The Roles of Coenzyme A Binding Pocket Residues in Short and Medium Chain Acyl-CoA Synthetases

Abstract

Short- and medium-chain acyl-CoA synthetases catalyze similar two-step reactions in which acyl substrate and ATP bind to form an enzyme-bound acyl-adenylate, then CoA binds for formation of the acyl-CoA product. We investigated the roles of active site residues in CoA binding in acetyl-CoA synthetase (Acs) and a medium-chain acyl-CoA synthetase (Macs) that uses 2-methylbutyryl-CoA. Three highly conserved residues, Arg¹⁹³, Arg⁵²⁸, and Arg⁵⁸⁶ of Methanothermobacter thermautotrophicus Acs (Acs_Mt), are predicted to form important interactions with the 5′- and 3′-phosphate groups of CoA. Kinetic characterization of Acs_Mt variants altered at each of these positions indicates these Arg residues play a critical role in CoA binding and catalysis. The predicted CoA binding site of Methanosarcina acetivorans Macs (Macs_Ma) is structurally more closely related to that of 4-chlorobenzoate:coenzyme A ligase (CBAL) than Acs. Alteration of Macs_Ma residues Tyr⁴⁶⁰, Arg⁴⁹⁰, Tyr⁵²⁵, and Tyr⁵²⁷, which correspond to CoA binding pocket residues in CBAL, strongly affected CoA binding and catalysis without substantially affecting acyl-adenylate formation. Both enzymes discriminate between 3′-dephospho-CoA and CoA, indicating interaction between the enzyme and the 3′-phosphate group is important. Alteration of Macs_Ma residues Lys⁴⁶¹ and Lys⁵¹⁹, located at positions equivalent to Acs_Mt Arg⁵²⁸ and Arg⁵⁸⁶, respectively, had only a moderate effect on CoA binding and catalysis. Overall, our results indicate the active site architecture in Acs_Mt and Macs_Ma differs even though these enzymes catalyze mechanistically similar reactions. The significance of this study is that we have delineated the active site architecture with respect to CoA binding and catalysis in this important enzyme superfamily.

1. Introduction

Acetyl-CoA synthetase (Acs) plays fundamental roles in the metabolism and physiology of cells from all three domains of life [1,2], and its regulation by acetylation is well studied [3]. Acs and other short and medium chain acyl-CoA synthetases catalyze a two-step reaction in which the first step Equation (1) requires acyl substrate and ATP but not CoA for formation of an enzyme-bound acyl-AMP intermediate with release of inorganic pyrophosphate (PP_i) as a product. In the second step Equation (2), the acyl group is transferred to the sulfhydryl group of CoA and the acyl-CoA and AMP products are released.

E + acyl substrate + ATP ⇆ E•acyl-AMP + PP_i

(1)

E•acyl-AMP + HSCoA ⇆ E + acyl-CoA + AMP

(2)

Structures of Acs from Saccharomyces cerevisiae (Acs_Sc; PDB ID 1RY2) and Salmonella enterica (Acs_Se; PDB ID 2P2F) [4,5] represent conformations of the enzyme in the acyl-adenylate-forming Equation (1) and thioester-forming Equation (2) steps, respectively. These structures indicate that the C-terminal domain rotates 140° toward the N-terminal domain in the transition between the two steps of the reaction. This domain alternation has been proposed to form the complete active site for proper positioning of CoA for nucleophilic attack on the acyl group of the intermediate during catalysis of the second half-reaction Equation (2) [5,6].

The 2.1 Å crystal structure of Macs_Ma (PDB ID 3ETC), a medium chain acyl-CoA synthetase from Methanosarcina acetivorans [7], revealed that in the absence of substrate this enzyme is in a similar conformation to that for thioester formation. This was surprising, given that the Acs_Se structure in this same conformation was obtained from enzyme crystallized in the presence of adenosine 5′propylphosphate, which mimics the acetyl-adenylate intermediate and CoA [5]. Recently, the structure of the Lathyrus sativus oxalyl-CoA synthetase was solved in the presence of ATP and oxalate but not CoA and was also found to adopt the thioester forming conformation [8].

Our characterization of the Methanothermobacter thermautotrophicus Acs (Acs_Mt) and Archaeoglobus fulgidus Acs (Acs_Af) revealed that these enzymes are more diverse in substrate utilization than previously thought [9]. Whereas the acyl substrate range for Acs_Mt is limited to acetate and propionate with a strong preference for acetate, Acs_Af has a broader acyl substrate range that includes butyrate, valerate, and the branched-chain isobutyrate, and has only a slight preference for acetate over propionate. The Pyrobaculum aerophilum Acs likewise has an expanded acyl substrate range [10].

