA C14-Acyl-CoA C16-Acyl-CoA C4-Ketoacyl-CoA C6-Ketoacyl-CoA C8-Ketoacyl-CoA C10-Ketoacyl-CoA C12-Ketoacyl-CoA C14-Ketoacyl-CoA C16-Ketoacyl-CoA. Free CoA. C4-ketoacyl-CoA (substrate). 0. 250. [Palmitoyl-CoA]CYT ȝ0

(39). C. D. j  ഥ |v σj |Ĭ Xj. ഥ |v |Ĭ X. 50% C6-acyl-CoA (substrate). 0% 0. 50. 100. 150. 200. 0. j  ഥ |v σj |Ĭ Xj. ഥ |v |Ĭ X. F. 50%. C8-enoyl-CoA (substrate). 0% 50. 100. 150. 50. 200. C4-Enoyl-CoA C6-Enoyl-CoA C4-Acyl-CoA C6-Acyl-CoA. 100. 150. 200. 250. vCPT2C16. Free CoA C4-Ketoacyl-CoA C6-Acyl-CoA C8-Acyl-CoA C10-Acyl-CoA C12-Acyl-CoA C14-Acyl-CoA C16-Acyl-CoA C8-Enoyl-CoA C10-Enoyl-CoA C12-Enoyl-CoA C14-Enoyl-CoA C16-Enoyl-CoA. 75%. 0. 250. [Palmitoyl-CoA]CYT ȝ0

(40). Free CoA. 25%. 200. C4-acyl-CoA (substrate). 250. vMTPC8. 100%. 150. C4-enoyl-CoA. [Palmitoyl-CoA]CYT ȝ0

(41). E. 100. vSCADC4. C4-Enoyl-CoA C6-Enoyl-CoA C8-Enoyl-CoA C10-Enoyl-CoA C12-Enoyl-CoA C4-Acyl-CoA C8-Acyl-CoA C10-Acyl-CoA C12-Acyl-CoA C6-Acyl-CoA. 75%. 25%. 50. [Palmitoyl-CoA]CYT ȝ0

(42). vMCADC6 100%. Free CoA C4-Acyl-CoA C6-Acyl-CoA C8-Acyl-CoA C10-Acyl-CoA C12-Acyl-CoA C14-Acyl-CoA C16-Acyl-CoA C4-Ketoacyl-CoA C6-Ketoacyl-CoA C8-Ketoacyl-CoA C10-Ketoacyl-CoA C12-Ketoacyl-CoA C14-Ketoacyl-CoA C16-Ketoacyl-CoA. Free CoA. 250. [Palmitoyl-CoA]CYT ȝ0

(43). Free CoA C4-Acyl-CoA C6-Acyl-CoA C8-Acyl-CoA C10-Acyl-CoA C12-Acyl-CoA C14-Acyl-CoA C16-Acyl-CoA C4-Acyl-carnitine C6-Acyl-carnitine C8-Acyl-carnitine C10-Acyl-carnitine C12-Acyl-carnitine C14-Acyl-carnitine C16-Acyl-carnitine. Free CoA. C16-acyl-carnitine (substrate). 0. 50. 100. 150. 200. 250. [Palmitoyl-CoA]CYT ȝ0

