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Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA
(RECEIVED May 17, 2006; FINAL REVISION July 17, 2006; ACCEPTED July 18, 2006)
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Abstract |
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4000-fold decrease in k cat along with a small decrease in K m. The decrease in catalytic efficiency of 2000-fold was a much smaller drop than that observed when Ala or Gly were substituted at position 102. The mechanism by which Thr can substitute for Ser in AP was further investigated by determining the X-ray structure of the S102T enzyme in the presence of the Pi (S102T_Pi), and after soaking the crystals with substrate (S102T_sub). In the S102T_Pi structure, the Pi was coordinated differently with its position shifted by 1.3 Å compared to the structure of the wild-type enzyme in the presence of Pi. In the S102T_sub structure, a covalent Thr-Pi intermediate was observed, instead of the expected bound substrate. The stereochemistry of the phosphorus in the S102T_sub structure was inverted compared to the stereochemistry in the wild-type structure, as would be expected after the first step of a double in-line displacement mechanism. We conclude that the S102T mutation resulted in a shift in the rate-determining step in the mechanism allowing us to trap the covalent intermediate of the reaction in the crystal. Keywords: X-ray crystallography; mutagenesis; side chain conformation; covalent intermediate; rate-determining step
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Introduction |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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The catalytic mechanism for the alkaline phosphatase reaction was proposed in 1991 and revised in 2000 (Kim and Wyckoff 1991; Stec et al. 2000). The reaction proceeds through a series of four steps: The enzyme binds the substrate to form a noncovalent complex (E•ROP); the deprotonated Ser102 hydroxyl group attacks the phosphorus of the substrate to form a covalent serine–phosphate intermediate (E–Pi) (Schwartz and Lipmann 1961; Murphy et al. 1997); a hydroxide ion coordinated to Zn1 attacks the phosphorus to form a noncovalent enzyme–phosphate complex (E•Pi); and finally, Pi is released, reforming the free enzyme. The rate-determining step of the reaction is pH dependent; at acidic pH, the hydrolysis of the covalent E–P complex is rate-limiting, while under basic conditions the rate-limiting step becomes the dissociation of Pi from the E•Pi noncovalent complex (Hull et al. 1976; Gettins and Coleman 1983).
In a variety of enzymes, Ser and Thr can often be interchanged for many catalytic functions, due to their structural similarity. Some hydrolytic enzymes, such as the asparaginases (Swain et al. 1993; Palm et al. 1996), the proteasome catalytic subunit (Löwe et al. 1995), and aspartyl glucosaminidase (Oinonen et al. 1995), use Thr as a nucleophile instead of Ser. These enzymes have a catalytic mechanism similar to that of the serine proteases, involving the formation of a covalent intermediate. The activation of a Thr nucleophile is different from a Ser nucleophile. Thr is typically activated by a primary amino group from the side chain of Lys or the N-terminal amino acid whereas Ser is activated as part of a catalytic triad.
Although there are no significant chemical differences between the O
atoms of Thr and Ser, the side chains of these residues differ substantially in available conformations and size. The side chain of Thr may have an advantage in certain cases, in that the C could help to exclude water from the active site (Dodson and Wlodawer 1998). Gijsbers et al. (2001) have also pointed out the structural and catalytic similarities between alkaline phosphatase and nucleotide pyrophosphatases/phosphodiesterases that utilize a Thr nucleotide in the reaction. Therefore, in this work we construct a mutant version of alkaline phosphatase that has the active site Ser replaced by Thr in order to investigate whether the enzyme can function with a Thr nucleophile. We combine functional characterization of the S102T alkaline phosphatase with two X-ray crystal structures (in the presence Pi and substrate) to understand how Thr functions as a catalytic group in the active site of alkaline phosphatase.