Characterization of Macs_Ma revealed that the preferred acyl substrate is the branched chain 2-methylbutyrate [11]. The enzyme has a broad acyl substrate range for the acyl-adenylate forming step of the reaction, with the ability to utilize propionate (C₃) to octanoate (C₈) as well as certain branched chain substrates; however, the acyl-adenylate formed with many of these substrates was not suitable for the thioester-forming second step of the reaction and was released in the absence of CoA. CoA inhibited acyl-AMP release and instead promoted its breakdown to AMP and the acyl group, which were released along with PP_i [11]. In the presence of 2-methylbutyrate, Macs_Ma did not release the acyl-AMP intermediate in the absence of CoA and in the presence of CoA completed the two-step reaction and released 2-methylbutyryl-CoA, AMP, and PP_i as products [11].

As Acs and Macs catalyze similar two-step reactions that differ only in the acyl substrate, it was expected that these enzymes would have similar active site architecture in which the acyl substrate binding pocket is expanded to accommodate larger substrates. We have shown that Trp⁴¹⁶ in Acs_Mt (Trp⁴¹⁴ in Acs_Se) plays an essential role in determining acyl substrate range and preference [12]. This Trp in almost completely conserved among Acs sequences but is replaced by Gly in medium chain acyl-CoA synthetases. Based on our results, other labs have engineered the acyl substrate pocket of Acs to utilize novel substrates to generate alternative acyl-CoA substrates for metabolic engineering [13,14,15].

Inspection of the Acs_Se and Macs_Ma crystal structures [5,7] and our analysis of site-directed variants altered in the acyl substrate pocket of Macs_Ma and Acs_Mt [11,12] indicate fundamental differences in the active site architecture of the two enzymes. Trp⁴¹⁶ of Acs_Mt is replaced by Gly in Macs_Ma, as would be expected, and an alternate Trp residue, Trp²⁵⁹, occupies a position similar to that of Trp⁴¹⁶ and was shown to be critical for substrate binding and catalysis [11,12].

ATP binding site determinants have been investigated in Acs [16,17] but not Macs. However, signature motif III (YXXGD) of the acyl-adenylate-forming enzyme superfamily [18], shown by Ingram-Smith et al. [16] to play a key role in ATP binding and catalysis in Acs, is well conserved in Macs_Ma as ⁴³¹YHTGD⁴³⁵. The Asp at the last position in motif III is invariant among superfamily members and interacts with one or both hydroxyl groups of the ribose moiety of ATP in all of the structures available thus far, including that of Macs_Ma [7], suggesting that residues in this motif may serve similar roles in ATP binding in both Acs and Macs.

Short- and medium-chain acyl-CoA synthetases are widespread in the archaea [9] and have provided a rich background for studying the structural and biochemical diversity within this family. Here we report our investigation of the CoA binding sites of Macs_Ma and Acs_Mt. As previously shown for acyl substrate binding and catalysis of the first step of the reaction, our results indicate that key residues involved in CoA binding and catalysis of the second step of the reaction in Acs_Mt are dispensable in Macs_Ma. Instead the CoA binding site of Macs_Ma more closely resembles that of 4-chlorobenzoate CoA ligase (CBAL), which catalyzes the formation of 4-chlorobenzoyl-CoA [19,20,21,22,23].

2. Materials and Methods

2.1. Site-Directed Mutagenesis

Site-directed alteration of the Macs_Ma and Acs_Mt gene was accomplished with the QuickChange kit (Stratagene, cat. 200519) and the altered sequences were confirmed by sequencing. Oligonucleotides for site-directed mutagenesis were purchased from Integrated DNA Technologies (www.idtdna.com).

2.2. Purification of Macs_Ma and Acs_Mt Enzymes

The Macs_Ma and Acs_Mt enzymes were heterologously produced in Escherichia coli Rosetta Blue (DE3) placI (EMD Millipore) as described previously [11,12]. Clarified cell lysate was applied to a 5 mL His-Trap column and purified protein was eluted using a linear gradient of increasing imidazole concentration in buffer. The purified enzymes were dialyzed against 25 mM Tris, 10% glycerol [pH 7.5], aliquoted, and stored at −20 °C. Protein concentrations were determined by the Bradford method [24] using Bio-Rad Protein Assay Kit II (Bio-Rad, cat. 5000002) according to the manufacturer’s instructions.

