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J. Biol. Chem., Vol. 276, Issue 48, 45059-45064, November 30, 2001

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Site-directed Mutational Analysis of Active Site Residues in the Acetate Kinase from Methanosarcina thermophila^*

Rebecca D. Miles, Prabha P. Iyer, and James G. Ferry

From the Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-4500

Received for publication, August 29, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acetate kinase catalyzes the magnesium-dependent transfer of the -phosphate of ATP to acetate. The recently determined crystal structure of the Methanosarcina thermophila enzyme identifies it as a member of the sugar kinase/Hsc70/actin superfamily based on the fold and the presence of five putative nucleotide and metal binding motifs that characterize the superfamily. Residues from four of these motifs in M. thermophila acetate kinase were selected for site-directed replacement and analysis of the variants. Replacement of Asp¹⁴⁸ and Asn⁷ resulted in variants with catalytic efficiencies less than 1% of that of the wild-type enzyme, indicating that these residues are essential for activity. Glu³⁸⁴ was also found to be essential for catalysis. A 30-fold increase in the magnesium concentration required for half-maximal activity of the E384A variant relative to that of the wild type implicated Glu³⁸⁴ in magnesium binding. The kinetic analysis of variants and structural data is consistent with nonessential roles for active site residues Ser¹⁰, Ser¹², and Lys¹⁴ in catalysis. The results are discussed with respect to the acetate kinase catalytic mechanism and the relationship to other sugar kinase/Hsc70/actin superfamily members.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acetate kinase, which catalyzes the magnesium-dependent transfer of the ATP -phosphate to acetate (Eq. 2), is a central enzyme in the energy-yielding metabolism of anaerobes.

(Eq. 1)

(Eq. 2)

In most fermentative procaryotes, the catabolic intermediate acetyl-CoA is converted to acetate by phosphotransacetylase (Eq. 1) and acetate kinase (Eq. 2), yielding ATP. In Archaea of the genus Methanosarcina, acetate kinase and phosphotransacetylase catalyze the ATP-dependent activation of acetate to acetyl-CoA in the first step of the conversion of acetate to methane and CO₂.

The discovery of acetate kinase in 1944 by Lipmann (1) and the subsequent isolation in 1954 by Ochoa and co-workers (2) initiated investigations of the Escherichia coli enzyme. Kinetic and biochemical studies between 1954 and 1980 have led to two distinct proposals for the catalytic mechanism, one of which involves the direct in-line transfer of the ATP -phosphate to acetate. This mechanism is consistent with the finding that the acetate kinase reaction proceeds with the net inversion of configuration, indicating an odd number of phosphoryl transfers (3). The second proposal is a covalent triple displacement mechanism (4), which reconciles the stereochemical evidence with the isolation of an unspecified phosphorylated glutamate residue that forms upon the incubation of acetate kinase with acetyl phosphate (5). In the triple displacement mechanism, the ATP -phosphate is transferred to two consecutive enzyme sites before transfer to acetate. The triple displacement mechanism was challenged when it was shown that phosphorylated acetate kinase from E. coli is a phosphoryl donor to enzyme I of the bacterial phosphotransferase system (6), which suggested an alternate function for phosphorylated acetate kinase in the transport of sugars.

The acetate kinase from Methanosarcina thermophila has been cloned and hyper-expressed in E. coli, enabling for the first time site-directed mutagenesis and structural analysis (7-10) to probe the catalytic mechanism. Although the acetate kinase was crystallized in the presence of ATP, only the - and -phosphates, in addition to the adenosine moiety, could be resolved in the proposed active site cleft between the two domains of the monomer (10). The -phosphate of ADP appears to be displaced, possibly as a result of charge repulsion from the sulfate molecule that is present in the active site (10). The sulfate lies in the proposed location of the ATP -phosphate (10). The structure identifies M. thermophila acetate kinase as a member of the sugar kinase/Hsc70/actin structural superfamily (10, 11). Acetate kinase shares no significant overall sequence similarity with sugar kinase/Hsc70/actin superfamily members; however, structural properties indicate that acetate kinase and other superfamily members share a core fold as well as five motifs (termed phosphate-1, phosphate-2, connect-1, connect-2, and adenosine) composed of both secondary structural elements and conserved amino acid residues (10, 12-14). In several superfamily members, site-directed mutagenesis has been used to implicate these motifs in either nucleotide binding, metal binding, or catalysis (15-18). In acetate kinase, the roles of residues in these analogous motifs have yet to be investigated. The adenosine moiety of ADP in the crystal structure of the M. thermophila enzyme is bound in part to the conserved adenosine motif of the sugar kinase/Hsc70/actin superfamily. Thus, the structural data are consistent with data from other superfamily members in supporting a role for this motif in adenosine binding (10). Identifying residues involved in binding the polyphosphate and the metal ion and facilitating phosphoryl transfer is less straightforward because of the apparent displacement of the ADP -phosphate and the lack of electron density that can be attributed to the ATP -phosphate or metal ions in the crystal structure (10).

