Sequence alignment indicates considerable conservation of motif III among the Acs sequences, with the first, fourth, and fifth positions highly or completely conserved, but the second and third positions showing a higher level of variability and an overall consensus of Y-X-S/T/A-G-D (). An alignment of Acs with other members of the adenylate-forming superfamily confirms that these positions are highly conserved throughout (). The Saccharomyces cerevisiae
ACS1 structure (AcsSc
; PDB 1RY2) contains AMP [20
] and is in a conformation thought to catalyze the first step of the Acs reaction in which acetate and ATP are bound and an enzyme-bound acetyl adenylate is formed with concomitant release of inorganic pyrophosphate. The Salmonella enterica
Acs structure (AcsSe
; PDB 2P2F) [19
] contains acetate, AMP, and CoA [8
] and is thought to be in the conformation for catalysis of the second step of the reaction, in which the C-terminal domain is repositioned near the active site to bring new residues into context for CoA binding and formation of the acetyl-CoA product with release of AMP [8
Alignment of motif III residues in Acs sequences.
Inspection of the AcsSe and AcsSc structures places motif III in the active site, regardless of which conformation of the enzyme. The positioning of motif III residues near the adenylate moiety of the bound AMP ligand suggests that residues in this motif may play a role in ATP binding and/or catalysis. Modeling of Acs1Mt on the S. enterica and S. cerevisiae Acs structures places the motif III residues in a similar position to interact with substrates ().
Figure 1 Position of motif III residues in the active sites of (a) AcsSc, (b) AcsSe, and (c) the Acs1Mt structural model. Acs1Mt modeled on the AcsSe structure (PDB:2P2F) using Accelrys DS Modeler and the stereo images were created using Accelrys DS ViewerPro (more ...)
In Acs1Mt, motif III has the sequence 498YTAGD502. We individually altered each position of motif III in Acs1Mt and determined the kinetic parameters of the purified enzyme variants. The residues in the highly conserved first, fourth, and fifth positions were changed to either Ala or a conservative amino acid replacement. The Thr residue at the more variable second position was changed to Ala, and the Ala residue in the third position was changed to Thr, as this is the residue found in many Acs sequences. The kinetic parameters for the purified enzyme variants were determined using the hydroxamate assay and are shown in .
Kinetic parameters for ACS1Mt wild-type and variant enzymes.
The hydroxamate assay measures activated acyl groups including both acetyl-AMP and acetyl-CoA by their conversion to the acyl hydroxamate and subsequently to a ferric hydroxamate complex. Wilson and Aldrich [21
] have shown that several stand-alone adenylating enzymes belonging to the same superfamily as Acs slowly release the acyl-adenylate intermediate in the absence of the native acceptor, and this released acyl-adenylate can react with hydroxylamine. Meng et al. [22
] also witnessed this phenomenon with a medium chain acyl-CoA synthetase (Macs) that favors 2-methylbutyrate as the acyl substrate. In this case, Macs released the acyl adenylate to varying degrees in the absence of the CoA acceptor when a less favored acyl substrate such as propionate was used. However, little to no release of the acyl-adenylate intermediate was observed in the absence of CoA with the favored 2-methylbutryate, suggesting that the acyl-adenylate intermediate is retained if the acyl moiety fits well in the active site but is more readily released in the absence of the native acceptor if the fit is suboptimal.
No activity was detected with Acs1Mt with acetate in the absence of HSCoA, indicating that the acetyl-AMP intermediate remains enzyme bound and that the bound intermediate is not reactive with hydroxylamine. Thus, the kinetic parameters shown in are for the overall reaction, although the Km values for ATP and acetate would likely be similar if just the first adenylation step of the reaction was measured.
Several of the variants were found to be inactive over a wide range of concentrations for each substrate and a range of enzyme concentrations. Enzymes that were inactive displayed similar behavior in both the ion exchange and hydrophobic interaction chromatography steps during purification, and gel filtration chromatography indicated the variants are dimeric as for the wild type enzyme, suggesting there are no gross structural alterations. Overall, alteration of any of the residues in motif III appeared to have a strong deleterious effect on catalysis, although substrate affinity was generally not impaired.