2.3. Assay for Acyl-CoA and Acyl-Adenylate Production

The hydroxamate assay [25,26] measures production of activated acyl groups, including both acyl-CoA and acyl-adenylate. Reaction mixtures (0.3 mL containing 100 mM Tris-HCl [pH 7.5] (Fisher Scientific, cat. BP152-5) and 600 mM hydroxylamine-HCl (Acros, cat. 270100010) [pH 7.0]) with varied concentrations of acyl substrate, MgATP (Fisher Scientific, cat. BP413-25), and CoA (Fisher Scientific, cat. BP25101). Reactions were stopped by the addition of two volumes (0.6 mL) stop solution [1 N HCl, 5% trichloroacetic acid (Acros, cat. 152130010), 1.25% FeCl₃ (Fisher Scientific, I88-500)]. The change in absorbance at 540 nm was measured and product formation was calculated by comparison to a standard curve. Reactions were performed at the optimal temperature for each enzyme (55 °C for Macs_Ma and 65 °C for Acs_Mt). For ethanol-soluble acyl substrates, the concentration of the stock solutions were adjusted such that the final ethanol concentration in the reaction was kept constant at 2%. All reactions were performed in triplicate.

For determination of apparent kinetic parameters, the concentration of each substrate was varied individually while the concentrations of the other substrates were held constant at a saturating level (~5–10 times the K_m for that substrate). The apparent kinetic parameters with their standard errors were calculated using non-linear regression to fit the data to the Michaelis-Menten equation. All reactions were performed in triplicate. Values are the mean ± standard deviation.

2.4. Assay for Inorganic Pyrophosphate Production

Pyrophosphate production by Macs_Ma was determined by the molybdate assay as described in Meng et al. [11]. Briefly, reactions (0.3 mL) were performed at 55 °C and reaction mixtures contained 50 mM Tris-HCl [pH 7.5], 4 mM MgCl₂, 10 mM dithiothreitol (Fisher Scientific, cat. BP172-25). The concentrations of ATP, CoA, and acyl substrate were varied for determination of kinetic parameters. Reactions were terminated at 10 min by addition of 0.08 mL H₂O, 0.05 mL 0.5 M 2-mercaptoethanol (Fisher Scientific, cat. O34461-100), 0.05 mL molybdate reagent [2.5% ammonium molybdate (Fisher Scientific, cat. A674-500) in 5 N H₂SO₄], and 0.02 mL Eikonogen reagent [25 mM sodium sulfite (Fisher Scientific, cat. S447-500), 13 mM 1-amino-2-naphthol-4-sulfonic acid (Themo Scientific, cat. B24339-22), 963 mM sodium meta-bisulfite (Fisher Scientific S244-500)]. The absorbance at 580 nm was measured after 10 min and compared to a PP_i standard curve. All reactions were performed in triplicate. Values are the mean ± standard deviation.

2.5. Assay for Acyl-CoA Thioester Bond Formation

Acyl-CoA thioester bond formation was measured as previously described [27]. Briefly, reactions (0.5 mL) were performed at 55 °C in 100 mM Tris-HCl (pH 7.5) with a range of substrate concentrations. Acyl-CoA thioester bond formation was measured spectroscopically at 233 nm. All reactions were performed in triplicate. Values are the mean ± standard deviation.

3. Results

3.1. Conserved Arg Residues in Acs Interact with CoA

Inspection of the Acs_Se structure reveals interaction between the negatively charged phosphate groups of CoA and two conserved Arg residues, Arg¹⁹¹ and Arg⁵⁸⁴, with Arg¹⁹¹ interacting with both the 5′-diphosphate and 3′-phosphate groups and Arg ⁵⁸⁴ interacting with just the 3′-phosphate of CoA [5]. An additional highly conserved Arg residue, Arg⁵²⁶, interacts with the phosphate group of the acyl-adenylate intermediate and has been predicted to play a role in stabilizing the thioester-forming conformation [5]. These three Arg residues are conserved in Acs_Mt as Arg¹⁹³, Arg⁵²⁸, and Arg⁵⁸⁶, respectively, and occupy similar positions relative to CoA (Figure 1).

