Escherichia coli aspartate transcarbamylase: the relation between structure and function.

The x-ray structures of the allosteric enzyme aspartate transcarbamylase from Escherichia coli have been solved and refined for both allosteric forms. The T form was determined in the presence of the heterotropic inhibitor cytidine triphosphate, CTP, while the R form was determined in the presence of the bisubstrate analog N-phosphonacetyl-L-aspartate. These two x-ray structures provide the starting point for an understanding of how allosteric enzymes are able to control the rates of metabolic pathways. Insights into the mechanisms of both catalysis and homotropic cooperativity have been obtained by using site-directed mutagenesis to probe residues thought to be critical to the function of the enzyme based on these x-ray structures.

Escherichia coli aspartate transcarbamoylase: the molecular basis for a concerted allosteric transition.

Aspartate transcarbamoylase from Escherichia coli has become a model system for the study of both homotropic and heterotropic interactions in proteins. Analysis of the X-ray structures of the enzyme in the absence and presence of substrates and substrate analogs has revealed sets of interactions that appear to stabilize either the 'T' or the 'R' states of the enzyme. Site-specific mutagenesis has been used to test which of these interactions are functionally important. By combining the structural data from X-ray crystallography, and the functional data from site-specific mutagenesis a model is proposed for homotropic cooperativity in aspartate transcarbamoylase that suggests that the allosteric transition occurs in a concerted fashion.

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.

AMP inhibition of pig kidney fructose-1,6-bisphosphatase.

Lys-112 and Tyr-113 in pig kidney fructose-1,6-bisphosphatase (FBPase) make direct interactions with AMP in the allosteric binding site. Both residues interact with the phosphate moiety of AMP while Tyr-113 also interacts with the 3'-hydroxyl of the ribose ring. The role of these two residues in AMP binding and allosteric inhibition was investigated. Site-specific mutagenesis was used to convert Lys-112 to glutamine (K112Q) and Tyr-113 to phenylalanine (Y113F). These amino acid substitutions result in small alterations in k(cat) and increases in K(m). However, both the K112Q and Y113F enzymes show alterations in Mg(2+) affinity and dramatic reductions in AMP affinity. For both mutant enzymes, the AMP concentration required to reduced the enzyme activity by one-half, [AMP](0.5), was increased more than a 1000-fold as compared to the wild-type enzyme. The K112Q enzyme also showed a 10-fold reduction in affinity for Mg(2+). Although the allosteric site is approximately 28 A from the metal binding sites, which comprise part of the active site, these site-specific mutations in the AMP site influence metal binding and suggest a direct connection between the allosteric and the active sites.

Use of silicate sol-gels to trap the R and T quaternary conformational states of pig kidney fructose-1,6-bisphosphatase.

Encapsulation of the homotetrameric pig kidney fructose-1,6-bisphosphatase (FBPase) in tetramethyl orthosilicate sol-gels was used to dramatically reduce the rate of the allosteric transition of the enzyme between the T and R allosteric states. When assayed in the absence of the allosteric inhibitor AMP, the enzyme encapsulated in the T-state exhibited little activity. The enzyme encapsulated in the R-state exhibited a 4-fold lower k(cat) and V(max) than the enzyme in solution, and the apparent K(m) for this enzyme was 350-fold higher than the corresponding value for the enzyme in solution. The [Mg(2+)](0.5) for the encapsulated enzyme was only 0.1 mM, compared to 0.54 mM for the normal enzyme. Magnesium activation, under both sets of conditions, was cooperative with a Hill coefficient of approximately 2. The activity of enzyme encapsulated in the R-state decreased to about 70% of initial activity within 1 min of adding AMP, it then decreased slowly to about 40% of initial activity over the following 7 h. Under the conditions tested, the encapsulated enzyme never became completely inactivated and AMP inhibition was no longer cooperative. For enzyme encapsulated in the T-state, activity was restored over approximately 7 h after removal of the AMP. The biphasic and slow responses to changing AMP levels suggest that encapsulated enzyme can be used to study the effects of local conformational changes distinct from the global quaternary conformational changes by slowing down the ability of the enzyme to carry out global rotations. The response to AMP exhibited by the encapsulated enzyme is consistent with the ability of AMP, at least partially, to directly influence the activity of the active site within each subunit.