(44).  Fig 5.Regulation of key reactions by their substrates and products. (A-F) ‡Žƒ–‹˜‡ƒ˜‡”ƒ‰‡ƒ„•‘Ž—–‡ ഥ ȁ࢜ ȁࢨ ࢄ. ࢐ ഥ ȁ࢜ ࢐ ȁࢨ ࢄ࢐. ‘–”‹„—–‹‘ǡ‹Ǥ‡Ǥ σ. ή ૚૙૙Ψ,‘ˆ‡–ƒ„‘Ž‹–‡•–‘–Š‡–”ƒ•‹–‹‘ˆ”‘–Š‡•–‡ƒ†›•–ƒ–‡ƒ–ͲǤͳɊ–‘–Š‡. ‹†‹ ƒ–‡† ‘ ‡–”ƒ–‹‘‘ˆ’ƒŽ‹–‘›ŽǦ‘ǡ ƒŽ —Žƒ–‡†ˆ‘”˜͸ȋ’ƒ‡ŽȌǡ˜Ͷȋ’ƒ‡ŽȌǡ ˜͸ȋ’ƒ‡ŽȌǡ˜Ͷȋ’ƒ‡ŽȌǡ˜ͺȋ’ƒ‡ŽȌǡ˜ʹͳ͸ȋ’ƒ‡Ž Ȍ..  Ž„‡‹– ‹†‹˜‹†—ƒŽŽ› Ž‡•• ‹’‘”–ƒ–ǡ ‘ŽŽ‡ –‹˜‡Ž› –Š‡ ‘–Š‡” ƒ ›ŽǦ‘ǯ• ‘ —’‹‡† ͵ͲǤͶΨ‘ˆ –Š‡ ƒ –‹˜‡ •‹–‡• —†‡” –Š‡ •ƒ‡ ‘†‹–‹‘Ǥ – –Šƒ– ’‘‹–ǡ –Š‡ ‡–‘ƒ ›ŽǦ‘ •—„•–”ƒ–‡• ‘ —’‹‡† ‘Ž›ͳǤ͸ΨƒŽ–‘‰‡–Š‡”Ǥ ‹ƒŽŽ›ǡ ™‡ ™‘†‡”‡† Š‘™ –Š‡ Ž‘‰‰‹‰ ‘ˆ ‡–ƒ„‘Ž‹–‡• ‹ –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Š ™ƒ• –”ƒ•‹––‡†–Š”‘—‰Š–Š‡‡–™‘”–‘‡˜‡–—ƒŽŽ›•Ž‘™†‘™–Š‡‡–‹”‡’ƒ–Š™ƒ›Ǥ‘–Š‹•‡† –Š‡‹–‡”ƒŽ‡–ƒ„‘Ž‹ ”‡‰—Žƒ–‹‘‘ˆǡǡƒ†ʹ™‡”‡ ‘’—–‡†Ǥƒ† ǡ –Š‡ ‡œ›‡• ƒ– –Š‡ ‰ƒ–‡™ƒ› ‘ˆ –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Šǡ ”‡•’‘†‡† „› ƒ •–”‘‰ ͷͳ. 2.