Each of these Arg residues was individually altered to Ala, Lys, and Gln in Acs_Mt and kinetic parameters were determined for the purified enzyme variants. Overall, alterations at Arg¹⁹³ had the most severe effect on the K_m value for CoA. The K_m values for CoA for the Arg¹⁹³Lys and Arg¹⁹³Gln variants increased 18.9- and 41.0-fold, respectively, and the Arg¹⁹³ Ala variant was unsaturable for CoA (

Table 1. Kinetics parameters for Acs_Mt wild-type and variant enzymes.

Enzyme	Km CoA (mM)	kcat (sec−1)	kcat/Km (sec−1 mM−1)
Wild-type a	0.19 ± 0.003	81.6 ± 0.7	423.7 ± 4.7
Arg193Ala	Unsaturable b
Arg193Lys	3.6 ± 0.37	0.51 ± 0.01	0.14 ± 0.01
Arg193Gln	7.8 ± 0.40	0.28 ± 0.004	0.036 ± 0.002
Arg528Ala	Unsaturable b
Arg528Lys	0.45 ± 0.03	0.25 ± 0.01	0.54 ± 0.02
Arg528Gln	1.67 ± 0.05	0.63 ± 0.04	0.38 ± 0.06
Arg586Ala	1.19 ± 0.08	2.13 ± 0.09	1.80 ± 0.06
Arg586Lys	0.33 ± 0.013	2.39 ± 0.016	7.34 ± 0.24
Arg586Gln	0.28 ± 0.009	0.12 ± 0.003	0.45 ± 0.028

^a Values are taken from [9]. ^b The enzyme was not saturable for CoA at concentrations up to 25 mM and kinetic parameters could not be determined.

). The Arg⁵⁸⁶ and the Arg⁵²⁸ variants generally showed much less of an effect on the K_m for CoA, with increases ranging from less than two-fold up to 8.8-fold except for the Arg⁵²⁸Ala variant, which was rendered unsaturable for CoA (Table 1).

The turnover rates for all the Arg variants were significantly impaired (Table 1), with 34- to 38-fold reductions in k_cat observed for the Arg⁵⁸⁶Lys and Arg⁵⁸⁶Ala variants, 160- to 291-fold reductions for the Arg¹⁹³Lys and Arg¹⁹³Gln variants, and 130- to 326-fold reductions for the Arg⁵²⁸Gln and Arg⁵²⁸Lys variants. The most severe reduction in catalysis was observed for the Arg⁵⁸⁶Gln variant, which displayed a 680-fold reduced k_cat. The effects on the overall catalytic efficiency with CoA ranged from a 58-fold reduction for the Arg⁵⁸⁶Lys variant to a nearly 12,000-fold reduction for the Arg¹⁹³Gln variant. Even the more conservative Arg¹⁹³Lys alteration resulted in ~3000-fold reduced catalytic efficiency, suggesting Arg¹⁹³ plays a critical role in catalysis as well as CoA binding.

The K_m values for ATP and acetate were also determined for each variant. Alterations at the targeted Arg residues had only minor effects on the K_m for ATP (Supplemental Table S1). The K_m for acetate for most of the variants was similar to that for the wild-type enzyme with the exception of the Arg¹⁹³Ala, Arg⁵²⁸Ala, and Arg⁵⁸⁶Gln variants which were unsaturable for acetate even at concentrations as high as 800 mM (Supplemental Table S1).

3.2. Interaction between Arg⁵⁸⁶ and the 3′-Phosphate Group of CoA Is Important for Substrate Binding and Catalysis

Based on the Acs_Se structure, Arg⁵⁸⁶ of Acs_Mt is predicted to interact with the 3′ phosphate group of CoA. To examine the contribution and nature of this interaction in CoA binding and catalysis, we examined whether the unaltered enzyme and the Arg⁵⁸⁶Ala and Arg⁵⁸⁶Lys variants could discriminate between CoA and 3′-dephospho CoA. The wild-type enzyme had over 10-fold higher K_m for 3′-dephospho CoA than for CoA but catalysis was not greatly reduced. The resulting 26.5-fold higher catalytic efficiency with CoA versus 3′-dephospho CoA (

Table 2. Discrimination between CoA and 3′-dephospho CoA (deCoA) for wild-type Acs_Mt and the Arg⁵⁸⁶ variants.

