acyl-CoA Analogs Research Articles

The Mechanism of Selective Recognition of Lipid Substrate by hDHHC20 Enzyme

S-acylation is a post-translational linkage of long chain fatty acids to cysteines, playing a key role in normal physiology and disease. In human cells, the reaction is catalyzed by a family of 23 membrane DHHC-acyltransferases (carrying an Asp-His-His-Cys catalytic motif) in two stages: (1) acyl-CoA-mediated autoacylation of the enzyme; and (2) further transfer of the acyl chain to a protein substrate. Despite the availability of a 3D-structure of human acyltransferase (hDHHC20), the molecular aspects of lipid selectivity of DHHC-acyltransferases remain unclear. In this paper, using molecular dynamics (MD) simulations, we studied membrane-bound hDHHC20 right before the acylation by C12-, C14-, C16-, C18-, and C20-CoA substrates. We found that: (1) regardless of the chain length, its terminal methyl group always reaches the "ceiling" of the enzyme's cavity; (2) only for C16, an optimal "reactivity" (assessed by a simple geometric criterion) permits the autoacylation; (3) in MD, some key interactions between an acyl-CoA and a protein differ from those in the reference crystal structure of the C16-CoA-hDHHS20 mutant complex (probably, because this structure corresponds to a non-native dimer). These features of specific recognition of full-size acyl-CoA substrates support our previous hypothesis of "geometric and physicochemical selectivity" derived for simplified acyl-CoA analogues.

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80-114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

Kinetic and Chemical Mechanism of α-Isopropylmalate Synthase from Mycobacterium tuberculosis

Mycobacterium tuberculosis alpha-isopropylmalate synthase (MtIPMS) catalyzes the condensation of acetyl-coenzyme A (AcCoA) with alpha-ketoisovalerate (alpha-KIV) and the subsequent hydrolysis of alpha-isopropylmalyl-CoA to generate the products CoA and alpha-isopropylmalate (alpha-IPM). This is the first committed step in l-leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and (1)H-NMR spectroscopy. The alpha-keto acid specificity of the enzyme is narrow, and the acyl-CoA specificity is absolute for AcCoA. In the absence of alpha-KIV, MtIPMS does not enolize the alpha protons of AcCoA but slowly hydrolyzes acyl-CoA analogues. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random bi-bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pK(a) values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of alpha-isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate-limiting, although, with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of alpha-isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. On the basis of these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed.

Profiling a Taxol Pathway 10β-Acetyltransferase: Assessment of the Specificity and the Production of Baccatin III by In Vivo Acetylation in E. coli

The 10beta-acetyltransferase on the biosynthetic pathway of the antineoplastic drug Taxol catalyzes the regiospecific transfer of the acetyl group of acetyl-coenzyme A (CoA) to 10-deacetylbaccatin III. We demonstrate that in addition to acetyl group transfer, the overexpressed enzyme also catalyzes the exchange of propionyl and n-butyryl from the corresponding CoA thioester to the hydroxyl group at C10 of the cosubstrate. Also, in vivo studies revealed that E. coli, producing endogenous acetyl-CoA and overexpressing the recombinant acetyltransferase, can convert exogenously supplied 10-deacetylbaccatin III to baccatin III. Potentially, this heterologous in vivo production method in bacteria could be optimized to couple various unnatural acyl-CoA analogs to myriad amino and/or hydroxyl acceptors by acyltransferase catalysis; conceivably, this process could facilitate the preparation of second-generation Taxols.

The use of Pseudomonas acyl-CoA synthetase to form acyl-CoAs from dicarboxylic fatty acids

Pseudomonas acyl-CoA synthetase is shown to act on saturated dicarboxylic acids with a chain length of C10 or greater to produce conjugates containing a single CoA unit. The synthetase can, therefore, be used to generate novel acyl-CoA analogues for studies on proteins that utilise, bind to, or are modulated by acyl-CoAs.