(45) Chapter 2 ”‡‰—Žƒ–‹‘ „› –Š‡‹” ’”‘†— –• ͶǦƒ† ͸Ǧ‡‘›ŽǦ‘ ȋ ‹‰ ͷ ƒ† ȌǤ Š‡•‡ ”‡‰—Žƒ–‹‘• ƒ”‡ ‘˜‡”ƒŽŽ‡‰ƒ–‹˜‡ƒ”‘—†–Š‡’‘‹–‘ˆˆŽ—š†‡ Ž‹‡ǡ•‹ ‡–Š‡‹” ‘ ‡–”ƒ–‹‘•‹ ”‡ƒ•‡ȋ ‹‰—”‡ ‹͵ ‹‰Ȍƒ†–Š‡›‹Š‹„‹––Š‡‡œ›‡ǤŠ‹•”‡‰—Žƒ–‹‘™ƒ••—’’‘”–‡†„›–Š‡ˆƒ ––Šƒ––Š‡ ƒ –‹˜‡•‹–‡•‘ˆƒ†™‡”‡’”‡†‘‹ƒ–Ž›‘ —’‹‡†„›–Š‡‹”’”‘†— –•ͶǦƒ†͸Ǧ ‡‘›ŽǦ‘ȋ ‹‰—”‡ƒ†‹ͺ ‹‰ȌǤŠ‡ƒ ›ŽǦ‘•—„•–”ƒ–‡•‘ˆƒ†‘ —’‹‡† ‘Ž›ƒ‹‘”ˆ”ƒ –‹‘‘ˆ–Š‡ƒ –‹˜‡•‹–‡•ƒ–•–‡ƒ†›•–ƒ–‡Ǥ‘”‡†‹•–ƒ–‡œ›‡•ƒ†ʹ ”‡•’‘†‡† –‘ –Š‡‹” Ž‘ ƒŽ •—„•–”ƒ–‡• ƒ† ’”‘†— –•ǡ „—– ƒŽ•‘ •–”‘‰Ž› ˜‹ƒ –Š‡ ‘‡ –‹‰ ‡–ƒ„‘Ž‹–‡ˆ”‡‡‘ȋ ‹‰ͷƒ† ȌǤ . Discussion Š‡ ‘ǦŽ‹‡ƒ” †›ƒ‹  ‘†‡Ž —•‡† ‹ –Š‹• ’ƒ’‡” ‹• –Š‡ ‘•– •–”— –—”‡† ƒ† “—ƒ–‹–ƒ–‹˜‡ ”‡’”‡•‡–ƒ–‹‘‘ˆ‘—” ‘ŽŽ‡ –‹˜‡‘™Ž‡†‰‡‘ Ǥ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‹• ‘’Ž‡–‡Ž› –”ƒ•’ƒ”‡–ǡ ƒ ’”‘’‡”–› –Šƒ– ™‡ ‡š’Ž‘‹–‡† –‘ †‹••‡ – ƒ ˜‹ ‹‘—• › Ž‡ Ž‡ƒ†‹‰ –‘ ˆŽ—š †‡ Ž‹‡ —’‘•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†ȋ ‹‰͸ȌǤ Palmitoyl-CoA. 5. Acyl-CoA. Mitochondrion 3.. MCKAT C4-C16 Ketoacyl-CoA. MSCHAD C4-C16 Keq = 0.0002. Acetyl-CoA 4.. CoASH. 2.. SCAD. MCAD. LCAD. C4-C6. C4-C12. VLCAD. 1.. Enoyl-CoA. NADH + H+ C4-ketoacyl-CoA NAD+ Hydroxyacyl-CoA. FAD FADH2. 2.. CROT. Acetyl-CoA. C4-C16. CoASH. MTP 5.. 2.. . Fig 6.Elucidating the mechanism of flux decline. 