Enzyme	Substrate	Km (mM)	kcat (sec−1)	kcat/Km (sec−1 mM−1)	(kcat/Km CoA)/ (kcat/Km deCoA)
Wild-type	CoA	0.19 ± 0.003	81.6 ± 0.7	423.7 ± 4.7	26.5
	deCoA	2.16 ± 0.50	34.5 ± 2.9	16.0 ± 2.5
Arg586Ala	CoA	1.19 ± 0.08	2.13 ± 0.09	1.80 ± 0.06	2.2
	deCoA	2.05 ± 0.40	1.66 ± 0.11	0.81 ± 0.11
Arg586Lys	CoA	0.33 ± 0.01	2.39 ± 0.02	7.34 ± 0.20	3.7
	deCoA	0.86 ± 0.04	1.72 ± 0.01	2.01 ± 0.09

) indicates that the interaction between the enzyme and the 3′-phosphate group plays an important role in CoA binding.

The Arg⁵⁸⁶Ala variant had a 6-fold higher K_m value for CoA but similar K_m value for 3′-dephospho CoA as the wild-type enzyme. Catalysis was greatly reduced with either substrate, resulting in just 2.2-fold difference in catalytic efficiency with CoA versus 3′-dephospho CoA (Table 2), indicating this variant can no longer discriminate well between the presence and absence of the 3′-phosphate group. Retention of a positive charge at position 586 in the Arg⁵⁸⁶Lys variant was not sufficient to restore discrimination between CoA and 3′-dephospho CoA. The K_m for CoA was less than 2-fold increased versus that of the wild-type enzyme. This variant had a lower K_m for 3′-dephospho CoA than the wild-type enzyme or the Arg⁵⁸⁶Ala variant, but k_cat was still greatly reduced resulting in only 3.7-fold preference for CoA versus 3′-dephospho CoA (Table 2).

3.3. Electrostatic Interaction between Macs_Ma and the 3′-Phosphate Group of CoA Is Important

To examine whether Macs_Ma also makes an electrostatic interaction with the 3′-phosphate group of CoA, the ability of wild-type enzyme to discriminate between CoA and 3′-dephospho CoA was determined. The enzyme displayed very low 2-methylbutyryl-CoA synthetase activity with 3′-dephospho CoA even at a concentration of 10 mM, whereas the activity observed with 10 mM CoA was over 5-fold higher (Figure 2A). Kinetic parameters could not be determined with 3′-dephospho CoA, so the level of discrimination could not be ascertained.

In the absence of CoA, wild-type Macs_Ma catalyzes synthesis and release of an acyl-adenylate when less favorable acyl substrates such as propionate are used, and the presence of CoA inhibits this activity [11]. Inhibition of the acyl-adenylate synthetase activity by CoA versus 3′-dephospho CoA was examined as another means for determining whether interaction between the enzyme and the 3′-phosphate group of CoA is important. The acyl-adenylate synthetase activity was inhibited by both CoA and 3′-dephospho CoA to a similar extent (Figure 2B), suggesting that interaction with the 3′-phosphate group is important for CoA binding for the second step of the reaction but does not play a role in interaction between CoA and the enzyme for the first step of the reaction or when the second step cannot occur.

3.4. The CoA Binding Pocket in Macs_Ma Resembles That in CBAL

The CoA nucleotide binding pocket in the Acs_Se and CBAL structures differs but the pantetheine tunnel is similar [5,20]. In CBAL, the aromatic residues Phe⁴⁷³ and Trp⁴⁴⁰ play key roles in CoA binding and catalysis by accommodating the adenine moiety of CoA. Alterations of these residues greatly reduced catalytic efficiency for the second step of the reaction while having little effect on first step [22]. Arg⁴⁷⁵ interacts with the CoA 3’-phosphate [5,20] and alteration reduced catalytic efficiency [22].

Comparison of the Macs_Ma, Acs_Se, and CBAL structures revealed that the CoA binding site of Macs_Ma more closely resembles that of CBAL [21]. In Macs_Ma, Tyr⁵²⁵ and Arg⁴⁹⁰ replace Phe⁴⁷³ and Trp⁴⁴⁰ of CBAL, respectively (Figure 3), although Tyr⁴⁶⁰ of Macs_Ma is also positioned such that it could function similarly to Trp⁴⁴⁰ of CBAL, which interacts with the adenine moiety of CoA [5,20]. Tyr⁵²⁷ of Macs_Ma occupies a similar location to Arg⁴⁷⁵ of CBAL but the side chain is positioned away from the 3′-phosphate and may instead interact with the ribose group of CoA via its benzoyl group [21]. Gly⁴⁵⁹, located in close proximity to the putative CoA binding site of Macs_Ma, is highly conserved among all members of the adenylate-forming enzyme superfamily and has been proposed to be necessary to open the pantetheine tunnel in the thioester-forming conformation [21].

