Biomedical subjects

E R Kantrowitz

Publications and source records attributed to E R Kantrowitz.

At least 37 records · Page 2Linked to original sources

PubMed1999

The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site-specific mutagenesis.

The proposed double in-line displacement mechanism of Escherichia coli alkaline phosphatase (AP) involving two-metal ion catalysis is based on NMR spectroscopic and X-ray crystallographic studies. This mechanism is further supported by the X-ray crystal structures of the covalent phospho-enzyme intermediate of the H331Q mutant AP and of the transition state complex between the wild-type enzyme and vanadate, a transition state analog. Kinetic and structural studies on several genetically engineered versions of AP illustrate the overall importance of the active site's metal geometry, hydrogen bonding network and electrostatic potential in the catalytic mechanism.

Alkaline Phosphatase↗

PubMed1999

A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase.

The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 A and 1.6 A resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when L-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces k(cat)/K(m) about 10,000-fold.

Aminopeptidases↗

PubMed1999

A model of the transition state in the alkaline phosphatase reaction.

A high resolution crystal structure of Escherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 A resolution. The vanadate ion takes on a trigonal bipyramidal geometry and is covalently bound by the active site serine nucleophile. A coordinated water molecule occupies the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the vanadate ion are stabilized by interactions with both Arg-166 and the zinc metal ions of the active site. This structural complex supports the in-line displacement mechanism of phosphomonoester hydrolysis by alkaline phosphatase and provides a model for the proposed transition state in the enzyme-catalyzed reaction.

Alkaline Phosphatase↗

PubMed1999

Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center.

Escherichia coli alkaline phosphatase catalyzes both the nonspecific hydrolysis of phosphomonoesters and a transphosphorylation reaction in which phosphate is transferred to an alcohol via a phosphoseryl intermediate. The rate-determining step for the wild-type enzyme is pH dependent. At alkaline pH, release of the product phosphate from the noncovalent enzyme-phosphate complex determines the reaction rate, whereas at acidic pH hydrolysis of the covalent enzyme-phosphate complex controls the reaction rate. When the lysine at position 328 was substituted with a cysteine (K328C), the rate-determining step at pH 8.0 of the mutant enzyme was altered so that hydrolysis of the covalent intermediate became limiting rather than phosphate release. The transphosphorylation activity of the K328C enzyme was selectively enhanced, while the hydrolysis activity was reduced compared to that of the wild-type enzyme. The ratio of the transphosphorylation to the hydrolysis activities increased 28-fold for the K328C enzyme in comparison with the wild-type enzyme. Several other mutant enzymes for which a positive charge at the active center is removed by site-specific mutagenesis share this characteristic of the K328C enzyme. These results suggest that the positive charge at position 328 is at least partially responsible for maintaining the balance between the hydrolysis and transphosphorylation activities and plays an important role in determining the rate-limiting step of E. coli alkaline phosphatase.

Alanine↗

PubMed1999

Amino acid substitutions at the subunit interface of dimeric Escherichia coli alkaline phosphatase cause reduced structural stability.

The consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic function of the dimeric enzyme alkaline phosphatase from Escherichia coli have been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse urea gradient gel electrophoresis. The kinetic data for the wild-type and altered alkaline phosphatases show comparable catalytic behavior with k(cat) values between 51.3 and 69.5 s(-1) and Km values between 14.8 and 26.3 microM. The ultracentrifugation profiles indicate that the wild-type enzyme is more stable than all the interface-modified enzymes. The wild-type enzyme is dimeric in the pH range of pH 4.0 and above, and disassembled at pH 3.5 and below. All the interface-modified enzymes, however, are apparently monomeric at pH 4.0, begin assembly at pH 5.0, and are not fully assembled into the dimeric form until pH 6.0. The results from transverse urea gradient gel electrophoresis show clear and reproducible differences both in the position and the shape of the unfolding patterns; all these modified enzymes are more sensitive to the denaturant and begin to unfold at urea concentrations between 1.0 and 1.5 M; the wild-type enzyme remains in the folded high mobility form beyond 2.5 M urea. Alkaline phosphatase H370A, modified at the active site and not at the dimer interface, resembles the wild-type enzyme both in ultracentrifugation and electrophoresis studies. The results obtained suggest that substitution of a single amino acid at the interface sacrifices not only the integrity of the assembled dimer, but also the stability of the monomer fold, even though the activity of the enzyme at optimal pH remains unaffected and does not appear to depend on interface stability.

Alkaline Phosphatase↗

PubMed1999

The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity.