Here, we report a site-directed mutational analysis of the M. thermophila acetate kinase to address the roles of active site residues in the phosphate-1, phosphate-2, connect-1, and connect-2 motifs that are highly conserved among acetate kinases. Our data have led us to hypothesize an alternate role for Glu³⁸⁴, previously postulated to be a phosphorylation site during catalysis (8). In addition, we have identified an active site aspartate (Asp¹⁴⁸) and an active site asparagine residue (Asn⁷) that are essential for catalysis. Finally, we have identified three active site residues (Ser¹⁰, Ser¹², and Lys¹⁴) that we propose to be involved in orienting the polyphosphate in a catalytically competent manner. Our results are discussed with respect to the acetate kinase catalytic mechanism and to data from other superfamily members.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Sequence Analyses-- Data bases were searched at the National Center for Biotechnology Information using the BLAST network server (19). ClustalX (20) was used for multiple protein sequence alignment.

Site-directed Mutagenesis-- Mutagenesis was performed according to the manufacturer's instructions using the QuikChange mutagenesis kit (Stratagene), which employs the oligonucleotide-directed in vitro mutagenesis method (21). A derivative of the T7-based expression vector pET15b (Novagen) containing the M. thermophila ack gene (9) was the target plasmid used for mutagenesis. In the resulting plasmids, a 60-nucleotide leader sequence with six tandem histidine codons was fused in-frame to the 5'-end of the wild-type and mutant ack genes. The mutations were verified by dye termination cycle sequencing using an ABI PRISM 377 DNA sequencer (Applied Biosystems) at the Nucleic Acid Facility at the Pennsylvania State University.

Heterologous Production and Purification of Acetate Kinase-- The wild-type and variant acetate kinases were overproduced in E. coli BL21(DE3) as described previously (9) and purified using a nickel-nitrilotriacetic acid silica spin kit (Qiagen) according to the manufacturer's instructions. The enzymes were eluted in buffer (pH 7.0) containing 50 mM NaH₂PO₄, 300 mM NaCl, and 250 mM imidazole. Protein purity was assessed by SDS-polyacrylamide gel electrophoresis (22) using 12% gels. Protein concentrations were determined by the Bradford method (23) using protein dye reagent (Bio-Rad) and bovine serum albumin as the standard.

Enzyme Activity Assays-- The hydroxamate assay, an adaptation of the method of Lipmann and Tuttle (24) and Rose et al. (2), indirectly detects the formation of acetyl phosphate from acetate and ATP. This previously described assay (25) was modified such that the final concentrations of the assay components were as follows (unless otherwise noted): 145 mM Tris (pH 7.4), 500 mM potassium acetate, 10 mM MgCl₂, 10 mM ATP, and 705 mM hydroxylamine hydrochloride. When indicated, 10 mM MnCl₂ was used in place of the MgCl₂. In the determination of the K_m for acetate, the concentration of ATP was 10 mM unless the K_m for ATP was determined to be higher than that for the wild-type enzyme, in which case the concentration was increased to 20 mM. Likewise, when determining the K_m for ATP, the acetate concentration was 500 mM unless the K_m for acetate was determined to be higher than that for the wild-type enzyme, in which case the concentration of acetate was increased to 1.5 or 2 M. In the determination of the magnesium concentration required for half-maximal activity, the potassium acetate concentration was 1 M.