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Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Structure of the S102T_Pi enzyme
The structure of the S102T in the presence of Pi was determined in order to elucidate the structure of the noncovalent E•Pi complex. Crystals of the S102T enzyme were soaked in a phosphate-containing buffer for 15 min before data collection. Analysis of 2F o –F c and F o –F c electron density maps indicated that the active sites in the S102T_Pi structure differed significantly from the corresponding wild-type structure (WT_Pi), particularly near the phosphate binding sites. The active site of the S102T_Pi structure is shown in Figure 1A. The side chain methyl group of Thr102 restricts the available conformations of the Thr side chain, so that the hydroxyl group cannot rotate as freely as was observed for the Ser hydroxyl group. The orientations of the Thr side chain are limited to three 
values: –60°, +180°, and +60° (Daley and Sykes 2003). In the S102T_Pi structure, the side chain of Thr102 adopts different conformations in the A-chain and B-chain. In the A-chain, the side chain of the Thr102 adopts the = –60° conformation, while in the B chain it adopts the = +180° conformation. In chain A, the methyl group of Thr102 points toward the midpoint of the two hydrogen bonds that form between a phosphorus oxygen and the side chain of the Arg166 in the WT_Pi structure (see Fig. 1C). If one compares the position of Thr102 in the S102T_Pi structure with the position of Pi and Arg166 in the WT_Pi structure, the distance between the methyl group and the phosphorus oxygen is 2.2 Å, while the distance between the methyl group and the Arg166 NH1 is 2.8 Å. This close contact between a hydrophobic group and two hydrophilic groups is energetically unfavorable, resulting in the displacement of Pi by 1.3 Å in the S102T_Pi structure, compared to its position in the WT_Pi structure. The new position prevents the Pi from forming two hydrogen bonds with Arg166, important interactions for the stabilization of Pi in the active site (Chaidaroglou et al. 1988; Chen et al. 1992). In addition, in the S102T_Pi structure, the Pi is stabilized by two coordinate bonds with Zn1, but has one fewer coordinate bond with Zn2.
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The S102T_sub structure—Trapping the covalent E–Pi intermediate
In an attempt to determine the structure of the alkaline phosphatase E•S complex, the X-ray structure of the S102T enzyme was determined using crystals of the S102T enzyme that were frozen after being soaked for a short time in buffer containing the substrate, p-nitrophenol phosphate. The 2.2 Å S102T_sub structure was refined to a R factor/R free of 0.192/0.229 in the I222 space group. In the active site of this structure, the 2F o –F c and F o –F c electron density maps showed continuous density between the phosphorus and the O of the Thr102, with a P–O bond distance of 1.6 Å in both the A and B chains (see Fig. 1B,E). The average distance of a P–O covalent bond is 1.7 Å. These two observations indicate that the structure was not of the E•S complex, but rather of the covalent E–Pi complex. To confirm this hypothesis, a phosphothreonyl residue (ThP102) was modeled into the active sites of both the A and B chains. The S102T_sub structure was then further refined with Thr102 replaced by ThP102. In the S102T_sub structure the active sites of the A and B chains are virtually identical. The average B factor for the side chain of ThP102 was 34.7 Å2, close to the average B factor for the side chains of the entire protein, 32.3 Å2 (Table 2). In the refined structure, one of the phosphate oxygens of ThP102 was coordinated to Zn1 while another oxygen was coordinated to Zn2. The stereochemistry of the phosphorus in the S102T_sub structure is inverted compared to the stereochemistry of the Pi in the structure of the wild-type noncovalent E•Pi structure, as would be expected after the first step of a double in-line displacement mechanism (see Fig. 1F).
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Arg166 is involved in transition state stabilization and the release of the Pi
Previous studies have suggested that Arg166 has two functions in the catalytic mechanism. First, it assists in substrate binding and the release of Pi and second, it stabilizes the charge on the covalent E–P intermediate and the trigonal bipyramidal transition state during the reaction (Chaidaroglou et al. 1988; Kim and Wyckoff 1991). Arg166 was observed to occupy different positions in the S102T_sub and S102T_Pi structures. In both chains of the S102T_sub structure, the guanidinium group of the Arg166 interacts with the phosphate moiety of ThP102 (see Fig. 1F), helping to stabilize the covalent E–Pi intermediate. In contrast, in both chains of the S102T_Pi structure, Arg166 rotates to a new position away from the Pi binding pocket. In this conformation Arg166 forms two fewer hydrogen bonds with Pi than are observed in the WT_Pi structure (see Fig. 1C,D). The loss of these interactions results in a dramatic increase in the average B factor of the Arg166 side chain to 44.9 Å2, an ~60% increase compared to the average B factors of the protein (Table 2). This significant increase in the average B factor of Arg166 side chain suggests that this side chain has considerably more flexibility in the S102T_Pi structure. In this structure, a water molecule interacts with Pi in the position normally occupied by Arg166 in the WT_Pi structure. However, the electron density for this water is weak (1.2 
), and there is no water at the equivalent position in chain B. With the loss of the interactions between Pi and Arg166, the B factors for the Pi are dramatically increased compared to the values observed for Pi in the wild-type structure (WT_Pi) (Table 2).