The X-ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl-L-aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain. To probe the function of Lys84, site-specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine. The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild-type enzyme, respectively. However, the K84A enzyme retained 12% of the activity of the wild-type enzyme. For each of these enzymes, the affinity for aspartate was reduced 5- to 10-fold, and the affinity for carbamoyl phosphate was reduced 10- to 30-fold. The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8. The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity. Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84. This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine. The DM enzyme has no cooperativity and exhibited 0.18% of wild-type activity, while the K83A enzyme exhibited 61% of wild-type activity. These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high-activity, high-affinity active site. Since low-angle X-ray scattering demonstrated that the mutant enzymes can be converted to the R-structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high-activity, high-affinity active site characteristic of the R-functional state of the enzyme.

Aspartate Carbamoyltransferase↗

PubMed1998

Threonine 82 in the regulatory chain is important for nucleotide affinity and for the allosteric stabilization of Escherichia coli aspartate transcarbamoylase.

The three-dimensional structure of Escherichia coli aspartate transcarbamoylase complexed with the allosteric effector CTP, shows an interaction between the hydroxyl of Thr-82 in the regulatory chain (Thr-82r) with the gamma-phosphate of CTP (R.P. Kosman, J.E. Gouaux, W.N. Lipscomb, Crystal structure of CTP-ligated T state aspartate transcarbamoylase at 2.5 A resolution: implications for aspartate transcarbamoylase mutants and the mechanism of negative cooperativity, Proteins Struct. Funct. Genet. 15 (1993) 147-176). In order to determine whether the Thr-82r interaction with the gamma-phosphate of CTP is important for either binding of the nucleotide effectors or their function, site-specific mutagenesis was employed. The mutant enzyme in which Thr-82r was replaced by Ala had almost the identical maximal observed specific activity as the wild-type enzyme; however, the mutant enzyme had a significantly increased [Asp]0.5, the aspartate concentration at one-half the maximal observed specific activity, as well as slightly increased homotropic cooperativity. The mutant enzyme was also activated more by ATP and inhibited less by CTP as compared to the wild-type enzyme. In addition, the nucleotide concentration required for one-half maximal effect was increased approx. 3-fold as compared to the corresponding values for the wild-type enzyme. The maximal inhibition of the mutant enzyme, in the presence of UTP and CTP was similar to that observed for the wild-type enzyme; however, higher concentrations of the nucleotides were required to achieve this level of inhibition. The reduced affinity of CTP, UTP and ATP induced by the mutation indicates that the hydrogen bonding interaction between the gamma-phosphate of the nucleotide effector and the side-chain hydroxyl of Thr-82r is important for the binding of the nucleotide effectors to the allosteric site. Furthermore, this interaction is important for the discrimination between CTP and CDP. Finally, the greater homotropic cooperativity, greater [Asp]0.5, diminished CTP inhibition and greater ATP activation of the mutant enzyme correlates with the X-ray structure of the mutant enzyme which shows that the unligated enzyme is in an 'extreme' T-state. These findings add support to the theory that the global stabilization of the enzyme is critical for both the homotropic and heterotropic properties of aspartate transcarbamoylase.

Adenosine Triphosphate↗

PubMed1998

A single mutation in the regulatory chain of Escherichia coli aspartate transcarbamoylase results in an extreme T-state structure.

Kinetic analysis of a mutant version of Escherichia coli aspartate transcarbamoylase in which Thr82 in the regulatory chain (Thr82r) was replaced by Ala results in a shift in the T <==> R equilibrium towards the T-state. In order to understand the structural determinants of this T-state stabilization, the X-ray structure of the unliganded Thr82r-->Ala enzyme was determined at 2. 6 A resolution and refined to a crystallographic residual of 0.175. The structure of the mutant r1 regulatory chain is more similar to that of the r6 regulatory chain than observed for the wild-type enzyme, resulting in a more symmetric structure. Furthermore, the structural changes in the mutant enzyme appears to occur only in the r1 chain, while the r6 chain is almost identical in structure to that of the r6 chain of the wild-type enzyme. The structure of the mutant enzyme exhibits alterations in the subunit interfaces between the regulatory and catalytic chains, as well as in the interface between the allosteric and zinc domains within the regulatory chain. Moreover, the regulatory dimers are rotated around their respective 2-fold axes approximately 1 degrees beyond the rotation which occurs in the wild-type T-state enzyme. The structural analysis indicates that the enzyme is an "extreme" T-state, in which a larger rotation of the regulatory dimers is required for the T to R transition compared to the wild-type enzyme. This extreme T-state structure correlates well with the kinetic parameters determined for the mutant enzyme, showing a stabilized T-state. Furthermore, the structural analysis of the mutant enzyme suggests that replacement of Thr82r with Ala alters the local conformation of the nucleotide binding pocket and therefore offers a plausible explanation for the reduced affinity of the enzyme for nucleotides.