Activity in the direction of ATP formation was assayed by the previously described enzyme-linked assay (25). The standard reaction mixture (500 µl) contained 100 mM Tris, pH 7.5, 0.2 mM dithiothreitol, 10 mM MgCl₂, 5.5 mM glucose, 1 mM NADP, 20 mM acetyl phosphate, 5 mM ADP, 10 units of hexokinase (yeast), and 10 units of glucose-6-phosphate dehydrogenase (yeast). In the determination of the K_m for acetyl phosphate, the ADP concentration used was 5 mM, whereas in the determination of the K_m for ADP, the acetyl phosphate concentration used was 20 mM. Kinetic constants were determined using non-linear regression to fit the data to the Michaelis-Menten equation using the program Kaleidagraph (Synergy Software, Reading, PA).

Circular Dichroism Spectroscopy-- Spectra were acquired at 25 °C with an Aviv circular dichroism spectrometer, model 62DS. Samples (1 to 10 µM) of acetate kinase in 25 mM Tris (pH 7.2) were placed in a cuvette with a 1-mm path length, and data points were obtained from 200 to 300 nm in 1.0-nm increments (5-s averaging time). The resulting spectra were normalized for direct comparison.

Molecular Mass-- The native molecular mass was determined by gel filtration chromatography using a Superose 12 gel filtration column (Amersham Pharmacia Biotech) calibrated with cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehydrogenase (150 kDa), and -amylase (200 kDa). Protein samples (1.0 ml) were loaded onto the column that had been pre-equilibrated with 145 mM Tris-HCl buffer, pH 7.0, containing 150 mM NaCl, and the column was developed with a flow rate of 0.5 ml/min.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Initial Characterization of the Wild-type and Variant Enzymes-- Acetate kinase and the sugar kinase/Hsc70/actin superfamily have in common five motifs that, in superfamily members other than acetate kinase, have been shown to be primarily involved in metal binding or catalysis. Four of these motifs are visualized in the ribbon diagram of the acetate kinase from M. thermophila, shown in Fig. 1. These four motifs contain 11 residues that are both highly conserved among acetate kinases from phylogenetically diverse species (Fig. 2) and present in the active site. Three of these residues (His²⁰⁸, Glu³⁸⁴, and Glu³⁸⁵) were previously investigated by site-directed mutagenesis and analyses of variants (7, 8). The active site locations of the remaining eight residues (in addition to Glu³⁸⁴) are shown in Fig. 3. These eight residues were individually replaced by site-directed mutagenesis. After a single step nickel-affinity purification, all of the variants were found to be homogeneous and have the same monomeric molecular mass as the wild type as determined by SDS-polyacrylamide gel electrophoresis (data not shown). In addition, gel filtration chromatography indicated that all of the variants were homodimers under native conditions (data not shown), as is the case with the wild-type enzyme (25). These results indicate no gross structural differences between the wild-type and variant acetate kinases.

View larger version (64K): [in this window] [in a new window]   Fig. 1.   Four motifs in the crystal structure of acetate kinase from M. thermophila that are conserved in the sugar kinase/Hsc70/actin superfamily. The motifs are highlighted as follows: phosphate-1 (red), phosphate-2 (blue), connect-1 (purple), and connect-2 (yellow). ADP (orange), and sulfate (green), which occupies the proposed location of the ATP -phosphate, are also shown.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Alignments of four of the putative nucleotide and metal binding motifs in acetate kinase sequences. The phosphate-1 fingerprint sequence, proposed to bind the adenine nucleotide -phosphate in members of the sugar kinase/Hsc70/actin superfamily, is also displayed (where X is any amino acid). Conserved residues targeted for site-directed replacement in the acetate kinase from M. thermophila in this study are shown in bold. MT, M. thermophila; EC, E. coli; CT, Clostridium thermocellum; TM, Thermotoga maritima; BS, Bacillus subtilis; BH, Bacillus halodurans.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Stereo view of the active site of M. thermophila acetate kinase with residues from four putative nucleotide and metal binding motifs displayed. ADP (orange) and sulfate (green) are also shown.

Kinetic Analyses of Variants in the Connect-1 and Connect-2 Motifs-- The catalytic efficiencies of variants D148A, D148N, and D148E were less than 0.1% of that for the wild-type enzyme, which indicated that Asp¹⁴⁸ is essential for catalysis (Table I). The circular dichroism spectra of the D148A variant, relative to that of wild type, varied by only 6.5% at 220 nm, confirming that replacement of Asp¹⁴⁸ with alanine resulted in no significant global conformational changes (data not shown). These results indicate that the inactivity of the D148A variant is the result of the loss of an essential functional group and not a gross structural change. The K_m values for ATP of the Asp¹⁴⁸ variants did not differ from wild type, and the K_m value for acetate of the D148A variant was only 3-fold higher than that of wild type (Table I).

