Biomedical subjects

E R Kantrowitz

Publications and source records attributed to E R Kantrowitz.

At least 55 records · Page 3Linked to original sources

PubMed1996

Glutamic acid residue 98 is critical for catalysis in pig kidney fructose-1,6-bisphosphatase.

Site-specific mutagenesis has been used to replace Glu-98 with flutamine in pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P(2)ase) in order to evaluate the role of this residue in catalysis. The combination of lower k(cat) and higher K(m) resulted in an approximately 12,000-fold reduction in the catalytic efficiency of the Glu-98-->Gln enzyme when compared to the wild-type enzyme. The affinity of the enzyme for Mg(2+) and for the allosteric inhibitor AMP was altered only slightly; however, cooperativity in the binding of both of these effectors was eliminated. In addition, AMP could not fully inhibit the Glu-98-->Gln enzyme. These data suggest a critical role for the carboxylate of Glu-98 both as a general base in the reaction, and in the mechanism of allosteric inhibition of the enzyme by AMP.

Amino Acid Sequence↗

PubMed1996

Kinetic and X-ray structural studies of a mutant Escherichia coli alkaline phosphatase (His-412-->Gln) at one of the zinc binding sites.

Site-specific mutagenesis has been used to replace His-412 with glutamine in Escherichia coli alkaline phosphatase. In the wild-type enzyme His-412 is a direct ligand to one of the catalytically important zinc atoms (Zn1) in the active site. The mutant enzyme (H412Q) exhibited about the same k(cat), but a 50-fold increase in K(m) compared to the corresponding kinetic parameters for the wild-type enzyme. Furthermore, the H412Q enzyme had a lower zinc content than the wild-type enzyme. In contrast to the wild-type enzyme, Tris was less effective in the transferase reaction and dramatically inhibited the hydrolysis reaction of the H412Q enzyme. The addition of zinc to the mutant enzyme increased the k(cat) value above that of the wild-type enzyme, partially restored the weak substrate and phosphate binding, and also alleviated the inhibition by Tris. The structure of the H412Q enzyme was also determined by X-ray crystallography. The overall structure of the H412Q enzyme was very similar to that of the wild-type enzyme; the only alpha-carbon displacements over 1 angstrom were observed near the mutation site. In the H412Q structure no phosphate was bound in the active site of the enzyme; however, two water molecules were observed where phosphate normally binds in the wild-type enzyme. Close examination of the active site of the H412Q structure revealed structural changes in Ser-102 as well as at the mutation site. For example, the carbonyl oxygen of the side chain of Gln-412 rotated away from the position of His-412 in the wild-type structure, although too far away (3.2 angstroms) to coordinate to Zn1. Studies on the H412Q enzyme, and a comparison of the H412Q and H412N structures, suggest that the structure and electostatics of the imidazole ring of histidine are critical for its function as a zinc ligand in alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1996

Crystal structures of the active site mutant (Arg-243-->Ala) in the T and R allosteric states of pig kidney fructose-1,6-bisphosphatase expressed in Escherichia coli.

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, we present the X-ray structures of the mutant version of the enzyme with Arg-243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose-1,6-bisphosphate was reduced 10-fold and affinity for the inhibitor fructose-2,6-bisphosphate was reduced 1,000-fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269:31404-31409). The X-ray structures show no major changes in the organization of the active site compared with wild-type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys-269 and Lys-274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase. Calculations using the X-ray structures of the Arg-243-->Ala enzyme suggest that the role of Arg-243 in the wild-type enzyme is predominantly electrostatic in nature.

Adenosine Monophosphate↗

PubMed1996

The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure.

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [S]0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme. The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used.

Adenosine Triphosphate↗

PubMed1996

Evidence for an active T-state pig kidney fructose 1,6-bisphosphatase: interface residue Lys-42 is important for allosteric inhibition and AMP cooperativity.