1. ’‘•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†™‹–Š ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ ‘ǡǯ•‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡••–ƒ”––‘ƒ ——Žƒ–‡Ǥ 2. Š‡—ˆƒ˜‘—”ƒ„Ž‡‡“—‹Ž‹„”‹— ‘•–ƒ–‘ˆ –Š‡ ’”‡ ‡†‹‰ ‡œ›‡ǡ ‡†‹—Ȁ•Š‘”–Ǧ Šƒ‹ Š›†”‘š›ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ǡ ™‘” ƒ• ƒ ƒ’Ž‹ˆ‹‡”ǡ Ž‡ƒ†‹‰–‘ƒ ——Žƒ–‹‘‘ˆ—’•–”‡ƒ‘‡•–‡”•ǡ‹ Ž—†‹‰ƒ ›ŽǦ‘‡•–‡”•Ǥ3.Š‡•‡ƒ ›ŽǦ‘‡•–‡”•ƒ”‡ƒ– –Š‡•ƒ‡–‹‡’”‘†— –•‘ˆƒ†‹Š‹„‹–‹–•ƒŽ”‡ƒ†›Ž‘™ƒ –‹˜‹–›ˆ—”–Š‡”Ǥ4. ‹ƒŽŽ›ǡ–Š‡ƒ ——Žƒ–‹‘ ‘ˆ ‘ ‡•–‡”• Ž‡ƒ†• –‘ ƒ •‡“—‡•–”ƒ–‹‘ ‘ˆ ˆ”‡‡ ‘Ǥ ‘ „‡‹‰ ƒ ‘ˆƒ –‘” ˆ‘” ǡ ‹–• •‡“—‡•–”ƒ–‹‘ Ž‹‹–‡†–Š‡ƒ –‹˜‹–›‡˜‡ˆ—”–Š‡”ǡ–Š—• ‘’Ž‡–‹‰–Š‡˜‹ ‹‘—• › Ž‡Ǥ5Ǥ‹ ‡‘‹•ƒŽ•‘ƒ•—„•–”ƒ–‡ ˆ‘” †‹•–ƒ– ‡œ›‡• ȋ•— Š ƒ• Ȍǡ ‹– ‡ˆˆ‹ ‹‡–Ž› ‘—‹ ƒ–‡• –Š‡ Ǯ–”ƒˆˆ‹  Œƒǯ ƒ–  –‘ –Š‡ ‡–‹”‡ ’ƒ–Š™ƒ›Ǥ.  •‹‰‡–ƒ„‘Ž‹ ‘–”‘ŽƒŽ›•‹•ƒ†”‡Žƒ–‡†ƒ’’”‘ƒ Š‡•ǡ™‡‹†‡–‹ˆ‹‡†ƒ’‹˜‘–ƒŽ”‘Ž‡ˆ‘”–Š‡ Žƒ•–‡œ›‡‘ˆ–Š‡’ƒ–Š™ƒ›ǡǡˆ‘”•‡“—‡•–”ƒ–‹‘‘ˆ‘ƒ† ‘’‡–‹–‹˜‡‹Š‹„‹–‹‘‘ˆ „›‹–•ƒ ›ŽǦ‘’”‘†— –•ǤŠ‹•ˆ‹†‹‰‹•‹•Šƒ”’ ‘–”ƒ•––‘‘—”‡ƒ”Ž‹‡”•’‡ —Žƒ–‹‘ –Šƒ––Š‡—–—ƒŽ ‘’‡–‹–‹‘„‡–™‡‡‡œ›‡substrates‹‰Š– ƒ—•‡–Š‡ˆŽ—š†‡ Ž‹‡ȏͺȐǤ Š‡ ‡Ž— ‹†ƒ–‡† ‡ Šƒ‹• ƒ› Šƒ˜‡ ‡†‹ ƒŽ ‹’Ž‹ ƒ–‹‘•ǡ „‘–Š ˆ‘” ’ƒ–‹‡–• ™‹–Š ‹„‘” †‡ˆ‡ –•‹–Š‡ ƒ†ˆ‘”—Ž–‹ˆƒ –‘”‹ƒŽ†‹•‡ƒ•‡•‹™Š‹ Š ‹•‹’Ž‹ ƒ–‡†Ǥ ‡Šƒ˜‡ ’”‡˜‹‘—•Ž› •Š‘™ –Šƒ–  †‡ˆ‹ ‹‡ › ƒ‡• –Š‡   ’ƒ–Š™ƒ› •—• ‡’–‹„Ž‡ –‘ „‡ ‘‡ ‘˜‡”Ž‘ƒ†‡†„›ƒŠ‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘ȏͻȐǤŠ‹• ƒ„‡—†‡”•–‘‘†‹–Š‡Ž‹‰Š–‘ˆ–Š‡ ˜‹ ‹‘—• › Ž‡Ǥ