Allosteric Site↗

PubMed1998

The influence of the regulatory chain amino acids Glu-62 and IIe-12 on the heterotropic properties of Escherichia coli aspartate transcarbamoylase.

In the structure of Escherichia coli aspartate transcarbamoylase with CTP bound [Kosman, R. P., Gouaux, J. E., and Lipscomb, W. N. (1993) Proteins, Struct. Funct. Genet. 15, 147-177] Lys-6 and Glu-62 form a salt-link between two regulatory chains. However, recent X-ray structural studies suggest that side chain and backbone interactions existed between Glu-62 and Ile-12. Thus the interaction between Glu-62 and Ile-12 may help to establish the correct conformation of the nucleotide binding site. The present study of two single-site mutant enzymes, Glu-62r-->Ala and Ile-12r-->Ala, was undertaken to investigate whether the role of Glu-62 is to maintain the stability of the interface between the regulatory chains in the dimer, or interacts with the side chain of Ile-12r to define the nucleotide binding site. For both the mutant enzymes, the maximal velocity, the aspartate saturation at half the maximal velocity, and Hill coefficient were close to wild-type values. The Glu-62r-->Ala enzyme showed enhanced regulatory effects with ATP, CTP, and UTP. As a result of this mutation the enzyme losses its ability to discriminate between CTP and UTP. For the Ile-12r-->Ala enzyme, the heterotropic properties were reduced or eliminated. The enhanced regulatory effects observed with the Glu-62r-->Ala enzyme do not seem to be consistent with the presence of a salt-link between Glu-62r and Lys-6r. However, based upon kinetic data of the unique but completely opposite heterotropic properties of the two mutant enzymes, it is suggested that the side chain interaction between Glu-62r and Ile-12r helps to define the conformation of the effector binding pocket. In this study, we report the properties of both the Glu-62r-->Ala and Ile-12r-->Ala enzymes and their importance for the heterotropic activation and inhibition of aspartate transcarbamoylase.

Allosteric Regulation↗

PubMed1998

Direct observation of an altered quaternary-structure transition in a mutant aspartate transcarbamoylase.

Time-resolved small-angle X-ray scattering (TR-SAXS) was used to monitor the structural changes that occur upon the binding of the natural substrates to a mutant version of the allosteric enzyme aspartate transcarbamoylase from Escherichia coli, in which the creation of a critical link stabilizing the R state of the enzyme is hindered. Previously, SAXS experiments at equilibrium showed that the structures of the unligated mutant enzyme and the mutant enzyme saturated with a bisubstrate analog are indistinguishable from the T and R state structures, respectively, of the wild-type enzyme (Tauc et al., Protein Sci. 3:1998-2004, 1994). However, as opposed to the wild-type enzyme, the combination of one substrate, carbamoyl phosphate, and succinate, an analog of aspartate, did not convert the mutant enzyme into the R state. By using TR-SAXS we have been able to study the transient steady-state during catalysis using the natural substrates rather than the nonreactive substrate analogs. The steady-state in the presence of saturating amount of substrates is a mixture of 60% T and 40% R structures, which is further converted entirely to R in the additional presence of ATP. These results provide a structural explanation for the reduced cooperativity observed with the mutant enzyme as well as for the stimulation by ATP at saturating concentrations of substrates. They also illustrate the crucial role played by domain motions and quaternary-structure changes for both the homotropic and heterotropic aspects of allostery.

Adenosine Triphosphate↗

PubMed1998

Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102.

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile.

Alanine↗

PubMed1998

The N-terminus of the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for both nucleotide binding and heterotropic effects.