3.1. Positioning of Tyr498 Plays an Important Role in Active-Site Architecture
Based on the two Acs structures, the highly conserved Tyr498 in the first position of motif III is part of a hydrogen bond network with Gln417 and through this hydrogen bond network may contribute to maintenance of the active-site architecture near the ATP binding site (Figures and ). In the Acs1Mt model (), there is an additional interaction between Ala500 and Gln417. To examine whether it is the hydrophobic and bulky nature of Tyr498 or its participation in this hydrogen bond network that plays the more important role in substrate binding and catalysis, this residue was altered to both Ala and Phe. The Tyr498Ala alteration in Acs1Mt did not significantly affect the Km for any substrate but reduced the turnover rate kcat 41-fold (). However, the Tyr498 Phe variant was soluble but inactive at all substrate and enzyme concentrations tested. These results suggest that although the size of Tyr498 is important in maintaining active-site architecture, the hydroxyl moiety plays a critical role in properly positioning this large side chain through hydrogen bonding with Gln417. Attempts at chemical rescue of the Tyr498 Ala variant with phenol were unsuccessful.
3.2. Thr499 and Ala500 Are Less Well Conserved and Play Lesser Roles
The second and third positions of motif III, represented by Thr499 and Ala500 in Acs1Mt, are less well conserved than the other positions (). Thr499 is replaced by Phe in AcsSe, AcsSc, and many other Acs sequences, but Leu is observed in that position in four of the five Acs sequences in Methanosaeta concilii as well as all four of the Acs sequences in Methanosaeta thermophila. Ser and Thr are commonly observed at the third position in motif III, although Ala is present at this position in eight of the nine total Acs sequences in M. concilii and M. thermophila.
These positions were individually altered to Ala and Thr, respectively, in Acs1Mt, and the purified variants were analyzed. The Km values for substrates showed only minor changes (less than threefold increase or decrease) versus the unaltered enzyme. However, the kcat value decreased 83-fold for the Thr499Ala variant and 44-fold for the Ala500 Thr variant (). Overall, these results suggest a less important role for these positions, which is consistent with the lower level of conservation observed.
3.3. Gly501 Is Highly Conserved and May Properly Position the Invariant Asp502
Gly501 in the fourth position of motif III is almost completely conserved within the acyl-adenylate-forming enzyme superfamily except for a few members most distantly related to Acs. Replacement of this residue by Ala resulted in two- to threefold reduced Km values for all three substrates; however, the turnover rate was over 200-fold reduced (). The strict conservation of this Gly and the reduced catalysis observed for the Gly501Ala variant are consistent with this residue playing a role in proper positioning of the critical Asp502 residue in the adjacent position.
3.4. Asp502 Plays a Critical Role in ATP Binding through Interaction with the 2′-OH of the Ribose Moiety
To investigate the role of the invariant Asp residue of motif III, Asp502 of Acs1Mt was altered to Ala and the more conservative residues Glu and Asn. Although the enzyme variants were soluble, each of these alterations eliminated all detectable enzymatic activity, regardless of substrate concentrations or concentration of enzyme used. The fact that even the most conservative changes inactivated the enzyme indicates that this Asp is absolutely critical for activity, as might be expected since Asp502 is completely conserved among all ACSs and throughout the superfamily.
Inhibition assays were performed as an indirect approach to delineate the interaction between Asp502
and ATP. Ribose completely inhibited enzyme activity at concentrations above 600
mM, and the Ki
was determined to be 53
mM (). The maximum adenosine concentration that could be reached in inhibition assays was 100
mM, which produced partial inactivation. However, extrapolation of the data indicated a Ki
mM for adenosine (). That the Ki
for ribose was approximately half the estimated Ki
for adenosine suggests that interaction between the enzyme and the ribose moiety plays an important role in ATP binding.
Figure 2 Inhibition of Acs1Mt. (a) Ribose, (b) adenosine, (c) 2′-deoxyadenosine, and (d) 3′-deoxyadenosine. Assays were performed with the indicated concentrations of inhibitor in the reaction, and results are plotted as a percentage of the activity (more ...)
To determine more precisely the interaction between Asp502
and the 2′- and 3′-OH groups of the ribose sugar of adenosine, inhibition by 2′- and 3′-deoxyadenosine was examined. Although only partial inhibition was observed with either compound, extrapolation of the results gave apparent Ki
values of 356
mM and 151
mM for 2′- and 3′-deoxyadenosine, respectively (Figures and ). These results suggest that interaction between Asp502
and adenosine is mediated primarily through the 2′-OH group of the ribose sugar, as the absence of the 3′-OH group had minimal effect.
The Acs1Mt model () predicts hydrogen bonds between Asp502 and both the 2′ and 3′-OH groups of the ribose moiety. However, these hydrogen bonds are eliminated in the Asp502Ala variant. The inhibition results and the complete impairment of enzymatic activity by alterations at Asp502 suggest that the interaction of Asp502 of motif III with the 2′-OH plays a key role although interaction with the 3′-OH is also important for achieving optimal activity.