A preliminary report on Glu³⁸⁴ from the M. thermophila acetate kinase had indicated that this residue was essential for catalysis (8). The authors concluded that the extremely low activities of the Glu³⁸⁴ variants precluded the determination of kinetic constants (8). We were able to purify E384A in sufficient quantity to determine the kinetic constants, enabling a further investigation into the role of Glu³⁸⁴ in catalysis. E384A had a catalytic efficiency that was less than 1% of that of the wild-type enzyme (Table I), consistent with the previous proposal that Glu³⁸⁴ is essential for catalysis. The K_m value for ATP of E384A was increased less than 3-fold relative to that of wild type (Table I). The K_m value for acetate could not be determined for E384A because of non-saturation kinetics with respect to acetate (Table I). The K_m values for ADP and acetyl phosphate did not increase relative to wild type for the D148A and E384A variants, whereas the catalytic efficiencies were less than 1% of the wild type (data not shown).

Kinetic Analyses of Variants in the Phosphate-1 and Phosphate-2 Motifs-- The catalytic efficiencies of the Asn⁷ variants were 1% or less than that of wild type, suggesting an essential role for Asn⁷ in catalysis (Table I). Variants of Ser¹⁰, Ser¹², and Lys¹⁴ also displayed reduced catalytic efficiencies, which were greater than 1% of the wild type, indicating important but not essential roles for these residues in catalysis. Variants of Asn⁷, Ser¹⁰, Ser¹², and Lys¹⁴ had K_m values for ATP similar to that of wild type (Table I). Non-saturation kinetics precluded determination of the K_m value for acetate of N7A. Replacement of Asn⁷ with aspartate resulted in a variant with a determinable K_m value for acetate that was 14-fold greater than that of the wild-type enzyme (Table I). Variants S10A, S12A, and K14A also displayed either high K_m values for acetate as compared with that of wild-type or non-saturation kinetics with respect to acetate (Table I). Conservative replacement of Ser¹⁰ or Ser¹² with threonine resulted in variants with higher catalytic efficiencies than those of S10A or S12A, indicating that the functional groups of Ser¹⁰ and Ser¹² are important for catalysis (Table I). In addition, variants S10T and S12T had K_m values for acetate that were elevated as compared with that of wild type (Table I). As compared with the K14A variant, K14R had a greater catalytic efficiency and a return to saturation kinetics with respect to acetate. These results are consistent with a role for the positive charge of Lys¹⁴ in catalysis (Table I). Variant S11A had a K_m value for ATP and catalytic efficiencies that were not far removed from those of wild type. The K_m value for acetate of variant S11A increased only 3-fold as compared with that of wild type. Replacement of Ser¹¹ with a bulky threonine residue resulted in a lower catalytic efficiency and an elevated K_m value for acetate (Table I) as compared with those of S11A, suggesting a size limitation for position 11. The functional group of residue Asn²¹¹ also appears to be unimportant for catalysis, as evidenced by the wild-type catalytic efficiencies of variant N211A (Table I).

The K_m values for ADP and acetyl phosphate did not increase relative to wild type for N7A, S10A, S11A, S12A, N211A, or K14A. However, the catalytic efficiencies for each variant were less than 2% of the wild type, except for S11A and N211A, which had values that were at least 21% of the wild type (data not shown).

Divalent Cation Requirements for the Wild-type and Variant Enzymes-- Several of the conserved motifs in other sugar kinase/Hsc70/actin superfamily members have been implicated in metal binding. Consequently, the magnesium concentration required for half-maximal activity was determined for the alanine variants (Table II) to implicate residues involved in metal coordination. All of the alanine variants except N211A and S11A showed an increase in the magnesium concentration required for half-maximal activity relative to that of wild type, particularly N7A, S12A, K14A, and E384A, suggesting that these residues contribute to the coordination of magnesium (Table II). Most significantly, Glu³⁸⁴ appears to be important for metal coordination, as evidenced by the 30-fold increase in the magnesium concentration required for half-maximal activity of the E384A variant relative to that of wild type.