Previous studies of a mutant alkaline phosphatase in which Asp101 was converted to Ser have shown that increased flexibility of the Arg166 side chain correlates with a faster dissociation of Pi and a high rate of catalysis (Chen et al. 1992). For the wild-type enzyme, Arg166 is positioned rigidly in a hydrogen bonding network that includes two hydrogen bonds with Pi, one with Asp101 and two with a water molecule (Kim and Wyckoff 1991; Stec et al. 2000). However, in the S102T_Pi structure, there is a repulsion between the methyl group of Thr102 and Pi, resulting in the inability of Pi to bind in the same position observed in the wild-type enzyme. The Pi shifts position until it is restabilized by Zn2. This positional shift of the Pi breaks the hydrogen bonding network of the active site, especially the part involving Arg166. Without these interactions, Arg166 becomes unstable in the position observed in the WT_Pi structure and therefore moves to a more stable position. Both the side chain of Arg166 and Pi have enhanced mobility in this structure, which results in a weakening of affinity for Pi and a faster release of the Pi from the noncovalent E•Pi complex.
Conclusions
Our ability to trap the covalent E–Pi intermediate in the S102T_sub structure indicates that the rate-determining step in the mechanism has changed for the S102T enzyme. In the case of the S102T enzyme, hydrolysis of the covalent E–Pi intermediate now becomes slow relative to release of Pi from the noncovalent E•Pi complex. Thus, in the S102T enzyme the chemical step of hydrolysis of the E–Pi complex becomes rate limiting, thereby allowing us to trap the covalent intermediate in the crystal.
The crystal structures of the S102T_sub and S102T_Pi enzymes suggest the manner in which the rate-determining step in the mechanism was changed by the mutation of Ser102 to Thr. The methyl group of Thr102 induces changes in the positions of active site residues, resulting in a weaker binding of the water molecule that acts as the nucleophile in the second step in the mechanism. This reduced availability of the entering nucleophile results in a significant decrease in the rate of the hydrolysis of the E–P covalent complex. Since the release of Pi is the rate-determining step for wild-type enzyme at alkaline pH, a slower hydrolysis rate of the E–P complex results in a shift in the rate-determining step from the dissociation of Pi from the E•Pi noncovalent complex to the hydrolysis of E–P covalent complex in the S102T enzyme. The reduced affinity of the catalytic water explains the 2000-fold decrease of the enzyme activity of the S102T enzyme as compared to the wild-type enzyme.
This S102T mutation in alkaline phosphatase has consequences that are similar to the Glu to Asp mutation in the active sites of triosephosphate isomerases. In both cases while the overall chemical mechanism is retained, the mutation changes the relative free energy of the transition states of the individual steps and results in a different rate-determining step (Raines et al. 1986).
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Materials and methods |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Construction of the S102T alkaline phosphatase by site-specific mutagenesis
The S102T mutation was introduced in the phoA gene using the procedure outlined by Stratagene in the QuikChange Mutagenesis kit protocol. The entire gene was sequenced to ensure that no other mutations had been introduced during the mutagenesis. The final plasmid containing the S102T mutation in the phoA gene was designated pEK625.
Expression of the S102T alkaline phosphatase
E. coli SM547 [
(phoA-phoC), phoR, tsx::Tn5, lac, galK, galU, leu, strr] was used as the host strain for the expression of the S102T alkaline phosphatase. Since the chromosomal phoA gene was deleted from the strain, all the alkaline phosphatase produced is expressed from the phoA gene on the plasmid. The mutation in the phoR regulatory gene results in constitutive synthesis of alkaline phosphatase.
Purification of the S102T alkaline phosphatase
The S102T enzyme was isolated from the SM547/pEK625 plasmid/strain combination by the method previously described (Chaidaroglou et al. 1988). The purity of the enzyme was checked by SDS-polyacrylamide gel electrophoresis (Laemmli 1970). The Bio-Rad version of Bradford's dye-binding assay was used to determine protein concentrations using the wild-type alkaline phosphatase as the standard (Bradford 1976).
Determination of enzymatic activity
Phosphatase activity was measured spectrophotometrically at 25°C utilizing p-nitrophenyl phosphate as substrate by monitoring the release of p-nitrophenylate at 410 nm (Garen and Levinthal 1960). The sum of the hydrolase and transferase activities was determined using 1.0 M Tris as the phosphate acceptor. The hydrolase activity was measured using 0.010 M Tris as the buffer. The ionic strength of the two Tris buffers was maintained constant using NaCl.
