Iso-coenzyme A

Abstract

Iso-coenzyme A is an isomer of coenzyme A in which the monophosphate is attached to the 2-carbon of the ribose ring. Although iso-CoA was first reported in 1959 (Moffatt, J. G., and Khorana, H. G. (1959) J. Am. Chem. Soc. 81, 1265-1265) to be a by-product of the chemical synthesis of CoA, relatively little attention has been focused on iso-CoA or on acyl-iso-CoA compounds in the literature. We now report structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and ␤-hydroxybutyryl-iso-CoA using mass spectrometry (MS), tandem MS, and homonuclear and heteronuclear NMR analyses. Although the 2-phosphate isomer of malonyl-CoA was recently identified in commercial samples, previous characterizations of iso-CoA itself have been based on chromatographic analyses, which ultimately rest on comparisons with the degradation products of CoA and NADPH or have been based on assumptions regarding enzyme specificity. We describe a high performance liquid chromatography methodology for separating the isomers of several CoA-containing compounds. We also report here the first examples of isoCoA-containing compounds acting as substrates in enzymatic acyl transfer reactions. Finally, we describe a simple synthesis of iso-CoA from CoA, which utilizes ␤-cyclodextrin to produce iso-CoA with high regioselectivity, and we demonstrate a plausible mechanism that accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. We anticipate that these results will provide methodology and impetus for investigating iso-CoA compounds as potential pseudo-substrates or inhibitors of the >350 known CoA-utilizing enzymes.Iso-coenzyme A is an isomer of coenzyme A in which the monophosphate is attached to the 2Ј-carbon of the ribose ring. Iso-CoA was first reported in 1959 by Moffatt and Khorana (1, 2) to be a by-product of the chemical synthesis of CoA. The final step of this synthesis entailed acid-catalyzed hydrolysis of cyclic coenzyme A (Scheme 1) to produce a 50:50 mixture of two products that were separated using epicholorohydrin triethanolamine (ECTEOLA) cellulose ion exchange chromatography (1, 2). Direct structural analysis was not readily available in 1959; consequently, the authors deduced the structure of isoCoA by establishing that, in contrast to CoA, iso-CoA was not a substrate for the enzyme phosphotransacetylase (1, 2). Moffatt and Khorana also used paper chromatography of phosphodiesterase hydrolysates to show that enzymatic hydrolysis of isoCoA produces adenosine-2Ј,5Ј-diphosphate, exclusively (1, 2). Since the original work of Moffatt and Khorana was done, four additional chemical syntheses of CoA have been reported (3-8), all of which produce cyclic CoA as the penultimate product; cyclic CoA is then hydrolyzed either chemically to produce a mixture of CoA and iso-CoA (1, 2, 4 -6, 8) or enzymatically with ribonuclease T2 to produce CoA regioselectively (3-7).Recently, Minkler et al. (9) observed the 2Ј-phosphate isomer of malonyl-CoA in commercial samples. These authors ...

LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters

“…The 2′-phosphate isomer of coenzyme A isomer is called isocoenzyme A [25]. Burns and coauthors [16] separated CoASH and iso-CoASH as well as several pairs of acylCoA and acyl-iso-CoA in commercially available standards via RPLC and characterized those using MS/MS and NMR. They discovered that iso-CoAs acted as substrates instead of CoAs in enzyme-catalyzed reactions.…”

“…Reversed phase liquid chromatography employing ion-pairing reagents (ion-pair RPLC) was addressed as the method of choice suitable for a broad spectrum of short-chain acyl-CoA analysis [11,12,13,14,15]. Alternately, a comparatively smaller selection of these target metabolites has been separated by reversed phase liquid chromatography without the use of ion-pairing reagents [16,17,18,19]. In studies where short-chain acyl-CoAs were measured within a comprehensive primary metabolome analysis, ion-pair RPLC was applied [20,21].…”

Metabolic Vulnerabilities in Endometrial Cancer

“…These data indicated that BrPA was directly interfering with CoA at its free thiol group. Using a previously published ionization pattern for CoA (29), we demonstrated by mass spectrometry that pyruvylation occurs at the sulfhydryl group of CoA (Supplementary Fig. S6).…”

Malonyl carba(dethia)‐ and Malonyl oxa(dethia)‐coenzyme A as Tools for Trapping Polyketide Intermediates

“…These species were absent in control samples and their MS/MS fragmentation pattern was consistent with that of coenzyme A derivatives. [43] While the assignment of m/z 922 (Figure 1 A) and 964 (Figure 1 B) to the diketide 21 a and the triketide 22 a, respectively, was clear-cut, two distinct peaks were found for m/z 1006 at 15.9 and 19.9 min (Figure 1 C and E). Their different MS/MS fragmentation patterns allowed their assignment to the nonhydrolyzable tetraketide 23 a (Figure 1 C) and to the dehydrated, possibly cyclized, natural tetraketide 25, respectively (Figure 1 E), with the latter showing the m/z 768 fragment diagnostic for a coenzyme A thioester.…”