The mechanism of synthesis of fatty acids by the pigeon liver enzyme system

Phillips, G. T.; Nixon, J.E.; Dorsey, Judith A.; Butterworth, Peter H. W.; Chesterton, C.J.; Porter, John W.

doi:10.1016/0003-9861(70)90360-7

Cited by 63 publications

(8 citation statements)

References 11 publications

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“…193,210 Moreover, the observation that acetyl and malonyl groups bound to the hydroxyl site of the animal (pigeon) FAS and formed stable intermediates indicates that the acyl moieties are, in a first stage, transferred to the catalytic serine of the MAT domain of FAS and, in a second stage, loaded into the ACP domain. 200,202 This serine-centered, two-stage transfer reaction is consistent with a ping-pong bi-bi mechanism 211 that is shared among the AT enzymes/domains of all FAS types. 47,186,191,193,212−215 Peptide mapping studies reported that the catalytic serine of the MAT occupies the position 581 in the animal (goat) FAS (unless otherwise stated the residue numbering always corresponds to the homologous human enzyme, UniProtKB: P49327), located within a highly conserved G-X-S-Y-G sequence that lies at a sharp turn between an α-helix and a βstrand (Figure 4B).…”

Section: The Catalytic Mechanism Of Animal Fasmentioning

confidence: 52%

Animal Fatty Acid Synthase: A Chemical Nanofactory

et al. 2021

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Fatty acids are crucial molecules for most living beings, very well spread and conserved across species. These molecules play a role in energy storage, cell membrane architecture, and cell signaling, the latter through their derivative metabolites. De novo synthesis of fatty acids is a complex chemical process that can be achieved either by a metabolic pathway built by a sequence of individual enzymes, such as in most bacteria, or by a single, large multi-enzyme, which incorporates all the chemical capabilities of the metabolic pathway, such as in animals and fungi, and in some bacteria. Here we focus on the multi-enzymes, specifically in the animal fatty acid synthase (FAS). We start by providing a historical overview of this vast field of research. We follow by describing the extraordinary architecture of animal FAS, a homodimeric multi-enzyme with seven different active sites per dimer, including a carrier protein that carries the intermediates from one active site to the next. We then delve into this multi-enzyme’s detailed chemistry and critically discuss the current knowledge on the chemical mechanism of each of the steps necessary to synthesize a single fatty acid molecule with atomic detail. In line with this, we discuss the potential and achieved FAS applications in biotechnology, as biosynthetic machines, and compare them with their homologous polyketide synthases, which are also finding wide applications in the same field. Finally, we discuss some open questions on the architecture of FAS, such as their peculiar substrate-shuttling arm, and describe possible reasons for the emergence of large megasynthases during evolution, questions that have fascinated biochemists from long ago but are still far from answered and understood.

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Section: The Catalytic Mechanism Of Animal Fasmentioning

confidence: 52%

Animal Fatty Acid Synthase: A Chemical Nanofactory

et al. 2021

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“…Second, the 'post-malonate' exchange may occur after the formation of RCH=CHCOSX in the cycle of synthesis. The final stages of the cycle are the formation of RCH2CH2-COSX and its transfer to another sulphydryl group in /COzH l c o s x the enzyme complex [5,16] and it is possible to formulate mechanisms for partial base-catalysed exchange of the methylene hydrogens indicated in boldface type during (or between) these two steps. An interesting (and experimentally testable) consequence would be that the surviving hydrogen from malonate would not necessarily occupy a specific stereochemical position in the product palmitate: for example, the -CH'H-groups in palmitate synthesised from HOZCC~HZCOSCOA would be partially racemised as a result of non-stereospecific hydrogen exchange.…”

Section: Nature Of 'Post-malonate' Exchangementioning

confidence: 99%

“…The thiol ester intermediates in Scheme 2 have been designated by the general formula RCOSX, where the thiol carrier X may be CoA, acyl-carrier protein (ACP) as in many plants [S] and in most bacterial cells [14,15], enzyme-bound thiol groups as in yeast [5] and avian liver [16,17] or model thiols such as panthetheine and N-acetyl-cysteamine which can be used for assays of many synthetase preparations in vitro [6,11,13,181.…”