Based on these structural comparisons, we investigated the role of Macs_Ma residues Gly⁴⁵⁹, Tyr⁵²⁵, Tyr⁴⁶⁰, Arg⁴⁹⁰ and Tyr⁵²⁷ in CoA binding and catalysis. Alterations were made at each of these residues and the recombinant enzyme variants were produced and purified. The Tyr⁴⁶⁰Ala and Arg⁴⁹⁰Ala variants were insoluble and were not characterized. Kinetic parameters were determined for the purified enzyme variants to examine the impact of the alterations on acyl-CoA synthetase activity. Alteration of Gly⁴⁵⁹ to Ala had little effect on enzymatic activity. The K_m and k_cat values showed just slight changes from those for the wild-type enzyme for the 2-methylbutyryl-CoA synthetase activity (

Table 3. Kinetic parameters for the 2-methylbutyryl-CoA synthetase activity for wild-type Macs_Ma and the Gly⁴⁵⁹, Lys⁴⁶¹, and Lys⁵¹⁹ variants.

Enzyme	kcat (sec−1)	Km CoA (mM)
Wild-type	2.15 ± 0.10	4.09 ± 0.45
Gly459Ala	0.41 ± 0.01	2.09 ± 0.06
Lys461Ala	0.55 ± 0.03	2.19 ± 0.18
Lys461Arg	0.46 ± 0.10	1.38 ± 0.14
Lys519Ala	*	*
Lys519Arg	1.07 ± 0.02	7.18 ± 0.29

* Activity was too low for determination of kinetic parameters.

and Supplemental Table S2).

Alterations at Tyr⁴⁶⁰, Tyr⁵²⁵, Tyr⁵²⁷, and Arg⁴⁹⁰ proved to be very deleterious to the acyl-CoA synthetase activity of Macs_Ma, with little 2-methylbutyryl-CoA synthetase activity observed even at high CoA concentrations. These variants displayed 15- to 80-fold reduced specific activity relative to the wild-type enzyme (Figure 4), and kinetic parameters could not be determined due to the low activity. These variants also had reduced propionyl-adenylate synthetase activity, with k_cat values reduced 4.5- to 21-fold (Supplemental Table S3). The K_m values for propionate and ATP were not substantially affected in these variants except for the Tyr⁵²⁵Ala variant for which the K_m value for propionate increased 6.0-fold and that for ATP decreased 14.7-fold (Supplemental Table S3).

To examine whether these alterations affected just the second step of the reaction in which CoA binding occurs or affected catalysis of the first step of the reaction as well, kinetic parameters were determined for the CoA-independent propionyl-adenylate synthetase activity of the enzyme. Except for the Tyr⁵²⁵Ala variant, the K_m values for propionate and ATP showed ~2-fold or less change from the values observed for the unaltered enzyme although the k_cat values were ~2–10 fold decreased (Supplementary Table S3). These results suggest that although the first step of the reaction is affected, the impact is not enough to account for the near lack of acyl-CoA synthetase activity and that CoA binding and/or catalysis of the second step of the reaction are specifically affected.

Because CoA inhibits the propionyl-adenylate synthetase activity of the wild-type enzyme [11], we examined the effect of a high concentration of CoA on this activity in the variants as a means for determining whether CoA can still bind even though the variants cannot catalyze the second step of the reaction. The presence of 15 mM CoA reduced activity of the wild-type enzyme by nearly 40% but had little to no inhibitory effect on activity of the variants (Figure 5). The presence of 15 mM CoA stimulated propionyl-adenylate synthetase activity of the Arg⁴⁹⁰Lys variant by nearly 25%. The reason for this is unknown and was not investigated further.

3.5. The Corresponding Lys Residues in Macs_Ma Do Not Play a Major Role in CoA Binding

To provide further confirmation that CoA binding in Macs_Ma more closely resembles that in CBAL than Acs, we also examined the role of Lys⁴⁶¹ and Lys⁵¹⁹, which are positioned similarly to Arg⁵²⁸ and Arg⁵⁸⁶ of Acs_Mt (Figure 6). These Lys residues were individually altered to Arg and Ala and the purified variants were characterized. Kinetic parameters were determined using 2-methylbutyrate, the preferred substrate for the acyl-CoA synthetase activity of these enzyme variants. The Lys⁴⁶¹Ala and Lys⁴⁶¹Arg variants showed just 1.9-fold and 3.0-fold decrease, respectively, in the K_m for CoA and a modest (less than 10-fold) decrease in the K_m value for 2-methylbutyrate (Table 3). The K_m values for 2-methylbutyrate and ATP were not substantially affected (Supplementary Table S2). These results suggest that Lys⁴⁶¹ in Macs_Ma does not play a role similar to the corresponding Arg in Acs as there was little impact on CoA binding and catalysis.