Synthesis of azidophospholipids and labeling of lysophosphatidylcholine acyltransferase from developing soybean cotyledons

A photoreactive substrate analog of lysophosphatidylcholine (LPC), 1-[(4-azidosalicyl)-12-amino]dodecanoyl- sn-glycerol-3-phosphocholine (azido-LPC) was synthesized. Fast atom bombardment mass spectrometry was employed to confirm the structures of azido-LPC and its intermediates. Azido-LPC was used to label putative acyl-CoA:LPC acyltransferase from microsomal membranes of developing soybean cotyledons. The synthesized substrate analog acts as a substrate for the target acyltransferases and phospholipases in the dark. When the microsomal membranes were incubated with the acyl acceptor analog and immediately photolyzed, LPC acyltransferase was irreversibly inhibited. Photoinactivation of the enzyme by the photoprobe decreased in the presence of LPC. Microsomal membranes were photolyzed with 125I-labeled azido-LPC and analyzed by SDS-PAGE followed by autoradiography. These revealed that the analog preferentially labeled 54- and 114-kDa polypeptides. Substrate protected the labeling of both the polypeptides. In our earlier report, the same polypeptides were also labeled with photoreactive acyl-CoA analogs, suggesting that these polypeptides could be putative LPC acyltransferase(s). These results demonstrated that the photoreactive phospholipid analog could be a powerful tool to label acyltransferases involved in lipid biosynthesis.

A Reversed Thioester Analogue of Acetyl-Coenzyme A: An Inhibitor of Thiolase and a Synthon for Other Acyl-CoA Analogues

We have previously reported a general synthetic approach to analogues of coenzyme A (CoA) and CoA esters using a combination of enzymatic and nonenzymatic reactions (Martin et al. J. Am. Chem. Soc....

Titration Calorimetric Analysis of AcylCoA Recognition by MyristoylCoA:Protein N-Myristoyltransferase

Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that catalyzes the transfer of myristic acid (C14:0) from myristoylCoA to the N-terminus of cellular proteins with a variety of functions. Nmts from an assortment of species display remarkable in vivo specificity for this rare acyl chain. To better understand the mechanisms underlying this specificity, we have used isothermal titration calorimetry as well as kinetic measurements to study the interactions of Nmt1p with acylCoA analogs having variations in chain length and/or conformation, analogs with alterations in the thioester bond, and analogs with or without a 3'-phosphate in their CoA moiety. MyristoylCoA binds to Nmt1p with a Kd of 15 nM and a large exothermic deltaH (-25 kcal/mol). CoA derivatives of C12:0-C16:0 fatty acids bind to Nmt1p with similar affinity, but with much smaller deltaH and a correspondingly less negative TdeltaS than myristoylCoA. Replacing the thioester carbonyl group with a methylene or removing the 3'-phosphate of CoA is each sufficient to prevent the low enthalpy binding observed with myristoylCoA. The carbonyl and the 3'-phosphate have distinct and important roles in chain length recognition over the range C12-C16. Acyltransferase activity parallels binding enthalpy. The naturally occurring cis-5-tetradecenoylCoA and cis-5,8-tetradecadienoylCoA are used as alternative Nmt substrates in retinal photoreceptor cells, even though they do not exhibit in vitro kinetic or thermodynamic properties that are superior to those of myristoylCoA. The binding of an acylCoA is the first step in the enzyme's ordered reaction mechanism. Our findings suggest that within cells, limitation of Nmt substrate usage occurs through control of acylCoA availability. This indicates that full understanding of how protein acylation is controlled not only requires consideration of the acyltransferase and its peptide substrates but also consideration of the synthesis and/or presentation of its lipid substrates.