Affinity and hydrophobic chromatography of Escherichia coli aspartate transcarbamoylase.

Chromatography of aspartate transcarbamoylase from Escherichia coli on agarose-immobilized dyes and alkyl-agaroses of differing carbon length were investigated. The bacterial aspartate transcarbamoylase was bound by Procoin red HE3B-agarose and Cibacron blue F3GA-agarose nearly completely under the conditions chosen relative to other agarose-coupled dyes. The aspartate transcarbamoylase holoenzyme was eluted from the Procion red HE3B-agarose slightly later than from the Cibacron blue F3GA-agarose during salt gradient elution. The catalytic trimer of the enzyme as well as its regulatory dimer were eluted by a lower salt concentration from both dye-agarose gels than the concentration required to elute the holoenzyme. The interaction of the catalytic trimer with the Procion red HE3B-agarose and Cibacron blue F3GA-agarose gels may be a determinant in the holoenzyme being retained on these resins. Of those alkyl-agaroses tested, the ethyl-, propyl- and hexyl-agarose gels bound the majority of aspartate transcarbamoylase activity. Chromatography of aspartate transcarbamoylase on ethyl-agarose found it to be eluted by a low salt concentration. A purification scheme for relatively small amounts of aspartate transcarbamoylase utilizing Procion red HE3B-agarose and ethyl-agarose is presented. This purification scheme is particularly useful for mutant versions of aspartate transcarbamoylase which cannot be purified by literature procedures.

Kinetic consequences of site-specific mutation of Glu-239----Gln in E. coli aspartate transcarbamylase: comparison with catalytic subunits and Phe-240 mutant enzyme.

The kinetic characteristics of E. coli aspartate transcarbamylase, altered by site-specific mutagenesis of Glu-239----Gln, have been determined by equilibrium isotope-exchange kinetics and compared to the wild-type system. In wild-type enzyme, residue Glu-239 helps to stabilize the T-state structure by multiple bonding interactions with Tyr-165 and Lys-164 across the c1-c4 subunit interface; upon conversion to the R-state, these bonds are re-formed within c-chains. Catalysis of both the [14C]Asp in equilibrium C-Asp and [32P]ATP in equilibrium Pi exchanges by mutant enzyme occurs at rates comparable to those for wild-type enzyme. Saturation with different reactant/product pairs produced kinetic patterns consistent with strongly preferred order binding of carbamyl-P prior to Asp and carbamyl-Asp release before Pi. The kinetics for the Gln-239 mutant enzyme resemble those observed for catalytic subunits (c3), namely a R-state enzyme (Hill coefficient nH = 1.0) and Km (Asp) approximately equal to 6 mM. The Glu-239----Gln mutation appears to destablize both the T- and R-states, whereas the Tyr-240----Phe mutation destablizes only the T-state.

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.

A revised mechanism for the alkaline phosphatase reaction involving three metal ions.

Here, X-ray crystallography has been used to investigate the proposed double in-line displacement mechanism of Escherichia coli alkaline phosphatase in which two of the three active-site metal ions have a direct role in catalysis. Two new X-ray crystal structures of the wild-type enzyme in the absence and presence of inorganic phosphate have been refined at 1.75 A to final working R-factors of 15.4% and 16.4%, respectively. In the refinement of both structures, residues in the active sites were treated anisotropically. The ellipsoids resulting from the partial anisotropic refinement show a clear route for the binding and release of substrate/product. In addition, a direct comparison of the refined structures with and without phosphate reveal a strong correlation between the occupancy of the third metal-binding site and the conformation of the Ser102 nucleophile. These findings clarify two important and unresolved aspects of the previously proposed catalytic mechanism, how Ser102 is activated for nucleophilic attack and why a magnesium ion in the third metal site is required for catalysis. Analysis of these results suggest that three metal-ion assisted catalysis is a more accurate description of the mechanism of the alkaline phosphatase reaction. A revised mechanism for the catalytic reaction of alkaline phosphatase is proposed on the basis of the two new X-ray crystal structures reported.

Mutations at positions 153 and 328 in Escherichia coli alkaline phosphatase provide insight towards the structure and function of mammalian and yeast alkaline phosphatases.