The Lys⁵¹⁹Arg alteration resulted in less than 2-fold change in the K_m for any substrate or the turnover rate for the 2-methylbutyryl-CoA synthetase (Table 3 and Supplementary Table S2) or the propionyl-adenylate synthetase (Supplementary Table S3) activities. In contrast, the Lys⁵¹⁹Ala variant had too little activity to determine kinetic parameters for either activity (Table 3 and Supplementary Tables S2 and S3).

4. Discussion

We have previously investigated substrate binding and catalysis in the short- and medium-chain acyl-CoA synthetases and identified residues important for acyl substrate binding in Acs and Macs and ATP binding in Acs. Here we examined CoA binding in Acs_Mt and Macs_Ma.

Inspection of the Acs_Sc and Acs_Se structures [4,5] revealed two conformations for the enzyme. In the first step of the reaction, the C-terminal domain is positioned out and away from the active site but then swings in toward the N-terminal domain for catalysis of the second step of the reaction. Three Arg residues, Arg¹⁹¹, Arg⁵²⁶, and Arg⁵⁸⁴ (Arg¹⁹³, Arg⁵²⁸, and Arg⁵⁸⁶ of Acs_Mt, respectively) were proposed to play an important role in CoA binding and catalysis of the second step. Arg¹⁹¹ interacts with both the 5′-diphosphate and the 3′-diphosphate groups of CoA. As this residue is on the N-terminal domain and already present in the active site before domain alternation, it may play an important role in initial binding of CoA. Arg⁵⁸⁴ enters the active site after domain alternation to interact with the 3′-phosphate group, and Arg⁵²⁶, also on the C-terminal domain, was proposed to stabilize the thioester-forming conformation through interaction with the phosphate group of the acyl-adenylate intermediate [5]. Although this residue was not proposed to directly interact with CoA, it may influence CoA binding and catalysis by locking the enzyme in the thioester-forming conformation and thus encasing CoA in the active site.

We altered each of these Arg residues individually in Acs_Mt and assessed each variant’s kinetic abilities. All the variants were impaired in catalysis, with k_cat values reduced by 34 to 680-fold. The effect of these alterations on the K_m for CoA varied, with alterations at Arg¹⁹³ being the most detrimental and replacements at Arg⁵²⁸ and Arg⁵⁸⁶ having more variable effects. As might be expected, substitution with Ala at each position was the most deleterious, likely due to loss of both side chain charge and size. In fact, the Arg¹⁹³Ala and Arg⁵²⁸Ala variants were not saturable for CoA or, surprisingly, for acetate.

Replacement of Arg⁵⁸⁶ had a lesser effect than replacement at Arg¹⁹³, most likely because Arg⁵⁸⁶ only contacts CoA at the 3′-phosphate rather than at both the 5′-diphosphate and the 3′-phosphate as for Arg¹⁹³. However, this single point of contact between Arg⁵⁸⁶ and CoA is important in CoA recognition and/or binding as shown by the fact that the Arg⁵⁸⁶ variants were unable to distinguish between CoA and 3′-dephosphoCoA.

Alteration of Arg⁵²⁸ increased the K_m for CoA and decreased k_cat despite Arg⁵²⁸ appearing to contact the phosphate group of the acyl-adenylate intermediate rather than direct contact with CoA, thus stabilizing the thioester-forming conformation of the enzyme. Substitution at this position would then be expected to reduce the ability of the enzyme to maintain proper positioning of the acyl-adenylate intermediate in the active site, thus affecting catalysis and influencing the ability to bind CoA as well. Our results suggest these three Arg residues are essential for CoA binding and catalysis, directly or indirectly. These residues may also influence acetate binding in the first step of the reaction, perhaps through an inability to fully control domain alternation.