X-ray crystallographic studies indicate that the N-terminal region of the regulatory chain in Escherichia coli aspartate transcarbamoylase resides close to the effector binding site. The proximity of the N-terminal region to the binding site suggests it may be important for nucleotide binding and, therefore, the heterotropic mechanism. The N-terminal region of the structure is not well-defined since the electron density in this region is weak, indicating a flexible and mobile region. Furthermore, alanine scanning mutagenesis of residues 2-7 indicated that the N-terminal region may be involved in nucleotide binding and the heterotropic mechanism, especially, UTP recognition [Dembowski, N., and Kantrowitz, E. R. (1994) Protein Eng. 7, 673-679]. In order to investigate further the role of the N-terminal region in the heterotropic mechanism, the first 10 N-terminal residues of the regulatory chain were deleted using site-specific mutagenesis. This mutant enzyme was compared to the wild-type enzyme, and both solubility and functional differences were observed. The mutant enzyme forms an insoluble aggregate which can be solubilized by the addition of nucleotides, such as CTP, suggesting that the exposed nucleotide binding site is involved in aggregate formation. Kinetic analyses of the mutant enzyme showed a lower maximal velocity and slightly lower aspartate affinity. Apparent binding constants determined for CTP, ATP, UTP, and CTP in the presence of UTP suggest the heterotropic response is also altered. This study suggests that the N-terminal region of the regulatory subunit is important for controlling nucleotide binding, creating the high-affinity and low-affinity effector binding sites, and coupling the binding sites within the regulatory dimer.

Adenosine Triphosphate↗

PubMed1997

Room temperature phosphorescence study of phosphate binding in Escherichia coli alkaline phosphatase.

The phosphorescence spectrum and decay of Trp109 in Escherichia coli alkaline phosphatase was measured for the enzyme in 10 mM Tris/HCl, pH 7.4, at 21 degrees C. Changes in the spectrum and decay from the steady-state in response to non-covalent phosphate binding suggested a phosphate-induced alteration in the local environment surrounding Trp109 which lies buried below the active site. The seemingly inflexible structure in the region of Trp109, as judged by its very long phosphorescence lifetime, appeared unaltered when the enzyme was symmetrically bound with phosphate. However, the protein with phosphate bound to only one site displayed a marked increase in flexibility that extended over both subunits. For ratios of phosphate/enzyme (mol/mol) between 1.0 and 2.0, the observation of exponential phosphorescence decays with lifetimes that are a function of dilution provided evidence for the rapid exchange between phosphate half-saturated and fully-saturated enzymes consistent with observed enzyme turnover rates. The lifetimes under these conditions result in the calculation of a Kd for the dissociation of phosphate from the doubly occupied enzyme of 1.1 +/- 0.1 microM. The non-exponential decays at P/Ed (phosphate/dimeric enzyme) ratios less than 1.0 revealed that the exchange of phosphate between phosphate-free and half-saturated enzymes was not occurring on the timescale of the phosphorescence decay times, which implied that the half-saturated molecule cannot be contributing significantly to catalysis under steady-state conditions. The observation that the phosphorescence decay at a P/Ed ratio of 1.0 is exponential with a lifetime characteristic of the half-saturated species indicates that the binding of the first phosphate is significantly greater than the second, or that the binding exhibits negative cooperativity.

Alkaline Phosphatase↗

PubMed1997

Importance of the dimer-dimer interface for allosteric signal transduction and AMP cooperativity of pig kidney fructose-1,6-bisphosphatase. Site-specific mutagenesis studies of Glu-192 and Asp-187 residues on the 190's loop.

The role of the 190's loop of fructose-1,6-bisphosphatase (Fru-1, 6-P2ase) in the allosteric regulation of Fru-1,6-P2ase has been investigated through kinetic studies on three mutant enzymes, Glu-192 --> Ala, Glu-192 --> Gln, and Asp-187 --> Ala. AMP is an allosteric inhibitor, which binds to the regulatory sites and induces the R- to T-state transition; for wild-type Fru-1,6-P2ase AMP inhibition is cooperative with a Hill coefficient of 2.0. The replacement of Asp-187, which forms an interaction across the C1:C2 monomer-monomer interface, with alanine did not change the catalytic efficiency, and it had no effect on the cooperativity of AMP inhibition; however, the apparent dissociation constant for AMP increased more than 4-fold as compared to the value for the wild-type enzyme. The replacement of Glu-192, which forms interactions across the C1:C4 dimer-dimer interface, with Ala and Gln lowered kcat from 21 s-1 for wild-type enzyme to 15 s-1 and 13 s-1, respectively, for the mutant enzymes, while their respective Km values were not changed. However, these replacements did have dramatic effects on AMP inhibition; first, cooperative AMP inhibition was lost; second, the AMP inhibition was biphasic, which can be interpreted as due to AMP binding to two classes of binding sites. The high affinity class of sites corresponds to the regulatory sites, while the low affinity class of sites may be the active sites. The results reported here, combined with the structural and kinetic results from the Lys-42 --> Ala enzyme, strongly suggest that the C1:C4 dimer-dimer interface, rather than the C1:C2 monomer-monomer interface, is critical for the propagation of the allosteric signal between the AMP sites on different subunits; in addition, cooperative AMP inhibition is essential for the enzyme to be fully inhibited by the binding of AMP to the allosteric site.