During the R-->T transition in the tetrameric pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P2ase, EC 3.1.3.11) a major change in the quaternary structure of the enzyme occurs that is induced by the binding of the allosteric inhibitor AMP (Ke HM, Liang JY, Zhang Y, Lipscomb WN, 1991, Biochemistry 30:4412-4420). The change in quaternary structure involving the rotation of the upper dimer by 17 degrees relative to the lower dimer is coupled to a series of structural changes on the secondary and tertiary levels. The structural data indicate that Lys-42 is involved in a complex set of intersubunit interactions across the dimer-dimer interface with residues of the 190's loop, a loop located at the pivot of the allosteric rotation. In order to test the function of Lys-42, we have replaced it with alanine using site-specific mutagenesis. The kcat and K(m) values for Lys-42-->Ala Fru-1,6-P2ase were 11 s-1 and 3.3 microM, respectively, resulting in a mutant enzyme that was slightly less efficient catalytically than the normal pig kidney enzyme. Although the Lys-42-->Ala Fru-1,6-P2ase was similar kinetically in terms of K(m) and kcat, the response to inhibition by AMP was significantly different than that of the normal pig kidney enzyme. Not only was AMP inhibition no longer cooperative, but also it occurred in two stages, corresponding to high- and low-affinity binding sites. Saturation of the high-affinity sites only reduced the activity by 30%, compared to 100% for the wild-type enzyme. In order to determine in what structural state the enzyme was after saturation of the high-affinity sites, the Lys-42-->Ala enzyme was crystallized in the presence of Mn2+, fructose-6-phosphate (Fru-6-P), and 100 microM AMP and the data collected to 2.3 A resolution. The X-ray structure showed the T state with AMP binding with full occupancy to the four regulatory sites and the inhibitor Fru-6-P bound at the active sites. The results reported here suggest that, in the normal pig kidney enzyme, the interactions between Lys-42 and residues of the 190's loop, are important for propagation of AMP cooperativity to the adjacent subunit across the dimer-dimer interface as opposed to the monomer-monomer interface, and suggest that AMP cooperativity is necessary for full allosteric inhibition by AMP.

Adenosine Monophosphate↗

PubMed1995

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.

Alkaline Phosphatase↗

PubMed1995

Fructose-1,6-bisphosphatase: arginine-22 is involved in stabilization of the T allosteric state.

A comparison of the X-ray crystallographic structures of the R and T allosteric states [Ke, H. M., Liang, J.-Y., Zhang, Y., & Lipscomb, W. N. (1991) Biochemistry 30, 4412-4420] of the pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) reveals major changes in the quaternary structure of the enzyme upon the binding of the allosteric inhibitor AMP. This change in quaternary structure involves the breaking of one set of interactions that stabilize the R state and the formation of another set of interactions that stabilize the T state of the enzyme. In particular, the interactions of Arg-22 with nearby amino acid residues are quite different in the R and T states of the enzyme. Although the crystallographic data suggest that intersubunit interactions such as those involving Arg-22 are important for stabilization of the R and/or T states, the X-ray structures do not provide direct evidence concerning the functional role of specific amino acid residues. Therefore, site-specific mutagenesis has been used to probe the function of Arg-22 in pig kidney fructose-1,6-bisphosphatase. The replacement of Arg-22 by Ala results in a mutant enzyme with enhanced catalytic efficiency compared to the wild-type, as indicated by a kinetic analysis showing a slightly lower Km and increased Vmax compared to the wild-type enzyme. In addition, the substitution enhances both substrate inhibition and the affinity of the inhibitor fructose 2,6-bisphosphate. Moreover, the replacement of Arg-22 by Ala results in a more than 10-fold loss of the ability of AMP to inhibit the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1995

Domain closure in the catalytic chains of Escherichia coli aspartate transcarbamoylase influences the kinetic mechanism.