In order to understand some of the differences between human placental, human, Saccharomyces cerevisiae and Escherichia coli alkaline phosphatases in specific activity, activation by magnesium, and pH versus activity profiles, the X-ray crystal structures of three mutant E. coli alkaline phosphatases have been determined. The aligned sequences of alkaline phosphatases from mammalian, yeast and E. coli show that 25 to 30% of the amino acids are absolutely conserved and the active site residues are completely conserved with the exception of residues 153, 328 and 155. The bacterial enzyme has a salt-bridge, Asp153/Lys328, near the third metal binding site which, based on sequence homology, is apparently absent in the yeast and mammalian enzymes. The human enzymes have histidine at positions 153 and 328, and the yeast enzyme has histidine at position 328. In the E. coli enzyme, Asp153 was replaced by histidine (D153H), Lys328 was replaced by histidine (K328H), and a double mutant (DM) was constructed containing both mutations. The structure of the K328H enzyme was refined using cross-validation to a resolution of 2.3 A with a working R-factor of 0.181 and a free R-factor of 0.249. The DM structure was determined to a resolution of 2.5 A with a working R-factor of 0.166 and a free R-factor of 0.233. The structure of the D135H enzyme, which has been reported to a resolution of 2.4 A, has been re-refined using cross-validation to a working R-factor of 0.179 and a free R-factor of 0.239 for controlled comparisons with the two new structures. In all three structures the most significant changes are related to the bound phosphate inhibitor and the identity of the metal ion in the third binding site. The changes in the position of the phosphate group and the alterations at the third metal binding site indicate the structural basis for the variations in the steady-state kinetic parameters previously reported for these enzymes.

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.

Structural consequences of the replacement of Glu239 by Gln in the catalytic chain of Escherichia coli aspartate transcarbamylase.

Low-angle X-ray scattering in solution has been used to probe the quaternary structure of a mutant version of Escherichia coli aspartate transcarbamylase in which Glu239 of the catalytic chain was replaced by glutamine by site-directed mutagenesis. X-ray crystallographic studies of the wild-type enzyme have shown that one set of intersubunit interactions involving Glu239 are lost, and are replaced by another set of intrachain interactions when the enzyme undergoes the allosteric transition from the T to the R state. Functional analysis of the mutant enzyme with glutamine in place of Glu239 indicates that homotropic co-operativity is lost without altering the maximal specific activity. The radius of gyration of the unligated mutant enzyme is larger than the unligated wild-type, indicating an alteration in quaternary structure of the mutant. However, the radius of gyration of the mutant enzyme in the presence of N-(phosphonoacetyl)-L-aspartate (PALA) is identical with the value for the wild-type enzyme in the presence of PALA. X-ray scattering at larger angles indicates that the mutant enzyme is in a new structural state different from the wild-type T and R structures. The scattering pattern in the presence of saturating concentrations of PALA is identical with that of the wild-type R structure. Saturating concentrations of carbamyl phosphate alone are sufficient to convert most of the mutant enzyme to the R structure, in the absence of aspartate. CTP shifts the scattering pattern of the mutant enzyme in the presence of saturating carbamyl phosphate towards the scattering curve of the unligated enzyme, but CTP has no effect on the scattering curve in the absence of carbamyl phosphate or in the presence of subsaturating PALA. However, in the presence of subsaturating PALA, ATP causes a strong shift towards the R structure. Neither ATP nor CTP has any effect on the activity of the mutant enzyme. These data suggest that the replacement of Glu239 by glutamine results in a new quaternary structure. These data also explain, on a structural basis, why co-operativity is lost in this mutant enzyme.

Characterization of heterodimeric alkaline phosphatases from Escherichia coli: an investigation of intragenic complementation.

