Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis.

The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1beta family of glycosyltransferases. This enzyme was used to prove, by site-directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity. Exchange of amino acids at the NRD site leads to a decrease of enzymatic activity, e.g. substitution of Glu368 by Asp. Glu368, which is a conserved amino acid in glycosyltransferases located at position 2 and is important for enzyme activity, does not serve as the nucleophile in the catalytic centre as proposed. When it is replaced by Ala, the resulting mutant enzyme E368A exhibits comparable activity as found for E368D in respect to vanillin. Enzyme activities of wild-type and E368A towards several substrates were not affected at the same level. His360 at position 1 of NRD1beta glycosyltransferases occupies a more crucial role as expected. When it is exchanged against other basic amino acids such as Lys or Arg the enzyme activity decreases approximately 1000-fold. Replacement of His360 by Glu leads to a mutant enzyme (H360E) with an approximately 4000-fold lower activity compared with the wild-type. This mutein still produces a beta-glucoside, not an alpha-glucoside and therefore indicates that generation of the typical E-E motif of NRD1alpha glycosyltransferases does not convert a NRD1beta enzyme into a NRD1alpha enzyme. The presented data do not support several suggestions made in the literature about catalytic amino acids involved in the glycosyltransfer reaction.     

Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis

Aminoacylase-1 from pig kidney (pAcy1) catalyzes the highly stereoselective acylation of amino acids, a useful conversion for the preparation of optically pure N-acyl-l-amino acids. The kinetic of this thermodynamically controlled conversion is determined by maximal velocities for synthesis (V_mS) and hydrolysis (V_mH) of the N-acyl-l-amino acid. To investigate which parameter affects maximal velocities, we focused on the proton acceptor potential of the catalytic base, E146, and studied the influence of the active site architecture on its contribution to the pKa of residue E146. The modeled structure of pAcy1 identified residue D346 as having the strongest impact on the electrostatic features of the catalytic base. Substitutions of D346 generally decreased enzymatic activities but also altered both the pH-dependency of hydrolytic activity and the V_mS/V_mH ratio of pAcy1. A reduced theoretical pKa value and a lowered experimental pH optimum of hydrolytic rates for the D346A mutant were associated with a 9-fold increase in V_mS/V_mH. This supports the importance of electrostatic contributions of D346 to the acid-base properties of E146 and demonstrates for the first time the possibility of engineering the V_mS/V_mH ratio of pAcy1.     

Mechanistic study of proton transfer and hysteresis in catalytic antibody 16E7 by site-directed mutagenesis and homology modeling

Antibody 16E7 catalyzes the carbon protonation of enol ether 2 to hemiacetal 3, and the carbon deprotonation of benzisoxazole 7 to phenol 8. This antibody shows an extreme case of hysteresis, requiring several hours to reach full activity. Antibody 16E7 was expressed as recombinant chimeric Fab in Escherichia coli. A model for the three-dimensional structure was produced by homology modeling and used for a docking procedure to obtain models for antibody-ligand complexes. Site-direct mutagenesis of GluL39, identified as a possible catalytic residue by the model, to either glutamine or alanine abolished catalysis, showing that both the protonation reaction of enol ether 2 and the deprotonation of benzisoxazole 7 are promoted by the same residue. The model furthermore suggested that substrate access to the catalytic site might be hindered by a flexible HCDR3 loop held in closed position by a hydrogen bond between SerH99 and GluL39, which could explain the observed hysteresis effect. In agreement with this model, mutagenesis of SerH99 to alanine, or deletion of this residue, was found to reduce hysteresis by approximately 50%.     

Probing the essential catalytic residues and substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-arabinose isomerase by site-directed mutagenesis

The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.     

Probing the mechanism of the hamster mitochondrial folate transporter by mutagenesis and homology modeling

The mitochondrial folate transporter (MFT) was previously identified in human and hamster cells. Sequence homology of this protein with the inner mitochondrial membrane transporters suggested a domain structure in which the N- and C-termini of the protein are located on the mitochondrial intermembrane-facing surface, with six membrane-spanning regions interspersed by two intermembrane loops and three matrix-facing loops. We now report the functional significance of insertion of the c-myc epitope into the intermembrane loops and of a series of site-directed mutations at hamster MFT residues highly conserved in orthologues. Insertional mutagenesis in the first predicted intermembrane loop eliminated MFT function, but the introduction of a c-myc peptide into the second loop was without effect. Most of the hamster MFT residues studied by site-directed mutagenesis were remarkably resilient to these mutations, except for R249A and G192E, both of which eliminated folate transport activity. Homology modeling, using the crystal structure of the bovine ADP/ATP carrier (AAC) as a scaffold, suggested a similar three-dimensional structure for the MFT and the AAC. An ion-pair interaction in the AAC thought to be central to the mechanism of membrane penetration by ADP is predicted by this homology model to be replaced by a pi-cation interaction in MFT orthologues and probably also in other members of the family bearing the P(I/L)W motif. This model suggests that the MFT R249A and G192E mutations both modify the base of a basket-shaped structure that appears to constitute a trap door for the flux of folates into the mitochondrial matrix.     