25] Synthesis of nonhydrolyzable acyl-coenzyme A analogs

This chapter describes the chemical synthesis of different precursors of the nonhydrolyzable acyl–CH 2 CoA derivatives. The final synthesis of various nonhydrolyzable acyl–CH 2 CoA derivatives is also described. The activity of pantetheine phosphate adenylyltransferase is assayed in the reverse direction by monitoring the production of ATP from dephospho–CoA and PP i . Dephospho–CoA kinase is assayed by monitoring the production of ADP from dephospho-CoA and ATP by using the standard pyruvate kinase/lactate dehydrogenase coupling enzyme system. The two key intermediates for the chemical synthesis of acyldethia-coenzyme A analogs are acyldethia(carba)pantetheine 4-phosphate and adenosine 2',3'-cyclophosphate 5'-phosphomorpholidate. The final steps in the synthesis of acyl–CH 2 CoA depend on whether its precursors are obtained by the chemical or the chemoenzymatic strategy. Nonhydrolyzable acyl–CoA analogs are useful as alternative substrates, inhibitors, and regulators of a large number of enzymatic reactions. Their enhanced stability may be advantageous not only in mechanistic investigations but also in drug development. The chapter describes some conventional chemical syntheses of nonhydrolyzable acyl–CoA analogs and also a more recent chemoenzymatic strategy for their preparation.

Coenzyme A Hemithioacetals as Easily Prepared Inhibitors of CoA Ester-Utilizing Enzymes

Hemithioacetals are formed by reactions of coenzyme A (CoA) with aldehydes in aqueous solution. Equilibria for hemithioacetal formation with four commercially available aldehydes and rate constants for hemithioacetal dissociation have been studied. The hemithioacetals are viewed as acyl-CoA analogs having a tetrahedral center in place of the planar trigonal thioester carbonyl carbon. These compounds may serve as mimics of the tetrahedral intermediate or transition state in the reactions of acyl-CoA dependent acyltransferase enzymes. The hemithioacetal generated by reaction of CoA with formaldehyde is a poor inhibitor of chloramphenicol acetyltransferase, with a Ki more than 6-fold higher than the Km for the substrate acetyl-CoA. The hemithioacetals formed by reaction of CoA with acetaldehyde and trifluroacetaldehyde are substantially better inhibitors, with Ki values approximately 2.4-fold and 10-fold lower than the Km values for acetyl-CoA, respectively. The hemithioacetal formed by reaction of CoA with succinic semialdehyde inhibits succinic thiokinase, with a Ki 4-fold lower than the Km for the substrate succinyl-CoA. The CoA hemithioacetals provide a novel readily accessible new class of acyl-CoA analogs for use in mechanistic and structural studies of CoA ester-utilizing enzymes.

Efficient Synthesis of Radiolabeled Propionyl-Coenzyme A and Acetyl-Coenzyme A

The efficient microscale synthesis of [1- 14C]propio-nyl-CoA from commercially available sodium [1- 14C]propionate using 1,1′-carbonyldiimidazole in yields of nearly 70% is reported for the first time. A substantial improvement in the process for making [1- 14C]acetyl-CoA from sodium [1- 14C]acetate was also achieved. Yields of greater than 90% were consistently obtained for the latter synthesis. The salt-free CoA-thioesters were obtained in homogenous form by reverse-phase HPLC. The products were judged to be pure by 1H NMR analysis: neither iso-CoA analogs nor contaminants frequently found in commercial samples could be detected. The samples of acetyl- and propionyl-CoA were shown to be radiochemically pure by HPLC and by analysis of the products of incubations with acetyl- and propionyl-CoA carboxylase. This highly efficient synthesis is a cost-effective method for the preparation of radiolabeled CoA thioesters and can easily be adapted to the production of other acyl-CoA analogs.

Photolabeling of soybean microsomal membrane proteins with photoreactive acyl-CoA analogs.