The closure of the two domains of the catalytic chains of Escherichia coli aspartate transcarbamoylase, which is critical for completion of the T-->R transition, is stabilized by salt-bridges between Glu-50 and both Arg-167 and Arg-234. Mutation of Glu-50 to Ala shifts the enzyme toward a low activity, low affinity state (Newton, C. J., and Kantrowitz, E. R. (1990) Biochemistry, 29, 1444-1451). Kinetic isotope effects (KIE) and equilibrium isotope exchange kinetics (EIEK) have been used to probe the dynamic properties of the Glu-50-->Ala enzyme. Unlike the behavior of the wild-type enzyme, the observed kinetic isotope effect for 13C versus 12C at the carbonyl group of carbamoyl phosphate (CP) increased upon the binding of ligands which promote the formation of the R-state (Asp, N-phosphonacetyl-L-aspartate (PALA), or ATP). The maximum rate for the [14C]Asp<-->Carbamoyl aspartate (CAsp) exchange with the Glu-50-->Ala enzyme was 500-fold slower than for the wild-type enzyme; however, the rate for the [14C]CP<-->CAsp exchange was only 50-fold slower, reversing the relative rates observed with the wild-type enzyme. In addition, upon variation of substrate pairs involving Asp or CAsp, loss of inhibition effects in the CP<-->CAsp exchange indicated that the Glu-50-->Ala substitution caused the kinetic mechanism for the mutant enzyme to shift from ordered to random. Computer simulations of the EIEK data indicate that the Glu-50-->Ala mutation specifically causes strong decreases in the rates of catalysis and association-dissociation for Asp and CAsp, with minimal effects on the CP and Pi on-off rates. With substrates bound, the Glu-50-->Ala enzyme apparently does not attain a full R-state conformation. The PALA-activated Glu-50-->Ala enzyme, however, exhibits substrate affinities comparable to those for the wild-type enzyme, but fails to restore the preferred order substrate binding. Unlike the wild-type enzyme, both the T and R-states of the Glu-50-->Ala enzyme contribute to catalysis. A third state, I, is proposed for the Glu-50-->Ala enzyme, in which random order substrate binding is exhibited, and the catalytic step contributes significantly to overall rate limitation.

Adenosine Triphosphate↗

PubMed1995

L-aspartate association contributes to rate limitation and induction of the T-->R transition in Escherichia coli aspartate transcarbamoylase. Equilibrium exchanges and kinetic isotope effects with a Vmax-enhanced mutant, Asp-236-->Ala.

Equilibrium isotope exchange kinetics (EIEK) and kinetic isotope effects have been used to determine the mechanistic basis for the altered kinetic characteristics of a mutant version of Escherichia coli aspartate transcarbamylase in which Asp-236 of the catalytic chain is replaced by alanine (Asp-236-->Ala). The [14C]Asp<--> N-carbamyl-L-aspartate (CAsp) and [14C]CP<-->CAsp exchange rates, observed as a function of various reactant-product pairs, exhibited dramatic increases in maximal rates, along with decreases in substrate half-saturation values and cooperativity. The carbon kinetic isotope effect, 13C versus 12C at the carbonyl group of carbamoyl phosphate, for the Asp-236-->Ala enzyme decreased toward unity as [Asp] increased, as observed for the wild-type enzyme. Both the kinetic isotope effects and EIEK results indicate that the Asp-236-->Ala enzyme operates by the same ordered kinetic mechanism as the wild-type enzyme. Although activation effects by ATP and N-phosphonacetyl-L-aspartate are lost, inhibition by CTP was apparent in equilibrium exchanges. Simulation of the EIEK data indicated that the best fit to the observed changes in saturation curves was obtained by preferentially increasing the rate of the T-->R transition, kappa T-->R, thereby destabilizing the T-state and increasing the equilbrium constant for the T<-->R transition. A multistep model for Asp bindng to aspartate transcarbamoylase is proposed, in which Asp induces the initial conformational changes that in turn trigger the T-->R transition, followed by stepwise filling of the remaining active sites.