(46) ‹–•‡Žˆǡ–Š‡‡œ›‡‹•‘–‡••‡–‹ƒŽǡ•‹ ‡”‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„› ƒ”‡–ƒ‡‘˜‡”„›ǡǡƒ†ȋ”‘†‡–•ǡ ‹‰ͳȌ‘”„›ƒ†ȋŠ—ƒ•Ȍ ȏͳ͸ȐǤ ‘™‡˜‡”ǡ–Š‡Žƒ––‡”ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡•Šƒ˜‡ƒ™‡ƒ‡”ƒˆˆ‹‹–›ˆ‘”–Š‡‡†‹—Ǧ ͷʹ.

(47) The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation Šƒ‹ ƒ ›ŽǦ‘ ‡•–‡”• –Šƒ  ȏͳ͹ǡͳͺȐǡ –Š‡”‡„› ‡Šƒ ‹‰ ƒ ›ŽǦ‘ ƒ ——Žƒ–‹‘ ƒ† ’”‘†— – ‹Š‹„‹–‹‘ ‘ˆ Ǥ ƒŽ‘‰‘—•Ž›ǡ –Š‡ ˜‹ ‹‘—• › Ž‡ ƒ› ’Žƒ› ƒ ”‘Ž‡ ‹ ‘„‡•‹–›Ǧ ”‡Žƒ–‡†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡•ǡ™Š‡”‡ƒ‹–‘ Š‘†”‹ƒŽ•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†Šƒ•„‡‡’”‘’‘•‡†–‘ Ž‡ƒ†–‘ƒ‡–ƒ„‘Ž‹ ‰”‹†Ž‘ ȏͳͻȐǤŠ‡˜‹ ‹‘—• › Ž‡™‘—Ž††‡ˆ‹‹–‡Ž›‡Šƒ ‡–Š‡”‹•‘ˆ•— Š ‡–ƒ„‘Ž‹  ‘’Ž‹ ƒ–‹‘•‘ˆŠ‹‰Š ‹” —Žƒ–‹‰ˆƒ––›Ǧƒ ‹† ‘ ‡–”ƒ–‹‘•‹‘„‡•‹–›Ǥ

(48) –Š‹••‡ –‹‘™‡™‹ŽŽ—•‡‘—”‡™‹•‹‰Š–•–‘„‡––‡”—†‡”•–ƒ†Š‘™–Š‡’ƒ–Š™ƒ›•–”— –—”‡ ƒ› ’Žƒ› ƒ ”‘Ž‡ ‹ †‹•‡ƒ•‡ǡ ’ƒ”–‹ —Žƒ”Ž› ”‡‰ƒ”†‹‰ –Š‡ Žƒ  ‘ˆ ”‘„—•–‡•• ‘ˆ ‰Ž— ‘•‡ ƒ† ‡–‘‡Ǧ„‘†› Š‘‡‘•–ƒ•‹• ‹ ’ƒ–‹‡–• ™‹–Š ‹Š‡”‹–‡†   †‡ˆ‡ –•Ǥ ”‘†— –• ‘ˆ –Š‡   ƒ”‡ ”‡“—‹”‡† ƒ• ‡‡”‰› •‘—” ‡ ˆ‘” de novo ‰Ž— ‘•‡ ’”‘†— –‹‘ ƒ† ƒ• •—„•–”ƒ–‡ ˆ‘” ‡–‘‰‡‡•‹•Ǥ‡Š›’‘–Š‡•‹œ‡–Š‡”‡ˆ‘”‡–Šƒ––Š‡ •–‡‡’ˆŽ—š†‡ Ž‹‡—’‘ •—„•–”ƒ–‡‘˜‡”Ž‘ƒ† ’Žƒ›• ƒ ‹’‘”–ƒ– ”‘Ž‡ ‹ –Š‹• ‡–ƒ„‘Ž‹  ˆ”ƒ‰‹Ž‹–›Ǥ ‡› ‡Ž‡‡–• ‘ˆ –Š‡ ‡Ž— ‹†ƒ–‡† ˜‹ ‹‘—• › Ž‡ ƒ”‡ ȋ‹Ȍ –Š‡ Š‹‰Š ˆŽ—š ‘–”‘Ž ‘ˆ  ƒ† ’”‘†— – ‹Š‹„‹–‹‘ ‘ˆ –Š‹• ‡œ›‡ „› ƒ ‘’‡–‹‰ •‡– ‘ˆ ƒ ›ŽǦ‘ ‡•–‡”•ǡ Ž‡ƒ†‹‰ –‘ ‡–‘ƒ ›ŽǦ‘ ƒ ——Žƒ–‹‘Ǣ ȋ‹‹Ȍ –Š‡ ƒ’Ž‹ˆ‹ ƒ–‹‘ ‘ˆ ‘Ǧ‡•–‡” ƒ ——Žƒ–‹‘ „› Ȁ  ƒ† –Š‡ ”‘Ž‡ ‘ˆ –Š‡ ȏ ΪȐȀȏ Ȑ ”ƒ–‹‘ –Š‡”‡‹Ǣ ƒ† ȋ‹‹‹Ȍ –Š‡ ”‡•—Ž–‹‰ †‡’Ž‡–‹‘ ‘ˆ –Š‡ ˆ”‡‡ ‘ ‘ ‡–”ƒ–‹‘ ™Š‹ Š ˆ—”–Š‡” Ž‹‹–•–Š‡ƒ –‹˜‹–›Ǥ‡™‹ŽŽ•–”— –—”‡‘—”†‹• —••‹‘ƒ”‘—†–Š‡•‡–Š”‡‡‡Ž‡‡–•ƒ† –Š‡‹” ‘‡ –‹‘ –‘ –Š‡ •—””‘—†‹‰ ‡–ƒ„‘Ž‹  ‡–™‘”Ǥ Š‹• ™‹ŽŽ ‰‹˜‡ ‹•‹‰Š– ‹–‘ Š‘™ ’‡”–—”„ƒ–‹‘• ‹ –Š‡ •—””‘—†‹‰ ‡–ƒ„‘Ž‹  ‡–™‘” ƒ› •–ƒ„‹Ž‹œ‡ ‘” †‡•–ƒ„‹Ž‹œ‡ –Š‡ ’ƒ–Š™ƒ›ƒ†–Š‡”‡„›ƒ––‡—ƒ–‡‘”ƒ‰‰”ƒ˜ƒ–‡–Š‡”‹•‘ˆ‡–ƒ„‘Ž‹ ˆƒ‹Ž—”‡Ǥ MCKAT – Š‡ ‡Ž— ‹†ƒ–‡† ˜‹ ‹‘—• › Ž‡ •–ƒ”–‡† ƒ– Ǥ – Ž‘™ ’ƒŽ‹–‘›ŽǦ‘ •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘• –Š‡ ˆ‹”•– ‡œ›‡ ‘ˆ –Š‡ ’ƒ–Š™ƒ›ǡ ͳ ‡š‡”–‡† ƒ Š‹‰Š ˆŽ—š ‘–”‘Žǡ ‹ ƒ‰”‡‡‡– ™‹–Š ‡ƒ•—”‡‡–• ‹ ”ƒ– Ž‹˜‡” ȏʹͲǡʹͳȐǤ ‘™‡˜‡”ǡ ‘ ‡ –Š‡ ’ƒ–Š™ƒ› „‡ ƒ‡ ‘˜‡”Ž‘ƒ†‡†™‹–Š ’ƒŽ‹–‘›Ž ‘ǡ  –‘‘‘˜‡” ‘–”‘Ž ‘˜‡” –Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ ˆŽ—š ‹ –Š‡ ‘†‡ŽǤ