Expansion of substrate specificity and catalytic mechanism of azoreductase by X-ray crystallography and site-directed mutagenesis

AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the Vmax value for p-methyl red 27-fold with a 3.8-fold increase of the Km value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The Vmax value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study.     

Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis

A three-dimensional structural model of fructosyl amine oxidase from the marine yeast Pichia N1-1 was generated using the crystal structure of monomeric sarcosine oxidase from Bacillus sp. B-0618 as template. The putative active site region was investigated by site-directed mutagenesis, identifying several amino acid residues likely playing important roles in the enzyme reaction. Asn354 was identified as a residue that plays an important role in substrate recognition and that can be substituted in order to change substrate specificity while maintaining high catalytic activity. While the Asn354Ala substitution had no effect on the V _max K _m⁻¹ value for fructosyl valine, the V _max K _m⁻¹ value for fructosyl-^ε N-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-^ε N-lysine.     

Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling

Protein C (PC) is activated to an essential anticoagulant enzyme (activated PC or APC) by thrombin (T) bound to thrombomodulin (TM), a membrane receptor present on the surface of endothelial cells. The understanding of this complex biological system is in part limited due to the lack of integration of experimental and structural data. In the work presented here, we analyze the PC-T-TM pathway in the context of both types of information. First, structural analysis of the serine protease domain of PC suggests that a positively charged cluster of amino acids could be involved in the activation process. To investigate the importance of these basic amino acids, two recombinant PC mutants were constructed using computer-guided site-directed mutagenesis. The double mutant had the K62[217]N/K63[218]D substitution and in the single mutant, K86[241] was changed to S. Both mutants were activated by free thrombin at rates equivalent to that of wild-type PC (wt-PC) and they demonstrated similar calcium-dependent inhibition of their activation. The K86[241]S mutant and wt-PC were activated by thrombin bound to soluble TM at a similar rate. In contrast, the K62[217]N/ K63[218]D mutant was activated by the T-TM complex at a 10-fold lower catalytic efficiency due to a lowering in k(cat) and increase in Km. Molecular models for PC and thrombin bound to a segment of TM were developed. The experimental results and the modeling data both indicate that electrostatic interactions are of crucial importance to orient PC onto the T-TM complex. A key electropositive region centered around loops 37[191] and 60[214] of PC is defined. PC loop 37[191] is located 7-8 A from the TM epidermal growth factor (EGF) 4 while the loop 60[214] is about 10 A away from TM EGF4. Both loops are far from thrombin. A key function of TM could be to create an additional binding site for PC. The Gla domain of PC points toward the membrane and away from thrombin or the EGF modules of TM during the activation process.     

Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain

The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.     

Probing the active site of cellodextrin phosphorylase from Clostridium stercorarium: kinetic characterization, ligand docking, and site-directed mutagenesis

Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl β-glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme's specificity, homology modeling and ligand docking were applied. Residue W168 has been found to form a hydrophobic stacking interaction with the substrate in subsite +2, and its importance has been examined by means of site-directed mutagenesis. The mutant W168A retains about half of its catalytic activity, indicating that other residues also contribute to the binding affinity of subsite +2. Finally, residue D474 has been identified as the catalytic acid, interacting with the glycosidic oxygen between subsites -1 and +1. Mutating this residue results in complete loss of activity. These results, for the first time, provide an insight in the enzyme-substrate interactions that determine the activity and specificity of cellodextrin phosphorylases.     

Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis

The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.     

Probing a putative active site of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 (PDP1c) by site-directed mutagenesis

The catalytic subunit of pyruvate dehydrogenase phosphatase 1 (PDP1c) is a magnesium-dependent protein phosphatase that regulates the activity of mammalian pyruvate dehydrogenase complex. Based on the sequence analysis, it was hypothesized that PDP1c is related to the mammalian magnesium-dependent protein phosphatase type 1, with Asp54, Asp347, and Asp445 contributing to the binuclear metal-binding center, and Asn49 contributing to the phosphate-binding sites. In this study, we analyzed the functional significance of these amino acid residues using a site-directed mutagenesis. It was found that substitution of each of these residues had a significant impact on PDP1c activity toward the protein substrate. The activities of Asp54, Asp347, and Asp445 mutants were decreased more than 1000-fold. The activity of Asn49 mutant was 2.5-fold lower than the activity of wild-type PDP1c. The decrease in activity of Asp54 and Asp347 came about, most likely, as a result of impaired magnesium binding. Unexpectedly, it was found that the Asp445 mutant bound Mg(2+) ions similarly to the wild-type enzyme. Accordingly, the Asp445 mutant was found to be active with the artificial substrate p-nitrophenyl phosphate (pNPP). Asp54 and Asp347 mutants did not demonstrate any appreciable activity with pNPP. Together, these observations strongly suggest that Asn49, Asp54, and Asp347 are important for the catalysis of the phosphatase reaction, contributing to the phosphate- and metal-binding centers of PDP1c. In contrast, Asp445 is not required for catalysis. The exact role of Asp445 remains to be established, but indirect evidence suggests that it might be involved in the control of interactions between PDP1c and the protein substrate pyruvate dehydrogenase.     