Synthesis of 32P-labeled 12-azidooleoyl-CoA and 125I-labeled 12-[(azidosalicyl)amino]dodecanoyl-CoA (ASD-CoA) was achieved. The synthesized radioactive, photoreactive reagents were tested as photoaffinity labels for acyl-CoA:lysophosphatidylcholine acyltransferase from the microsomal membranes of developing soybean cotyledons. When a mixture of microsomal membranes and the azidooleoyl-CoA or ASD-CoA were incubated in the dark, the analogs were recognized as substrate and competitive inhibitor, respectively. The enzyme preferentially utilizes unsaturated acyl-CoAs rather than saturated acyl-CoAs. Incubation of microsomal membranes with acyl-CoA analogs and immediately followed by photolysis resulted in an irreversible inhibition of lysophosphatidylcholine acyltransferase activity. Analysis of photolyzed microsomal membranes by SDS/PAGE and autoradiography revealed that azidooleoyl-CoA preferentially labeled eight acyl-CoA binding proteins, but ASD-CoA labeled only three polypeptides with molecular masses of 110, 90 and 32 kDa that are commonly labeled by both the analogs. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme against photoinactivation by azidooleoyl-CoA and ASD-CoA, respectively. The protection was profound in 110-kDa polypeptide indicating that this protein could be lysophosphatidylcholine acyltransferase. These results demonstrate that the photoaffinity of acyl-CoA analogs makes them potential probes to identify and characterize lipid biosynthetic enzymes.

Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA

Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.

Reductive half-reaction in medium-chain acyl-CoA dehydrogenase: modulation of internal equilibrium by carboxymethylation of a specific methionine residue.

Pig kidney medium-chain acyl-CoA dehydrogenase is specifically alkylated at a methionine residue by treatment with iodoacetate at pH 6.6. This residue corresponds to Met249 in the human medium-chain acyl-CoA dehydrogenase sequence [Kelly, D. P., Kim, J. J., Billadello, J. J., Hainline, B. E., Chu, T. W., & Strauss, A. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4068-4072]. The S-carboxymethylated dehydrogenase shows a drastically lowered affinity for octanoyl-CoA (from submicromolar to 65 microM), but retains about 23% of the maximal activity of the native enzyme. In addition, alkylation perturbs the internal redox equilibrium: E.FADox.octanoyl-CoA K2 in equilibrium with E.FAD2e.octenoyl-CoA K2 ranges from about 9 for the native enzyme to about 0.2 for the homogeneously modified protein. This effect is not due to a significant change in the redox potential of the free enzyme upon alkylation. Rather, carboxymethylation weakens the preferential binding of enoyl-CoA product to the reduced enzyme (K3) compared to octanoyl-CoA binding to the oxidized dehydrogenase (K1) that is required to pull the substrate thermodynamically uphill. Thus, the ratio of dissociation constants, K1/K3, decreases from about 15,000 for the native enzyme to only 330 upon carboxymethylation of Met249. Binding studies with a variety of acyl-CoA analogues and manipulation of enzyme redox potentials by substitution of the natural prosthetic group by 8-Cl-FAD confirm the thermodynamic effects of alkylation.

The reductive half-reaction in acyl-CoA oxidase from Candida tropicalis: Interaction with acyl-CoA analogues and an unusual thioesterase activity

A series of acyl-CoA analogues has been used to probe the substrate binding site and reductive half-reaction of acyl-CoA oxidase from the alkane utilizing yeast Candida tropicalis. Alkyl-SCoA thioethers, from octyl- to hexadecyl-SCoA, bind to the oxidase with progressively larger spectral perturbation of the flavin chromophore and with an incremental binding energy of about 260 cal/methylene group. The hydrocarbon binding subsite for acylCoA oxidase appears extensive and only weakly hydrophobic. CoA binding per se appears to contribute about 2.8 kcal to the observed binding energy. A number of acyl-CoA analogues such as 3-thia-acyl-, 3-oxa-acyl-, trans-3-enoyl-, and 3-keto-acyl-CoA derivatives form charge transfer complexes with the oxidase, but these long wavelength bands are both less pronounced and much less stable than those encountered with the acyl-CoA dehydrogenases. This instability reflects an intrinsic thioesterase activity of the oxidase which is observed with those ligands forming enolate to oxidized flavin charge-transfer complexes, but not with normal substrates such as palmitoyl-CoA. Chemical precedent suggests that these enzyme-bound enolates eliminate CoA via a ketene intermediate. The differences in behavior between acyl-CoA oxidase and dehydrogenase toward the ligands used in this work are discussed in terms of the need to exclude oxygen from productive encounters with substrate-reduced dehydrogenase.