Alanine↗

PubMed1995

Weakening of the interface between adjacent catalytic chains promotes domain closure in Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp-100-->Ala mutation was introduced into the Glu-50-->Ala holoenzyme, a mutant in which domain closure is impaired. The Glu-50/Asp-100-->Ala enzyme is fourfold more active than the Glu-50-->Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp-100-->Ala mutation restores the ability of the Glu-50-->Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA). At subsaturating concentrations of aspartate, the Glu-50/Asp-100-->Ala enzyme is activated more by ATP than the Glu-50-->Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50-->Ala enzyme. As opposed to the wild-type enzyme, the Glu-50/Asp-100-->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu-50/Asp-100-->Ala enzyme by solution X-ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild-type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu-50/Asp-100-->Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50-->Ala and wild-type enzymes. Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100-->Ala enzyme.

Adenosine Triphosphate↗

PubMed1995

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)

Alkaline Phosphatase↗

PubMed1995

Overexpression and purification of the trimeric aspartate transcarbamoylase from Bacillus subtilis.

A procedure has been developed for the overexpression and purification of milligram quantities of the Bacillus subtilis aspartate transcarbamoylase. The plasmid pEK171, carrying the B. subtilis pyrB structural gene under the control of the Escherichia coli pyrBI promoter, was transformed into the E. coli strain EK1104 and the enzyme overexpressed to approximately 50% of total soluble protein under extreme derepression of the pyrimidine pathway. The enzyme was subsequently purified by means of ammonium sulfate fractionation, anionic exchange chromatography using Q-Sepharose Fast Flow resin, negative chromatography on Matrex Gel Red A agarose, and hydrophobic interaction chromatography using Matrex Phenyl Cellufine. The purification yields approximately 60 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties very similar to those of the enzyme purified from B. subtilis cells.

Aspartate Carbamoyltransferase↗

PubMed1994

Shared active sites of fructose-1,6-bisphosphatase. Arginine 243 mediates substrate binding and fructose 2,6-bisphosphate inhibition.

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, Arg-243 was replaced by alanine using techniques of site-specific mutagenesis and the cloned pig kidney enzyme expressed in Escherichia coli. Compared with wild-type enzyme, kinetic parameters of the altered enzyme characterizing catalytic efficiency, magnesium binding, and inhibition by AMP differed but by less than an order of magnitude; affinity for substrate fructose 1,6-bisphosphate was 10-fold poorer, and affinity for inhibitor fructose 2,6-bisphosphate was 1000-fold poorer. Molecular dynamics simulations were undertaken to determine possible alterations in active sites of the enzyme due to replacement of Arg-243 by Ala and suggested that in the mutant enzyme loss of one cationic group leads to reorganization of the active site especially involving lysine residues 269 and 274. The differences in properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase and confirm on a functional basis the shared active site in this important metabolic enzyme.

Adenosine Monophosphate↗

PubMed1994

Mutations at histidine 412 alter zinc binding and eliminate transferase activity in Escherichia coli alkaline phosphatase.

His-412 in wild-type Escherichia coli alkaline phosphatase is a direct ligand to one of the two zinc atoms critical for the function of the enzyme. To investigate the function of this residue, site-specific mutagenesis was used to substitute His-412 with asparagine and alanine, generating mutant enzymes H412N and H412A, respectively. Both mutant enzymes show a 5-fold decrease in kcat and 30-fold increase in Km when compared to the corresponding kinetic parameters for the wild-type enzyme. In contrast to the wild-type enzyme, Tris and ethanolamine inhibit both the mutant enzymes by inhibiting the hydrolysis reaction and not participating in the transferase reaction; furthermore, both mutants have lower zinc and phosphate content than the wild-type enzyme. The addition of Zn2+ to the H412N and H412A enzymes restores catalytic activity to within 2-fold of the value for the wild-type enzyme, but more importantly the presence of Zn2+ completely restores substrate affinity. The similarity in the kinetic parameters for the H412N and H412A enzymes in the absence and presence of zinc suggests that the asparagine side chain does not play a significant role in coordinating zinc. Furthermore, both the asparagine and alanine substitutions reduce the affinity of the resulting enzymes for zinc. The pH profiles for the two mutant enzymes are different than the pH profile observed for the wild-type enzyme, suggesting that the amino acid substitutions may have altered the pKa of the zinc coordinated water molecule that is critical in the second step of the mechanism. These data suggest that His-412 does not directly participate in the catalytic mechanism but is mainly involved in zinc binding, and therefore is also indirectly involved in substrate binding and product release.