In contrast to our results, Reger et al. [6] reported that alteration of Arg⁵²⁶ and Arg⁵⁸⁴ of Acs_Se resulted in just a 2-fold decrease in catalysis. The K_m for CoA increased for each of the variants, ranging from 4-fold for the Arg⁵²⁶Ala variant to 7 to 8-fold for the Arg⁵⁸⁴Ala and Arg⁵⁸⁴Glu variants [6]. However, no alterations were made at Arg¹⁹¹, the equivalent to Arg¹⁹³ of Acs_Mt. Reger et al. [6] determined the kinetic parameters for CoA and ATP using 20 mM acetate in the reaction mixture for all enzyme variants. Given that the wild-type enzyme has a reported K_m for acetate of 6.05 mM, the kinetic constants for ATP and CoA may have been determined at subsaturating acetate concentrations. These inconsistencies between our results and those of Reger et al. [6] may thus reflect differences between the two Acs enzymes, which share only 49% sequence identity at the amino acid level, or the experimental conditions. Such differences among Acs enzymes were already noted for acyl substrate selection [9,10].

In the Macs_Ma structure, the enzyme was found to be in a similar conformation to that observed for Acs_Se, as if poised for the second step of the reaction even in the absence of substrates [7]. Comparison of the CBAL [21] and Acs_Se structures [5] in the conformation for the second step of the reaction revealed that the binding pockets for CoA nucleotide moiety in these enzymes are significantly different [21], with more interactions with the N-terminal domain in Acs but with the C-terminal domain for CBAL. Superposition of the Macs_Ma structure with the CBAL and Acs_Se structures indicates that the CoA binding site more closely resembles that of CBAL [7]. The recent structure of a 2-hydroxyisobutyric acid CoA ligase shows CoA binding in a substantially bent conformation in the thioester conformation [28]. This contrasts with the more stretched conformations of CoA observed in the thioester forming conformations of CBAL, Macs, and Acs.

We investigated five residues in Macs_Ma predicted to interact with CoA based on comparison with the CBAL structure. Tyr⁴⁶⁰, Arg⁵⁹⁰, Tyr⁵²⁵, or Tyr⁵²⁷ variants displayed greatly reduced 2-methylbutyryl-CoA synthetase activity, and propionyl-adenylate synthetase activity was also reduced but to a much lesser extent. Alteration at Gly⁴⁵⁹, which is strictly conserved in the acyl-adenylate-forming superfamily [7] reduced the turnover rate for both enzymatic activities but did not substantially affect the K_m values for substrates.

In order to examine whether the alterations in the putative CoA binding pocket residues affected just catalysis or also affect CoA binding, we took advantage of the fact that CoA inhibits the propionyl-adenylate synthetase activity of Macs_Ma [11]. In each case, the variant showed less inhibition of the propionyl-adenylate synthetase activity by CoA than for the wild-type enzyme, suggesting that CoA cannot bind as well and supporting that these four residues play a key role in CoA binding as well as catalysis by Macs_Ma.

Macs_Ma lacks each of the three Arg residues investigated in Acs_Mt. However, Arg⁵²⁸ and Arg⁵⁸⁶ of Acs_Mt are replaced by Lys residues at the corresponding positions (residues 461 and 519) in Macs_Ma. Structurally, although these residues are in the vicinity of the predicted CoA binding pocket of Macs_Ma, they are more remote from CoA than the Arg residues of Acs. Our kinetics results for Macs_Ma variants altered at these Lys residues suggest that Lys⁴⁶¹ does not play a role in CoA binding or catalysis. Although Lys⁵¹⁹ may play some role, maintenance of the positive charge at this position is sufficient. Alterations at these positions (with the exception of a Lys⁵¹⁹Ala alteration) resulted in only mild reductions in k_cat (5-fold or less) for either the acyl-CoA synthetase activity or the propionyl-adenylate synthetase activity. Per residue binding free energy decomposition had previously identified Lys⁴⁶¹ as a residue important in 2-methyl butyrate binding and catalysis [29]. Our alteration of Lys⁴⁶¹ to alanine and arginine had only 7.5-fold and 9-fold reduction on K_m, respectively.

Overall, although Acs and Macs have similarities in active site architecture for substrate binding and catalysis of the first step of the reaction, our results strongly suggest that the active site architecture for CoA binding and catalysis of the second step has diverged greatly. Although structural comparison between Acs_Se and Macs_Ma revealed distinct differences in the CoA binding pocket [7], it appears that electrostatic interaction with the 3′-phosphate group of CoA is important for both enzymes; however, this interaction occurs with disparate residues in each enzyme. Differences in acyl substrate binding sites among acyl-CoA synthetase family members is not surprising as the enzymes must accommodate substrates of different lengths that may be branched or unbranched. However, the diversity in CoA binding sites among family members was unexpected.