Synthesis and characterization of inhibitors of myristoyl-CoA:Protein N-myristoyltransferase

Several substrate and product analogs were synthesized and tested as in vitro inhibitors of bovine brain N-myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97). At 40 μM, the acyl CoA analog, S-(2-ketopentadecyl)-CoA, completely inhibited NMT in the presence of 80 μM myristoyl CoA. Decreasing but marked inhibition was also observed with the acyl CoA analogs, S-(2-bromo-tetradecanoyl)-CoA and S-(3-(epoxymethylene)dodecanoyl)-CoA, and the multisubstrate derivative N-(2- S-CoA-tetradecanoyl)glycinamide in the presence of 40 μm myristoyl CoA. Inhibition was also observed with the non-coenzyme A myristoyl analog, 1-bromo-2-pentadecanone. All of the above compounds exhibited reversible competitive inhibition kinetics with respect to myristoyl CoA with K i, values of 0.11 to 24 μM. Two additional acyl CoA analogs, S-( cis-3-tetradecenoyl)-CoA and S-(3-tetradecynoyl)-CoA, functioned as alternative substrates for NMT.

Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA:protein N-myristoyltransferase

The importance of myristoylation for the proper biological functioning of many acylated proteins has generated interest in the enzymes of the myristoylation pathway and their interactions with substrates and inhibitors. Previous observations that S-(2-oxopentadecyl)-CoA, a nonhydrolyzable methylene-bridged analogue of myristoyl-CoA, was a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT) [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1989) J. Med. Chem. 32, 1665] prompted a closer examination of the effect of substituents at the 2-position on the interactions of myristic acid and myristoyl-CoA analogues with NMT. As an initial approach, three myristic acid derivatives bearing different substituents at the 2-position, 2-fluoromyristic acid, 2-bromomyristic acid, and 2-hydroxymyristic acid, were selected for study. Both 2-bromomyristic acid and 2-hydroxymyristic acid were available commercially; 2-fluoromyristic acid was prepared synthetically. All three compounds were found to be only weak inhibitors of NMT in vitro. Of the three, 2-bromomyristic acid was the most potent (Ki = 100 microM). In cultured cells, however, 2-hydroxymyristic acid was by far the more effective inhibitor of protein myristoylation. Neither 2-hydroxymyristic acid nor 2-bromomyristic acid significantly inhibited protein palmitoylation in cultured cells, indicating that inhibition was not occurring at the level of acyl-CoA synthetase. Activation of the 2-substituted myristic acid derivatives to their corresponding acyl-CoA thioesters by acyl-CoA synthetase resulted in inhibitors of greatly increased potency. The 2-substituted acyl-CoA analogues, 2-hydroxymyristoyl-CoA, 2-bromomyristoyl-CoA, and 2-fluoromyristoyl-CoA, were synthesized and shown to be competitive inhibitors of NMT in vitro (Ki's = 45, 450, and 200 nM, respectively). These data suggested that the enhanced inhibitory potency of 2-hydroxymyristic acid seen in cells was most probably a result of its metabolic activation to the CoA thioester. The presence of substituents at the 2-position also affected the ability of the acyl group to be transferred by NMT to a peptide substrate. Of the three acyl-CoA analogues, only 2-fluoromyristoyl-CoA served as a substrate for NMT.

Substrate specificity of acetyl coenzyme A synthetase.