3.5. Interaction between the Invariant Asp and the Ribose Moiety of ATP in Other Members of the Enzyme Superfamily
The active-site architecture in AcsSc
is similar in the vicinity of the AMP ligand (Figures and ). However, Asp559
of motif III in AcsSc
interacts with both the 2′- and 3′-OH groups and with Arg574
, whereas Asp500
of motif III in AcsSe
hydrogen bonds with the 2′-OH group and Trp413
and the 3′-OH interacts with Gln415
]. The AcsSc
structure is proposed to be that for the enzyme poised to catalyze the first step of the reaction, whereas the AcsSe
structure has the C-terminal domain shifted inward toward the active-site to bring additional amino acid residues into context for substrate binding and catalysis of the second step of the reaction [8
]. Whether these differences are due to slight changes in active-site architecture between two different enzymes or a movement of this Asp residue as the enzyme converts from one conformation to the other during catalysis of the two steps is unknown, as structures in both conformations are not available for either enzyme.
Structures have been determined for a number of enzymes spanning the adenylate-forming enzyme superfamily, including short, medium, and long-chain acyl-CoA synthetases, the aryl-CoA synthetases CBL and benzoyl-CoA ligase, several NRPS adenylation domains, and luciferase. These structures have revealed that domain alternation between the first and second steps of the reaction is universal among the superfamily [1
]. Inspection of those structures with bound ligands indicates that in each case the invariant Asp in motif III/A7 interacts with one or both hydroxyl groups of the ribose moiety of ATP [6
Three other members of the superfamily have structures in both the adenylate-forming and thioester-forming conformations. In 4-chlorobenzoate:CoA ligase (CBL), Asp385
hydrogen bonds with just the 2′-OH, whereas the 3′-OH interacts with Arg400
in the adenylate-forming conformation, whereas in the thioester-forming conformation, Asp385
maintains its hydrogen bond with the 2′-OH and now also interacts with 3′-OH group along with Arg400
]. In DltA, the D-alanine:D-alanyl carrier protein ligase, Asp383
, interacts with both hydroxyl groups in both conformations. The 2′-OH also interacts with Tyr394
in the adenylate-forming conformation, and the 3′-OH also interacts with Arg396
in the thioester-forming conformation [25
]. Most recently, structures for the human medium-chain acyl-CoA synthetase ACSM2A in both conformations have been reported [28
interacts with both the 2′-OH and 3′-OH in both the adenylate-forming and thioester-forming conformations for this enzyme. Thus, different enzymes interact with the ribose moiety of ATP in different ways. In some cases, the interaction changes slightly after domain alternation. However, in all cases, the invariant Asp of motif III interacts with at least the 2′-OH, suggesting that this is the most important interaction.
3.6. Role of the Invariant Asp in Other Members of the Enzyme Superfamily
The role of this invariant Asp has been studied biochemically in only a few members of the superfamily. In 3-chlorobenzoate-CoA ligase, alteration of this Asp to Val essentially eliminated all catalytic activity [30
]. Gocht and Marahiel [31
] reported that for gramicidin synthetase 1, replacement of this Asp residue with Asn or Ser reduced activity by 22% and 88%, respectively. Pavela-Vrancic et al. [32
] observed with this same enzyme that replacement of ATP in the reaction with 2′-dATP resulted in a 20% reduction in activity versus a 74% reduction in activity when ATP was replaced with 3′-dATP. These results suggest that for gramicidin synthetase 1, as for Acs, interaction between the invariant Asp and the hydroxyl groups of the ribose moiety of ATP is important, with the interaction with the 2′-OH playing the most important role.
In CBL, the invariant Asp385
of motif III hydrogen bonds with the 2′-OH group. Alteration of this residue to Ala greatly reduced the overall rate of catalysis, primarily due to a reduced rate for the first step of the reaction, and resulted in increased Km
values for both ATP and 4-chlorobenzoate [7
]. In D-alanyl carrier protein ligase, the invariant Asp hydrogen bonds with both the 2′- and 3′-OH groups of the ribose moiety [27
]. Alteration of this residue to Asn reduced the rate of catalysis of the adenylation reaction only twofold but resulted in a 75-fold increased Km
value for ATP, leading the authors to conclude that this Asp plays a major role in tight binding of ATP and the adenylate intermediate [25