Also consistent with roles for Glu³⁸⁴ and Asn⁷ in magnesium coordination was the change in the preferred cation of the E384A and N7A variants relative to that of wild type (Table III). Alteration of the divalent cation preference of an enzyme by replacement of a metal binding residue has been used to implicate residues in metal binding (26, 27). The specific activity of variant E384A was increased by 120% in the presence of manganese versus magnesium, whereas the specific activity of wild type was not significantly altered (Table III). Variants N7A, S12A, and D148A also displayed increases in specific activity with manganese versus magnesium (56, 83, and 58%, respectively) (Table III). Wild-type acetate kinase utilizes manganese or calcium in place of magnesium but is inactive with copper or zinc (25). The alanine variants of Asn⁷, Ser¹⁰, Ser¹¹, Ser¹², Lys¹⁴, Asp¹⁴⁸, Asn²¹¹, and Glu³⁸⁴ did not show a change in preference for the divalent cation relative to the wild-type enzyme when magnesium was replaced with copper, zinc, or calcium in the activity assay (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acetate kinase is proposed to be the newest member of the sugar kinase/Hsc70/actin structural superfamily and possibly the urkinase (10, 11). The proposed membership is based on a common fold and the presence of five analogous motifs in other members of the superfamily that are involved in catalysis, nucleotide binding or metal binding (28). Here, we report a site-directed mutational analysis of active site residues in four of the shared motifs to further define the relationship of acetate kinase to other members of the superfamily and to address the roles for active site residues in catalysis.

In several sugar kinase/Hsc70/actin superfamily members, the phosphate-1 motif has been implicated in binding the nucleotide -phosphate (15, 16, 18). Replacement of Asn⁷, Ser¹⁰, Ser¹¹, Ser¹², or Lys¹⁴ from the phosphate-1 loop of acetate kinase had little effect on the K_m value for ATP or ADP relative to that of wild type. However, the k_cat values for Asn⁷ and Ser¹⁰ variants indicated that Asn⁷ and Ser¹⁰ are important for catalysis. What has been proposed for hexokinase, and seems applicable to acetate kinase, is that polyphosphate-protein interactions are primarily involved in the stabilization of the transition state (16, 17). For example, replacement of the Asn⁷ equivalent in hexokinase resulted in a 1000- to 200-fold decrease in the k_cat value with little change in the K_m value (16), similar to the results reported here for Asn⁷. The results presented here suggest a degree of variability in the roles of residues from the phosphate-1 loop among superfamily members. For example, replacement of residue Lys¹⁴ of acetate kinase had significantly less effect on catalysis than did replacement of the analogous residue (R539) in human brain hexokinase. Variant R539I had a 114-fold decrease in the k_cat value, whereas variant R539K exhibited a 12-fold reduction in the k_cat value relative to that of the wild-type enzyme (15, 17). Neither the R539I nor the R539K variant of the hexokinase displayed a significant change in the K_m value for ATP relative to that of the wild-type enzyme (15, 17), similar to the results reported here for Lys¹⁴ of acetate kinase. In another sugar kinase/Hsc70/actin superfamily member, Hsc70, the residue equivalent to Lys¹⁴ of acetate kinase is absent; instead, a potassium ion is proposed to provide an equivalent electrostatic interaction with the polyphosphate (30). It should be noted that in the crystal structure of acetate kinase, residues of the phosphate-1 loop are not within hydrogen bonding distance of the nucleotide polyphosphate or the sulfate molecule proposed to occupy the location of the ATP -phosphate (10). However, these data alone do not preclude the involvement of these residues in binding the polyphosphate. Members of the sugar kinase/Hsc70/actin superfamily are proposed to undergo a substrate-induced interdomain hinge motion. In hexokinase, for example, the binding of glucose causes partial closure of the cleft between the two domains, and the subsequent binding of ATP is proposed to cause even greater closure (13, 31). Thus, it has been proposed that the phosphate-1 loop of acetate kinase is likely to come into closer proximity of the polyphosphate during catalysis (10).