Adenosine Monophosphate↗

PubMed1997

The road less travelled: taming phosphatases.

Phosphatases are important in signal transduction, bacterial pathogenesis and several human diseases. So far, however, it is their opposite numbers, the kinases, that have received more attention from chemists. Recent progress in inhibitor development offers hope that new probes of cellular processes, and perhaps novel therapeutic agents, may soon become available.

Affinity Labels↗

PubMed1997

Trapping and visualization of a covalent enzyme-phosphate intermediate.

Using a mutant version of E. coli alkaline phosphatase, we succeeded in trapping and determining the structure of the phospho-enzyme intermediate. The X-ray structure also revealed the catalytic water molecule, bound to one of the active site zinc ions, positioned ideally for the apical attack necessary for the hydrolysis of the intermediate.

Alkaline Phosphatase↗

PubMed1996

Engineered complementation in Escherichia coli aspartate transcarbamoylase. Heterotropic regulation by quaternary structure stabilization.

Escherichia coli aspartate transcarbamoylase regulates pyrimidine biosynthesis by altering its activity homotropically in response to one of its substrates and heterotropically in response to nucleotide effectors. The mechanism of this regulation involves two structurally and functionally different forms of the enzyme, one with low activity and low affinity for substrates (T state) and the other with high activity and high affinity for substrates (R state). Heterotropic regulation may be due to the direct transmission of a regulatory "signal" between the regulatory site and the active site some 60 A away and/or may involve altering the relative stability of the two forms of the enzyme. By combining a T state-stabilized mutant version of the enzyme, previously thought to have a defect in a heterotropic transmission pathway, with a known R state-stabilized mutant enzyme, we were able to restore some properties of the wild-type enzyme. These data imply that the relative stabilization of the T and R states of the enzyme is in part responsible for the homotropic and heterotropic properties of aspartate transcarbamoylase and that direct pathways for transmission of the heterotropic signals are unlikely.

Aspartate Carbamoyltransferase↗

PubMed1996

Kinetic and structural consequences of replacing the aspartate bridge by asparagine in the catalytic metal triad of Escherichia coli alkaline phosphatase.

In each subunit of the homodimeric enzyme Escherichia coli alkaline phosphatase, two of the three metal cofactors Zn2+ and Mg2+, are bound by an aspartate side-chain at position 51. Using site-specific mutagenesis, Asp51 was mutated both to alanine and to asparagine to produce the D51A and D51N enzymes, respectively. Over the range of pH values examined, the D51A enzyme did not catalyze phosphate ester hydrolysis above non-enzymic levels and was not activated by the addition of millimolar excess Zn2+ or Mg2+. Replacement of Asp51 by asparagine, however, resulted in a mutant enzyme with reduced activity and a higher pH optimum, compared with the wild-type enzyme. At pH 8.0 the D51N enzyme showed about 1% of the activity of the wild-type enzyme, and as the pH was raised to 9.2, the activity of the D51N enzyme increased to about 10% of the value for the wild-type enzyme. Upon the addition of excess Mg2+ at pH 9.2, the D51N enzyme was activated in a time-dependent fashion to nearly the same level as the wild-type enzyme. The affinity for phosphate of the D51N enzyme decreased tenfold as the concentration of Mg2+ increased. Under optimal conditions, the k(cat)/K(m) ratio for the D51N enzyme indicated that it was 87% as efficient as the wild-type enzyme. To investigate the molecular basis for the observed kinetic differences, X-ray data were collected for the D51N enzyme to 2.3 angstroms resolution at pH 7.5, and then to 2.1 angstroms resolution at pH 9.2 with 20 mM MgCl2. The two structures were then refined. The low magnesium, low pH D51N structure showed that the third metal site was unoccupied, apparently blocked by the amide group of Asn51. At this pH the phosphate anion was bound via one oxygen atom, between the zinc cations at the first and second metal sites, which strongly resembled the arrangement previously determined for the D153H enzyme at pH 7.5. In the high magnesium, high pH D51N structure, the third metal site was also vacant, but the phosphate anion bound closer to the surface of the enzyme, coordinated to the first metal site alone. Electron density difference maps provide evidence that magnesium activates the D51N enzyme by replacing zinc at the second metal site.

Alkaline Phosphatase↗