(49)  ƒ ‘’”‡Š‡•‹˜‡ Ž‹–‡”ƒ–—”‡ ”‡˜‹‡™ ƒ„‘—– ‘–”‘Ž ‘ˆ –Š‡ ȾǦ‘š‹†ƒ–‹‘ ’ƒ–Š™ƒ›ǡ ƒ–‘ ȏʹͳȐ •Š‘™‡† –Šƒ–  ‹‰Š– ‹†‡‡† Ž‹‹– –Š‡   ˆŽ—š ‹ Ž‹˜‡” ‹–‘ Š‘†”‹ƒ —†‡” ‡”–ƒ‹ ‘†‹–‹‘•Ǥ Š‹• ‘ Ž—•‹‘ ™ƒ• „ƒ•‡† ‘ •–—†‹‡• ™‹–Š  ‹Š‹„‹–‘”• ™Š‹ Š ™‡”‡ ˆ‘—† –‘ ƒˆˆ‡ – –Š‡   ˆŽ—š ȏʹʹǡʹ͵ȐǤ  ‡š’‡”‹‡–ƒŽ “—ƒ–‹ˆ‹ ƒ–‹‘ ‘ˆ ˆŽ—š ‘–”‘Ž•Š‘™‡†–Šƒ–•–ƒ”–‡†–‘ ‘–”‘Ž–Š‡ˆŽ—š‘Ž›ƒ–Š‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘• ȏʹͶȐǤŠ‹• ‘ˆ‹”•‘—””‡•—Ž––Šƒ– ‘–”‘Ž•Š‹ˆ–•†‡’‡†‹‰‘–Š‡•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘ ƒ†–Š‡”‡„›‰‹˜‡•ˆ—”–Š‡” ”‡†‹„‹Ž‹–›–‘–Š‡ ‘’—–ƒ–‹‘ƒŽ‘†‡ŽǤ ‡ ‡–Ž›ǡ –Š‡ ‹–‘ Š‘†”‹ƒŽ Š  ‹•‘ˆ‘” ’Ͷ͸Š  ™ƒ• ‹†‡–‹ˆ‹‡† ƒ• ƒ •’‡ ‹ˆ‹ ǡ ‡†‘‰‡‘—• ‹Š‹„‹–‘” ‘ˆ  ȏʹͷȐǤ Š  ‘ †‘™ ‹ ”‡ƒ•‡† ‹–‘ Š‘†”‹ƒŽ ’ƒŽ‹–ƒ–‡ ‘š‹†ƒ–‹‘ǡ ˆ—”–Š‡” ‘””‘„‘”ƒ–‹‰ –Š‡ ‘–‹‘ –Šƒ–  ƒ› Ž‹‹– –Š‡ ˆŽ—š in vivoǤ