Crystallization
The vapor diffusion method was used to crystallize the enzyme using hanging drops of 2 µL. The enzyme solution was dialyzed against a protein-stabilization buffer of 100 mM Tris (pH 9.5), 10 mM MgCl2, 0.01 mM ZnCl2, and 0.85 M (NH4)2SO4 before being concentrated to 40 mg/mL. Within 1 mo, crystals formed in the drops with ammonium sulfate concentrations between 2.0 M and 2.2 M. Crystal sizes varied from 0.2 x 0.2 x 0.1 mm3 to 1.2 x 0.6 x 0.3 mm3.
Crystal mounting and data collection
Crystals were initially transferred into a crystal stabilization buffer composed of 3.0 M (NH4)2SO4, 100 mM Tris (pH 7.5), 10 mM MgCl2, and 2 mM ZnCl2 for at least 24 h. A 0.3 x 0.25 x 0.2 mm3 crystal was soaked in a p-nitrophenylphosphate (PNPP) crystal stabilization buffer (10 mM PNPP, 3.0 M (NH4)2SO4, 100 mM Tris, 10 mM MgCl2, 2 mM ZnCl2 at pH 7.5) for 15 min before it was soaked into a mixture containing 80% p-nitrophenyl phosphate crystal stabilization buffer and 20% glycerol for 1 min (S102T_sub crystal). Another 0.2 x 0.1 x 0.1 mm3 crystal was first soaked in a Pi crystal stabilization buffer (2 mM NaH2PO4, 3.0 M (NH4)2SO4, 100 mM Tris, 10 mM MgCl2, 2 mM ZnCl2 at pH 7.5) for 15 min before it was soaked into a mixture of 80% Pi crystal stabilization buffer and 20% glycerol for 1 min (S102T_Pi crystal). Crystals were picked up in cryo-loops and frozen in liquid nitrogen.
The diffraction data were collected at the Crystallographic Facility in the Chemistry Department of Boston College, using a Rigaku RU-200 rotating anode generator, operating at 50 kV and 100 mA, equipped with a Rigaku/MSC R-axis IV++ detector. Diffraction data were collected to 2.20 Å and 2.00 Å, respectively, for the S102T_sub and S102T_Pi crystals (Table 3). The data sets were integrated, scaled, and averaged by using d*TREK (Rigaku/MSC) (Pflugrath 1999).
Structure refinement
The initial models for both the S102T_sub and the S102T_Pi structures were the coordinates of wild-type E. coli alkaline phosphatase with Co2+ in all the metal sites determined to 1.60 Å (PDB code 1Y6V; Wang et al. 2005). This initial coordinate was chosen because the S102T structures are in the same space group I222 and have almost identical unit cell dimensions. Before refinement, all ligands and waters were removed from the model and the Ser102 residue was replaced by Thr in XtalView (McRee 1999). Refinements were carried out using the Crystallography & NMR System (CNS) (Brunger et al. 1998). After the first rigid body refinement, simulated annealing (Brunger et al. 1997), energy minimization, and B-factor refinement, initial maps were inspected and manual rebuilding was performed in XtalView. After each round of refinement, R free was used to avoid overfitting and the F o –F c electron density map was visually inspected for alternate positions (Kleywegt and Brunger 1996). When the R factor fell below 25%, waters were added on the basis of peaks in the F o –F c electron density maps that were at or above the 3 level, and were checked and retained only when they could be justified by hydrogen bonding interactions. With approximately 520 and 560 waters, the S102T_sub and the S102_Pi structures were refined to an R factor/R free of 19.2%/22.9% and 20.5%/24.2% at a resolution of 2.20 Å and 2.00 Å, respectively (see Table 3).
Stereochemical quality of the two structures was determined from the Ramachandran plot using PROCHECK (Laskowski et al. 1993). Both structures have more than 90% of the residues within the most favored region. None of the active site residues were in disallowed regions. The details of data processing and refinement statistics for both structures are reported in Table 3. The atomic coordinates of the S102T_Pi and S102T_sub structures have been deposited in the Protein Data Bank as entries 2G9Y and 2GA3, respectively.
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Footnotes |
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Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062351506.
Abbreviations: PNPP, p-nitrophenylphosphate; S102T, the mutant version of E. coli alkaline phosphatase with Ser102 replaced by Thr; WT_Pi, wild-type E. coli alkaline phosphatase with inorganic phosphate bound in the active site (PDB code 1ED8); S102T_Pi, S102T mutant alkaline phosphatase with inorganic phosphate bound in the active site (PDB code 2G9Y); S102T_sub, S102T mutant alkaline phosphatase soaked in PNPP crystal stabilization buffer (PDB code 2GA3).
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Acknowledgments |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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References |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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