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confidence: 99%

The Biosynthesis of Long-Chain Fatty Acids. Stereochemical Differentiation in the Enzymic Incorporation of Chiral Acetates

Sedgwick

Cornforth

1977

Eur J Biochem

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1. Acetyl-CoA carboxylase and fatty acid synthetase were purified from chicken liver. Fatty acid synthetase was also purified from bakers' yeast.2. The corresponding CoA thiol esters were prepared enzymically from R-[2H1, 3H~]acetate, S-[2H1, 3Hl]acetate and [3Hl]acetate, using a linked acetate kinase/phosphotransacetylase system. [2-14C]Acetate was added to the tritiated specimens to provide doubly-labelled products, which were purified by column chromatography on DEAE-cellulose.3. The three acetyl-CoA specimens thus obtained were incubated separately in a combined acetylCoA carboxylase/fatty acid synthetase system containing the cofactors necessary for synthesis of long-chain fatty acids.4. The fatty acids formed were extracted into n-pentane, methylated, separated by gas-liquid chromatography and the 3H/14C isotope ratio determined.5. In this manner it was shown, using the chicken liver synthetase, that in palmitic acid a higher proportion of tritium was retained from S-[2-I4C, 2H1, 'Hl]acetyl-CoA than from the R-(2-14C, 2 H ,, 3H1)-labelled thiol ester, the non-chiral (2-14C, 3H1)-labelled substrate giving an intermediate result.6. This result was confirmed using the fatty acid synthetase preparation purified from bakers' yeast in place of the corresponding chicken liver enzyme.7. In a separate experiment it was shown that the intramolecular tritium isotope effect in the carboxylation of [3Hl]acetyl-CoA is normal, but small. The slightly smaller, normal deuterium isotope effect calculated from this was shown to be in good agreement with the value expected, based on the experiments on tritium incorporation from chiral acetyl-CoA.8. It is demonstrated that partial exchange of carbon-bound hydrogen must occur on the synthetase during the enzymic synthesis of fatty acids from malonyl thiol esters and that the extent of this exchange differs in synthetases of different origin.9. A theoretical treatment of the effect of partial hydrogen exchange on the chirality of asymmetrically labelled methylene groups is given.10. The results indicate the existence of an overall stereospecificity in the reactions involved in the de novo biosynthesis of long-chain fatty acids in different cell types. ~~~Abbreviations.

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“…The assignment of a Ping Pong mechanism to the f,-oxoacyl synthetase reaction is based on the assumption that Euglena FAS, like the pigeon liver and yeast enzymes, has a 'peripheral' thiol site which covalently binds acetyl residues (Chesterton et al, 1968;Joshi et al, 1970;Lynen et al, 1967;Phillips et al, 1970a,b) and is essential for condensation to occur (Nixon et al, 1970;Phillips et al, 1970b). Condensation in E. coli proceeds by a two part reaction in which an acetyl (or acyl) group is transferred from acetyl-(or acyl-) ACP to a covalent binding site on the f,-oxo acyl-ACP synthetase enzyme, followed by condensation with malonyl- ACP (D'Agnolo et al, 1975;Greenspan et al, 1969;Toomey & Wakil, 1966).…”

Section: Discussionmentioning

confidence: 99%

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris

Walker¹,

Jonak²,

Worsham³

et al. 1981

Biochemical Journal

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A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.

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The mechanism of synthesis of fatty acids by the pigeon liver enzyme system

Cited by 63 publications

References 11 publications

Animal Fatty Acid Synthase: A Chemical Nanofactory

Animal Fatty Acid Synthase: A Chemical Nanofactory

The Biosynthesis of Long-Chain Fatty Acids. Stereochemical Differentiation in the Enzymic Incorporation of Chiral Acetates

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris

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