(50) –‡”‡•–‹‰Ž›ǡ Š  ‘ †‘™ƒŽ•‘Ž‡†–‘ƒ–‡†‡ ›‘ˆ‹ ”‡ƒ•‡†Ž‹ˆ‡•’ƒ‹‹ ‡ȏʹͷȐƒ†–Š‡ ‘””‡•’‘†‹‰ SHC1Ž‘ —•Šƒ•„‡‡ƒ••‘ ‹ƒ–‡†™‹–ŠŽ‘‰‡˜‹–›‹ƒǤƒ•‡†‘‘—””‡•—Ž–•ǡ–Š‹•ƒ›„‡†—‡ –‘ ‹ ”‡ƒ•‡†   ’‡” •‡ ‘” –‘ ƒ ”‡Ž‹‡ˆ ‘ˆ ȋ–‘š‹ Ȍ ‘Ǧ‡•–‡” ƒ ——Žƒ–‹‘Ǥ

(51) – ™‹ŽŽ „‡ ‹–‡”‡•–‹‰ –‘ ‹˜‡•–‹‰ƒ–‡ ‹ˆ –Š‡ ‡š’”‡••‹‘ ‘ˆ –Š‡ ‰‡‡• ‡ ‘†‹‰  ȋʹȌ ‘” ‹–• ‹Š‹„‹–‘” SHC1 ‹‰Š– †‹ˆˆ‡”‡–‹ƒ–‡ „‡–™‡‡ Ǧ†‡ˆ‹ ‹‡– ’ƒ–‹‡–• –Šƒ– ’”‡•‡–‡† ™‹–Š Š›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒƒ†–Š‘•‡–Šƒ–‡˜‡”†‹†Ǥ M/SCHAD and the mitochondrial redox state – Š‡ ‹–‘ Š‘†”‹ƒŽ ȏΪȐȀȏ Ȑ ”ƒ–‹‘ ‘‡ –• –Š‡   ’ƒ–Š™ƒ› –‘ –Š‡ ”‡•’‹”ƒ–‘”› Šƒ‹ ƒ† –‘ ‘–Š‡”   ’”‘†— ‹‰ ’ƒ–Š™ƒ›•Ǥ‡•Š‘™‡†–Šƒ––Š‡ȏΪȐȀȏ Ȑ”ƒ–‹‘ƒˆˆ‡ –•–Š‡ƒ ——Žƒ–‹‘‘ˆ‘‡•–‡”• ˜‹ƒ –Š‡ Ž‘•‡Ǧ–‘Ǧ‡“—‹Ž‹„”‹— ”‡ƒ –‹‘ Ȁ Ǥ

(52) – ‹• ’Žƒ—•‹„Ž‡ǡ –Š‡”‡ˆ‘”‡ǡ –Šƒ– ‹†‹˜‹†—ƒŽ †‹ˆˆ‡”‡ ‡•‹–Š‡ƒ –‹˜‹–›‘ˆ–Š‡”‡•’‹”ƒ–‘”› Šƒ‹‘†—Žƒ–‡–Š‡”‹•‘ˆ•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†Ǥ. ͷ͵. 2.

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