Unexpectedly, replacement of several conserved residues in the phosphate-1 loop (Asn⁷, Ser¹⁰, Ser¹², and Lys¹⁴) resulted in either large increases in the K_m value for acetate relative to that of the wild-type enzyme or non-saturation kinetics with respect to acetate. Although these results are consistent with a role for Asn⁷, Ser¹⁰, Ser¹², and Lys¹⁴ in the affinity for acetate, other factors unrelated to acetate binding could contribute to the elevated K_m values. An acetate binding site for acetate kinase has been proposed based on sequence alignments of acetate kinases and butyrate kinases as well as the glycerol and glucose binding sites of glycerol kinase and hexokinase, respectively (10). Furthermore, site-directed mutational analyses support the proposed acetate binding site.¹ Given that the distance between this acetate binding site and the phosphate-1 loop is at least 6.4 Å, it seems unlikely that residues Asn⁷, Ser¹⁰, Ser¹², or Lys¹⁴ are directly involved in the binding of acetate. Nonetheless, the location of the phosphate-1 loop may in part explain the elevated K_m values for acetate. The adenosine moiety of the nucleotide binds in the opposite domain to that of the phosphate-1 loop (10). Consequently, binding of the polyphosphate by the phosphate-1 loop may contribute to the interdomain hinge motion. Indeed, ATP is proposed to form a "structural bridge" between the two domains in actin and other members of the superfamily (13, 32). The elevated K_m values for acetate of the acetate kinase variants may be the consequence of a mechanism in which binding of the ATP polyphosphate is required for domain closure and formation of the ternary complex. The K_m values for acetyl phosphate did not increase for N7A, S10A, S12A, or K14A relative to wild type, a result consistent with a role for these residues in catalysis and not substrate binding. Dramatic effects on the K_m value for the co-substrate because of the site-directed replacement of phosphate-1 loop residues have not been found for hexokinase or glycerol kinase from the sugar kinase/Hsc70/actin superfamily (15, 16, 18). Glucose binding is proposed to be both necessary and sufficient for domain closure in hexokinase, whereas ATP binding is neither (14).

The phosphate-2 motif has been proposed to bind the ATP -phosphate in other members of the sugar kinase/Hsc70/actin superfamily (28, 33). This motif contains several conserved amino acid residues, although they vary somewhat depending upon the particular protein family. In hexokinase and glycerol kinase, the phosphate-2 sequence is GT, whereas in Hsc70 and actin, the consensus sequence is DXG (where X is any amino acid). Site-directed replacement of Asp²¹⁹ from the DXG motif of human E-type ATPase, a proposed member of the superfamily, indicated that this residue has an important role in enzymatic activity (33). Replacement of Asp²¹⁹ with alanine or asparagine resulted in the loss of 90-98% of the ATPase activity and the loss of 100% of the ADPase activity (33). However, variant D219E had ATPase and ADPase activities that were similar to wild type (33), suggesting a requirement for a carboxyl group at position 219. Acetate kinase exhibits yet another variation in the consensus sequence of the phosphate-2 motif (GNG²¹² in the M. thermophila enzyme). In the crystal structure of M. thermophila acetate kinase, the backbone amides of residues Asn²¹¹ and Gly²¹² hydrogen-bond to the polyphosphate (10), which is consistent with results presented here, indicating no role for the functional group of Asn²¹¹ in catalysis.

Residue Asp¹⁴⁸ in the connect-1 motif was found to be essential for acetate kinase activity, a result consistent with kinases of the sugar kinase/Hsc70/actin superfamily in which the carboxyl group of the equivalent aspartate is proposed to function in base catalysis or metal binding (18, 28, 34, 35). For example, essential residue Asp²⁰⁵ in hepatic glucokinase has been proposed to deprotonate the glucose 6-hydroxyl group, thus facilitating the nucleophilic attack of this group on the -phosphate of ATP (35). In acetate kinase, Asp¹⁴⁸ may serve to activate acetate by abstraction of the carboxyl proton; however, based on the intrinsic pK_a of acetic acid (4.7), it is not obvious why this would be necessary. One possible scenario is that the microenvironment of the bound acetate significantly increases the pK_a of the carboxyl group, necessitating deprotonation. It seems more likely that Asp¹⁴⁸ in acetate kinase has an alternative role in catalysis.