Probing the ubiquinol-binding site in cytochrome bd by site-directed mutagenesis

To probe the structure of the quinol oxidation site in loop VI/VII of the Escherichia coli cytochrome bd, we substituted three conserved residues (Gln249, Lys252, and Glu257) in the N-terminal region and three glutamates (Glu278, Glu279, and Glu280) in the first internal repeat. We found that substitutions of Glu257 by Ala or Gln, and Glu279 and Glu280 by Gln, severely reduced the oxidase activity and the expression level of cytochrome bd. In contrast, Lys252 mutations reduced only the oxidase activity. Blue shifts in the 440 and 630 nm peaks of the reduced Lys252 mutants and in the 561 nm peak of the reduced Glu257 mutants indicate the proximity of Lys252 to the heme b(595)-d binuclear center and Glu257 to heme b(558), respectively. Perturbations of reduced heme b(558) upon binding of aurachin D support structural changes in the quinol-binding site of the mutants. Substitutions of Lys252 and Glu257 caused large changes in kinetic parameters for the ubiquinol-1 oxidation. These results indicate that Lys252 and Glu257 in the N-terminal region of the Q-loop are involved in the quinol oxidation by bd-type terminal oxidase.     

Alteration of catalytic properties of chymosin by site-directed mutagenesis

Artificial mutations of chymosin by recombinant DNA techniques were generated to analyze the structure--function relationship in this characteristic aspartic proteinase. In order to prepare the mutant enzymes in their active form, we established procedures for purification of correctly refolded prochymosin from inclusion bodies produced in Escherichia coli transformants and for its subsequent activation. Mutagenesis by linker insertion into cDNA produced several mutants with an altered ratio of milk clotting activity to proteolytic activity and a different extent of stability. In addition to these mutants, several mutants with a single amino acid exchange were also constructed by site-directed mutagenesis and kinetic parameters of these mutant enzymes were determined by using synthetic hexa- and octa-peptides as substrates. Exchange of Tyr75 on the flap of the enzyme to Phe caused a marked change of substrate specificity due to the change of kcat or Km, depending on the substrate used. Exchange of Val110 and Phe111 also caused a change of kinetic parameters, which indicates functional involvement of these hydrophobic residues in both the catalytic function and substrate binding. The mutant Lys220----Leu showed a marked shift of the optimum pH to the acidic side for hydrolysis of acid-denatured haemoglobin along with a distinct increase in kcat for the octa-peptide in a wide pH range.     

Probing the unfolding region of ribonuclease A by site-directed mutagenesis.

Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent beta-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in DeltaG can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule.     

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.     

Probing the role of arginines and histidines in the catalytic function and activation of yeast 3-phosphoglycerate kinase by site-directed mutagenesis

A cluster of conserved histidines and arginines (His-62, His-167, Arg-21, Arg-38, and Arg-168) in 3-phosphoglycerate kinase (PGK) has been implicated as possibly involved in the binding of 3-phosphoglycerate (3-PG) and/or stabilization of the negatively charged transition state. The role of these residues in the catalytic function of yeast PGK and in the substrate- and sulfate-dependent activation was investigated by site-directed mutagenesis. The following substitutions, R21A, R21Q, H62Q, H167S, and R168Q, produced functional enzymes. In contrast, the R38A and R38Q mutations resulted in a complete loss of catalytic activity. These results demonstrate that of the basic residues studied, only arginine 38 is essential for the catalytic function of PGK. A moderate decrease in the catalytic efficiency as the result of the R21A, H167S, and R168Q mutations and an increased catalytic efficiency of the H62Q mutant rule out a possible role of a positive charge at these positions in the mechanism of phosphoryl transfer reaction. In contrast to the wild type PGK and the H62Q mutant, both of which are activated at low and inhibited at high sulfate concentration, the H167S, R168Q, and R21A mutants exhibited a progressive inhibition with increased concentration of sulfate. The activation observed at high concentration of either ATP or 3-PG as a variable substrate in the steady-state kinetics of wild type PGK was abolished as the result of the latter three mutations. The results of this work support the hypothesis that PGK has two binding sites for anionic ligands, the catalytic and regulatory sites for each substrate and the activatory and inhibitory sites for sulfate, and suggest that arginine 21, arginine 168, and histidine 167 are located in the activatory anion binding site, common for sulfate, 3-PG, and ATP. The increased Km values for both substrates and decreased specific activities of the mutants suggest that this regulatory site is close to the catalytic site.