Acetyl coenzyme A synthetase (EC 6.2.1.1) has been examined for its ability to accept various carboxylic acids as substrates in place of acetic acid. The activity of the enzyme with these substrates was monitored using a coupled enzyme assay and high pressure liquid chromatography (HPLC) analysis. Short chain carboxylic acids were found to be active including: propionic, acrylic, fluoroacetic, methacrylic, 3-chloropropionic, 3-bromopropionic, and propiolic. The kinetic parameters, Km and % Vmax of the carboxylic acid substrates, are reported and show that these acids are poorer substrates than acetic acid. Several of the acyl CoAs were synthesized on a preparative scale using enzyme catalysis, purified using preparative HPLC, and characterized using proton NMR spectroscopy. In the course of the NMR identification, a complete and fully resolved spectral assignment for all the protons of coenzyme A was made and is reported. The acyl-CoA analogs should be useful as substrate analogs and as potential affinity labels for enzymes that bind acetyl-CoA.

Regulation of enzymes by fatty acyl coenzyme A. Interactions of short and long chain spin-labeled acyl-CoA with the acetyl-CoA site on pig heart citrate synthase.

The binding of two similar spin-labeled fatty acyl-CoA analogues, one short chain, 6-doxyloctanoyl-CoA (S-(2-(5-carboxybutyl)-2-ethyl-4, 4-dimethyl-3-oxazolidinyl-N-oxyl)-CoA) and one long chain, 6-doxylstearoyl-CoA (S-(2-(5-carboxybutyl)-2-dodecyl-4, 4-dimethyl-3-oxazolidinyl-N-oxyl)-CoA) to pig heart citrate synthase (citrate oxaloacetate-lyase (pro-3S-CH2COO- leads to acetyl-CoA) EC 4.1.3.7) has been compared. The binding of the short chain analogue could be satisfactorily fit by a classical treatment (independent, noninteracting sites) with well defined stoichiometry: 2 mol of spin label bound per mol of dimeric enzyme. Binding of the long chain analogue was complex and in excess of 2 mol/dimer. Competitive binding experiments using either analogue in the presence of various nucleotides and substrates revealed differences in the binding of the long and short chain analogues. These additional studies, together with kinetic measurements, implied isosteric binding of acyl-CoA, ATP, NADPH, NADH, NADP+, acetyl-CoA, and partial isosteric binding of the long chain acyl-CoA. Binding of NADPH and NADP+ to the same form of the enzyme, perhaps through overlapping sites, was kinetically verified even though these nucleotides had differing effects on the binding of the spin-labeled analogues. Oxalacetate was shown to decrease the binding of the long chain analogue but to have no effect on the binding of the short chain. This result was supported by kinetic measurements. The competitive binding experiments with the long chain analogue suggested that its complex isotherm resulted from binding in two classes of sites, i.e. two cooperative nucleotide sites and other sites. An empirical mathematical model employing this rationale provided a satisfactory fit for the binding of fatty acyl-CoA to citrate synthase. A spin-labeled fatty acid which was not bound by the native enzyme was appreciably bound in the presence of additional palmitoyl-CoA. This binding might be identified with one of the two sets of binding sites proposed in the model. These and previous results on acyl-CoA binding were correlated with the properties of the CoA binding site defined crystallographically (Remington, S., Wiegand, G., and Huber, R. (1982) J. Mol. Biol. 158, 111-152).

Interaction of long-chain acyl-CoA analogs with pig kidney general acyl-CoA dehydrogenase.

The interaction of two long-chain acyl-CoA analogs with pig kidney general acyl-CoA dehydrogenase (EC 1.3.99,3) was examined. The effect of S-heptadecyl-CoA and heptadecan-2-onyl-dethio-CoA on the flavo-protein was observed spectrophotometrically using the flavin as an active-site probe. The S-heptadecyl thioether analog bound strongly to the enzyme (Kd = 17 nM) and was a powerful competitive inhibitor (Ki less than 40 nM). In contrast to the thioether analog, the dethiocarba derivative, heptadecan-2-onyl-dethio-CoA, was a substrate inthe standard assay system being dehydrogenated at about 60% of the rate shown by palmitoyl-CoA. These results support the proposal that alpha-carbanion formation is an early event in the dehydrogenation of acyl-CoA substrates.