The results reported here are consistent with the involvement of Glu³⁸⁴, and to a lesser extent Asn⁷, in chelating the divalent metal of the metal-nucleotide complex. The results are consistent with data from several superfamily members that implicate the residues analogous to Asn⁷ in metal binding (16, 36). The elevated magnesium concentration required for half-maximal activity of S10A, K14A, and especially S12A relative to that of wild type may be attributable to the fact that the nucleotide polyphosphate is most likely a metal ligand. Consequently, replacement of residues from the phosphate-1 loop, proposed to bind the nucleotide -phosphate, resulted in variants requiring a greater concentration of magnesium for activity relative to that of wild type. Whereas the results with Asn⁷ were predictable because of similarities with members of the superfamily, the results for Glu³⁸⁴ from the connect-2 motif were unexpected. Although conserved alanine and glycine residues in the connect-1 and connect-2 motifs of sugar kinase/Hsc70/actin superfamily members have been proposed to form key contact points between these two motifs (13, 14), to our knowledge no other residues from the connect-2 motif have been implicated in catalysis. In particular, the results reported here for Glu³⁸⁴ represent the first proposed function for any analogous residue in the connect-2 motif of the superfamily. The reason for the non-saturation kinetics with respect to acetate of variant E384A is uncertain. The location of Glu³⁸⁴ relative to the proposed acetate binding site in the crystal structure (10) does not indicate a role for this residue in the binding of acetate. As proposed above, it is possible that nucleotide binding contributes to the domain closure mechanism, which in turn influences the K_m for acetate. Glu³⁸⁴ may participate in the postulated domain closure mechanism by binding the magnesium of the metal-nucleotide complex; thus, Glu³⁸⁴ may indirectly influence the K_m for acetate.

The controversy over the catalytic mechanism of acetate kinase between a direct in-line phosphoryl transfer mechanism and a triple displacement mechanism involving two phosphoenzyme intermediates is still unresolved. Nevertheless, the data implicating Glu³⁸⁴ in the coordination of magnesium are inconsistent with Glu³⁸⁴ as a site for phosphorylation in a triple displacement mechanism, as previously hypothesized (8). The data do not exclude the possibility that essential Asp¹⁴⁸ acts as a phosphorylation site during catalysis. However, this scenario seems unlikely given the conservation of this residue in at least two other sugar kinase/Hsc70/actin superfamily members, hexokinase and glycerol kinase, for which the catalytic mechanisms do not proceed via phosphoenzyme intermediates. Other site-directed mutational analyses of the M. thermophila enzyme have eliminated the possibility of the only other active site residues (His¹²³, His¹⁸⁰, and His²⁰⁸) that could potentially function as phosphorylation sites in catalysis (7), which is also consistent with a direct transfer mechanism.

No density attributable to a metal ion was detected in the crystal structure for the acetate kinase (10). The results reported here, which implicate Glu³⁸⁴ and Asn⁷ in metal coordination, represent the first step toward understanding the metal binding site in any acetate kinase. The functional groups of Asn⁷ and Glu³⁸⁴ are within ~7 Å of each other in the active site cleft, consistent with these residues forming a metal coordination pocket (10). Although shown to be essential for activity for the E. coli acetate kinase (25), the role of magnesium during catalysis has not been investigated. Blättler and Knowles (3) proposed a direct in-line phosphoryl transfer mechanism for E. coli acetate kinase based on the net inversion of configuration of the transferred phosphate. In the proposed associative-type mechanism, the structure of the transition state is trigonal bipyramidal with three negatively charged equatorial oxygens. Three electron-deficient residues (positive charges or hydrogen bond donors) are necessary to stabilize this transition state (3). Active site arginine residues, Arg⁹¹ and Arg²⁴¹, have been shown to be essential for activity and are proposed to be two of these stabilization sites (9). If a direct in-line mechanism is operable, the third stabilization site may be the magnesium ion of the metal-nucleotide complex.

	ACKNOWLEDGEMENTS

We thank David Sanders, Andrea Gorrell for suggestions on the manuscript and Laura Van Zant for technical assistance. We also thank Dr. C. Robert Matthews for the use of a circular dichroism spectrometer and the members of Dr. Matthews' laboratory for technical assistance.

	FOOTNOTES

^* This work was supported by Department of Energy, Basic Energy Sciences Grant DE-FG02-95ER20198 (to J. G. F.) and by National Science Foundation Fellowship Grant DBI-9602232 (to R. D. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 814-863-5721; Fax: 814-863-6217; E-mail: jgf3@psu.edu.

Published, JBC Papers in Press, September 18, 2001, DOI 10.1074/jbc.M108355200

¹ C. Ingram-Smith, P. P. Iyer, and J. G. Ferry, manuscript in preparation.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1.	Lipmann, F. (1944) J. Biol. Chem. 155, 55-70
2.	Rose, I. A., Grunberg-Manago, M., Korey, S. R., and Ochoa, S. (1954) J. Biol. Chem. 211, 737-756
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