Alkaline Phosphatase↗

PubMed1994

Glutamic acid 86 is important for positioning the 80's loop and arginine 54 at the active site of Escherichia coli aspartate transcarbamoylase and for the structural stabilization of the C1-C2 interface.

Glu-86, which interacts with the side chain of Arg-54 across the C1-C2 interface of Escherichia coli aspartate transcarbamoylase, tethers the end of the flexible 80's loop, which moves into the active site during the T to R transition. In order to determine whether this interaction is important for the correct positioning of the 80's loop and Arg-54 at the active site and also for the structural stabilization of the enzyme, a mutant version was created in which Glu-86 was replaced by Gln (Glu-86-->Gln). Although the mutant holoenzyme exhibits almost normal homotropic cooperativity, both the holoenzyme and catalytic subunit exhibit substantial reductions in activity and affinity for aspartate and carbamyl phosphate. Furthermore, the mutant holoenzyme shows a marked decrease in the activation by ATP and by the bisubstrate analog N-(phosphonoacetyl)-L-aspartate, reduced inhibition by CTP, as well as reduced affinities for these ligands. Results from molecular dynamics simulations of the Glu-86-->Gln and Glu-86-->Ala enzymes suggest that the positions of the 80's loop and Arg-54 are significantly perturbed by the introduction of these mutations. Taken together, these results indicate that the interaction between Glu-86 and Arg-54 is important for the formation of the high affinity, high activity form of the enzyme by stabilizing the correct position of the 80's loop and Arg-54 at the active site. Heat inactivation experiments also demonstrated that Glu-86 plays a significant role in the structural stabilization of the C1-C2 interface, since the temperature required for loss of half of the activity of the Glu-86-->Gln catalytic subunit is reduced by 5 degrees C relative to the wild-type catalytic subunit.

Aspartate Carbamoyltransferase↗

PubMed1994

Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis.

In the X-ray structure of Escherichia coli alkaline phosphatase at 2.0-A resolution, His-372 was found only 3.8 A away from the zinc and forms a hydrogen-bonding interaction with Asp-327, a bidentate ligand of the zinc at the M1 site. However, His-372 does not directly interact with the zinc atom at the M1 site. In order to investigate the role of the side chain of His-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase, site-directed mutagenesis was used to convert His-372 to alanine. The fact that the His-372-->Ala enzyme has similar zinc binding affinity as the wild-type enzyme indicates that His-372 is not involved in zinc binding at the M1 site. However, the altered kinetic behavior of the mutant enzyme compared to the wild-type enzyme suggests that the imidazole ring of His-372 plays an indirect role in the catalytic mechanism of the enzyme. The hydrolysis activity of the His-372-->Ala enzyme at pH 8.0 is 10-fold lower than that of the wild-type enzyme. In the presence of a phosphate acceptor at pH 8.0, the mutant enzyme is approximately 80% as active as the wild-type enzyme. Therefore, the His-372-->Ala mutation selectively enhances the transphosphorylation activity of the enzyme. The His-372-->Ala enzyme also exhibits 4- and 30-fold decreases in Km as compared to the wild-type enzyme in 0.1 M MOPS buffer and 1.0 M Tris, buffer at pH 8.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1994

Glu-50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure.

Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high-activity high-affinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu-50 (Glu-50-->Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). A study of the structural consequences of replacing Glu-50 by alanine using solution X-ray scattering is reported here. Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so-called T quaternary conformation as is the wild-type enzyme. In the presence of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), the mutant enzyme is in the same, so-called R quaternary conformation as the wild-type enzyme. However, the Glu-50-->Ala enzyme differs from the wild-type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate. Addition of ATP under these conditions does result in a slight shift toward the R structure. Steady-state kinetic studies indicate that, in contrast to the wild-type enzyme, the Glu-50-->Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu-50 are critical for the creation of the high-activity, high-affinity R state of the enzyme.

Alanine↗

PubMed1994

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.

Alanine↗