Escherichia coli alkaline phosphatase (EC 3.1.3.1) belongs to a rare group of enzymes that exhibit intragenic complementation. When certain mutant versions of alkaline phosphatase are combined, the resulting heterodimeric enzymes exhibit a higher level of activity than would be expected based upon the relative activities of the parental enzymes. Nine previously identified alkaline phosphatase complementation mutants were re-examined in this work in order to determine a molecular explanation of intragenic complementation in this experimental system. The locations of these mutations were determined by DNA sequence analysis after PCR amplification of the phosphatase-negative phoA gene. Most of the mutations involved ligands to metal-binding sites. Each of the mutant enzymes was re-created by site-specific mutagenesis, expressed, purified, and kinetically characterized. To investigate cooperativity between the two subunits, we analyzed heterodimeric forms of some of the site-specific mutant enzymes. To enable the isolation of the heterodimeric alkaline phosphatase in pure form, the overall charge of one subunit was altered by replacing the C-terminal Lys residue with three Asp residues. This modification had no effect on the kinetic properties of the enzyme. Heterodimeric alkaline phosphatases were created using two methods: (1) in vitro formation by dissociation at acid pH followed by reassociation at slightly alkaline pH conditions in the presence of zinc and magnesium ions; and (2) in vivo expression from a plasmid carrying two different phoA genes. Increases in k(cat), as well as a large reduction in the p-nitrophenyl phosphate K(m) were observed for certain combinations of mutant enzymes. These results suggest that the structural assembly of E. coli alkaline phosphatase into the dimer induces cooperative interactions between the monomers necessary for the formation of the functional form of the holoenzyme.

Involvement of tryptophan 209 in the allosteric interactions of Escherichia coli aspartate transcarbamylase using single amino acid substitution mutants.

Five mutant versions of aspartate transcarbamylase have been isolated, all with single amino acid substitutions in the catalytic chain of the enzyme. A previously isolated pyrB nonsense mutant was suppressed with supB, supC, supD and supG to create enzymes with glutamine, tyrosine, serine or lysine, respectively, inserted at the position of the nonsense codon. Each of these enzymes was purified to homogeneity and kinetically characterized. The approximate location of the substitution was determined by using tryptic fingerprints of the wild-type enzyme and the enzyme obtained with a tyrosine residue inserted at the position of the nonsense codon. By first cloning the pyrBI operon, from the original pyrB nonsense strain, followed by sequencing of the appropriate portion of the gene, the exact location of the mutation was determined to be at position 209 of the catalytic chain. Site-directed mutagenesis was used to generate versions of aspartate transcarbamylase with tyrosine and glutamic acid at this position. The Tyr209 enzyme is identical with that obtained by suppression of the original nonsense mutation with supC. The two enzymes produced by site-directed mutagenesis were purified using a newly created overproducing strain. Kinetic analysis revealed that each mutant has an altered affinity for aspartate, as judged by variations in the substrate concentration at one-half maximal activity. In addition, the mutants exhibit altered Hill coefficients and maximal activities. In the wild-type enzyme, position 209 is a tryptophan residue that is involved in the stabilization of a bend in the molecule near the subunit interface region. The alteration in homotropic cooperativity seems to be due to changes induced in this bend in the molecule, which stabilizes alternate conformational states of the enzyme.

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.

Analysis of two purified mutants of Escherichia coli aspartate transcarbamylase with single amino acid substitutions.

Two mutant versions of Escherichia coli aspartate transcarbamylase have been purified and analyzed kinetically. Each of these mutant enzymes contains a single amino acid different from the wild-type enzyme, which was introduced by suppression of a nonsense codon within the E. coli pyrB gene. These enzymes exhibited alterations in both homotropic and heterotropic interactions with little change in specific activity. Depending upon the site of the substitution, the allosteric interactions have been either enhanced or diminished over the wild-type enzyme. Carbamyl phosphate saturation curves indicate that aspartate and carbamyl phosphate homotropic co-operativity are separable. Experiments employing the allosteric effectors indicate that the transmission of the regulatory effect is dependent upon the structure of the catalytic subunit, and that CTP inhibition can be partially decoupled from ATP activation. The kinetics of one of the mutants is unusually sensitive to dissociation at elevated temperatures. This sensitivity may be due to weakened interactions between the subunits of the enzyme.

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.

Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.

The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site.

Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.

The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase.

As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site-specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5'-triphosphate (PTP), 6-chloropurine 5'-triphosphate (Cl-PTP), 6-mercaptopurine 5'-triphosphate (SH-PTP), 6-methylpurine 5'-triphosphate (Me-PTP), and 1-methyladenosine 5'-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysis was performed on the wild-type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP. PTP, Cl-PTP, and SH-PTP each activate the enzyme at subsaturating concentrations of L-aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP. These experiments suggest that the interaction between N6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response. Me-PTP and Me-ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer. The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2' and 3' ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site. 2'-dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2-carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP. Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect.