The quaternary structure of Escherichia coli inorganic pyrophosphatase is essential for phosphorylation

The hexameric inorganic pyrophosphatase (PPase) is irreversibly inactivated by phosphoric acid monoesters. The inactivation kinetics are consistent with the formation of a dissociable complex of the phosphoric acid monoester with the enzyme, followed by phosphorylation of the dicarboxylic amino acid of its active site. PPi and its analogues, binding at the regulatory site, release the inhibitor from the active site and thus restore PPase activity. Chemically identical subunits in the hexameric PPase interact, promoting their cooperativity in a reaction with phosphoric acid monoesters. The trimeric and monomeric PPase, exhibiting full catalytic activity, form a dissociable complex with the phosphoric acid monoesters but, in contrast to the hexameric PPase, do not form a covalent bond with them. This indicates that the native hexameric structure is essential for the irreversible inactivation of Escherichia coli PPase by phosphoric acid monoesters. Possible nontraditional pathways for activity regulation of PPase are discussed.     

Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function

The fluorescence and phosphorescence properties of the tryptophan residues in glutamate dehydrogenase were utilized to probe the conformation of the macromolecule at various states of aggregation of its subunits (hexamer, trimer, and monomer) in guanidine hydrochloride. According to the phosphorescence lifetime no gross alteration in the conformation of the protein follows from complete dissociation of the hexamer into native monomer, implying that the native fold is stabilized exclusively by intrasubunit bonding. Although modest concentrations of denaturant induce a change in configuration in the enzyme, a comparison with the macromolecule cross-linked into the hexameric form by glutaraldehyde confirms that this alteration in structure is not the result of subunit dissociation. Inhibition of catalysis by the denaturant is found to be considerably smaller than anticipated from the extent of hexamer dissociation. Furthermore, this inhibition is in no way prevented by cross-linking the enzyme in its hexameric form. This finding together with the ability of the trimer to bind the coenzyme and to undergo the characteristic structural changes induced by the effectors ADP and GTP suggests that, contrary to what is generally believed, the smallest functional unit of glutamate dehydrogenase is not the hexameric form.     

Isolation of the hexameric form of purine nucleoside phosphorylase from E. coli. Comparative study of trimeric and hexameric forms of the enzyme

The presence of two forms (high and low molecular weight ones) of purine nucleoside phosphorylase II (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was demonstrated. The high molecular weight form of the enzyme was purified, and the properties of both forms were compared. The enzyme forms were shown to differ in their quaternary structure (trimeric and hexameric), molecular weight of the native enzyme and its subunits (85,000 and 28,000 for the trimer, 150,000 and 25,000 for the hexamer, respectively) as well as substrate specificity (the trimer is specific for all major purine nucleosides, while the hexamer does not cleave adenine nucleosides). Adenosine is a competitive inhibitor of the hexameric form with respect to deoxyguanosine (Ki = 1.16 X 10(-3) M); the Km value for deoxyguanosine is 9.85 X 10(-5) M. The isoelectric point for the both forms of the enzyme in the presence of 9 M urea is about 5.5. Both forms have a pH optimum of phosphorolytic activity between 6.5 and 7.0.     

The role of Asp42 in Escherichia coli inorganic pyrophosphatase functioning.

Excess of Mg2+ ions is known to inhibit the soluble inorganic pyrophosphatases (PPases). In contrast, the mutant Escherichia coli inorganic pyrophosphatase Asp42-->Asn is three times more active than native and retains its activity at high Mg2+ concentration. In this paper, another two mutant variants with Asp42 replaced by Ala or Glu were investigated to characterize the role of Asp42 in catalysis. pH-independent kinetic parameters of MgPPi hydrolysis and the dissociation constants for the activating and inhibitory Mg2+ ions were calculated. It was shown that Mg2+ inhibition of MgPPi hydrolysis by native PPase exhibited uncompetitive kinetics under the saturating substrate concentration. All three substitutions of Asp42 lead to a sharp decrease of inhibitory Mg2+ affinity to the enzyme. These findings allow determination of the sites of inhibitory and substrate Mg2+ ions binding to PPase. Common features of these mutants allow the conclusion that the function of Asp42 is to accurately coordinate the residues implicated in the substrate and the inhibitory Mg2+ ion binding to PPase active site. Structural analysis of PPase complexed with Mg2+ compared with PPase complexed with Mn2+ and reaction products confirms this supposition.     

Structure of the arginine repressor from Bacillus stearothermophilus.

The arginine repressor (ArgR) is a hexameric DNA-binding protein that plays a multifunctional role in the bacterial cell. Here, we present the 2.5 A structure of apo-ArgR from Bacillus stearothermophilus and the 2.2 A structure of the hexameric ArgR oligomerization domain with bound arginine. This first view of intact ArgR reveals an approximately 32-symmetric hexamer of identical subunits, with six DNA-binding domains surrounding a central oligomeric core. The difference in quaternary organization of subunits in the arginine-bound and apo forms provides a possible explanation for poor operator binding by apo-ArgR and for high affinity binding in the presence of arginine.     

Reciprocal effects of substitutions at the subunit interfaces in hexameric pyrophosphatase of Escherichia coli. Dimeric and monomeric forms of the enzyme

A homohexameric molecule of Escherichia coli pyrophosphatase is arranged as a dimer of trimers, with an active site present in each of its six monomers. Earlier we reported that substitution of His(136) and His(140) in the intertrimeric subunit interface splits the molecule into active trimers (Velichko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyyti?, T., Goldman, A., Cooperman, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemistry 37, 734-740). Here we demonstrate that additional substitutions of Tyr(77) and Gln(80) in the intratrimeric interface give rise to moderately active dimers or virtually inactive monomers, depending on pH, temperature, and Mg(2+) concentration. Successive dissociation of the hexamer into trimers, dimers, and monomers progressively decreases the catalytic efficiency (by 10(6)-fold in total), and conversion of a trimer into dimer decreases the affinity of one of the essential Mg(2+)-binding sites/monomer. Disruptive substitutions predominantly in the intratrimeric interface stabilize the intertrimeric interface and vice versa, suggesting that the optimal intratrimeric interaction is not compatible with the optimal intertrimeric interaction. Because of the resulting "conformational strain," hexameric wild-type structure appears to be preformed to bind substrate. A hexameric triple variant substituted at Tyr(77), Gln(80), and His(136) exhibits positive cooperativity in catalysis, consistent with this model.     

Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes.

We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria. Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism. This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties. Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis. The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K. This latter loop is present only in type II citrate synthase sequences. Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network. Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer. The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases. This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation. Another surprising observation from the structure of type II E. coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site. Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur. Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure. Thus, acetylcoenzyme A binding to type II E. coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite. We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases. Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.     

Flexibility of the rings: structural asymmetry in the DnaB hexameric helicase

DnaB is the primary replicative helicase in Escherichia coli and the hexameric DnaB ring has previously been shown to exist in two states in the presence of nucleotides. In one, all subunits are equivalent, while in the other, there are two different subunit conformations resulting in a trimer of dimers. Under all conditions that we have used for electron microscopy, including the absence of nucleotide, some rings exist as trimers of dimers, showing that the symmetry of the DnaB hexamer can be broken prior to nucleotide binding. Three-dimensional reconstructions reveal that the N-terminal domain of DnaB makes two very different contacts with neighboring subunits in the trimer of dimers, but does not form a predicted dimer with a neighboring N-terminal domain. Within the trimer of dimers, the helicase domain exists in two alternate conformations, each of which can form symmetrical hexamers depending upon the nucleotide cofactor used. These results provide new information about the modular architecture and domain dynamics of helicases, and suggest, by comparison with the hexameric bacteriophage T7 gp4 and SV40 large T-antigen helicases, that a great structural and mechanistic diversity may exist among the hexameric helicases.     

Estimation of binding constants for the substrate and activator of Rhodobacter sphaeroides adenosine 5'-diphosphate-glucose pyrophosphorylase using affinity capillary electrophoresis

Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5'-triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5'-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using affinity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buffer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a approximately 15-fold decrease in apparent affinity for F6P compared to that of WT while ACE indicated an affinity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent affinity for ATP in the presence of F6P. The ACE results were in good agreement with the activity assay data, confirming the increased affinity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study different binding sites/ligand interactions in allosteric enzymes.     

A functionally important Tyr-89 residue in Saccharomyces cerevisiae pyrophosphatase. II. A possible role in the mechanism of enzyme action]

Earlier it has been demonstrated that inactivation of inorganic pyrophosphatase (PPase) of S. cerevisiae by 7-chloro-4-nitronbenzofurasane is due to modification of Tyr89. The effect of pH and active center ligands on this reaction has been studied. It was found that pK for Tyr89 does not exceed 8.5; the phosphate-metal complex binding to the high affinity center protects Tyr89 from inactivation. Activating ions (Mg2+ and Zn2+) do not influence the inactivation, whereas the PPase inhibitor, Ca2+, enhances this process after saturation of the high affinity binding site. Saturation of two binding sites with Ca2+ has a protective effect on the enzyme. An increase in the rate of Tyr89 binding to the inhibitor in the presence of low concentrations of Ca2+ is due to the decrease of Tyr89 pK. The data obtained suggest that Tyr89 is located near the high affinity binding site for phosphate. The high reactivity of Tyr89 and its tight binding in the active center point to the presence of a hydrogen bondage with the substrate and suggest a role of a proton donor whose acceptor is the product of the enzymatic reaction, i.e., phosphate.     

RNA channelling by the archaeal exosome

Exosomes are complexes containing 3' --> 5' exoribonucleases that have important roles in processing, decay and quality control of various RNA molecules. Archaeal exosomes consist of a hexameric core of three active RNase PH subunits (ribosomal RNA processing factor (Rrp)41) and three inactive RNase PH subunits (Rrp42). A trimeric ring of subunits with putative RNA-binding domains (Rrp4/cep1 synthetic lethality (Csl)4) is positioned on top of the hexamer on the opposite side to the RNA degrading sites. Here, we present the 1.6 A resolution crystal structure of the nine-subunit exosome of Sulfolobus solfataricus and the 2.3 A structure of this complex bound to an RNA substrate designed to be partly trimmed rather than completely degraded. The RNA binds both at the active site on one side of the molecule and on the opposite side in the narrowest constriction of the central channel. Multiple substrate-binding sites and the entrapment of the substrate in the central channel provide a rationale for the processive degradation of extended RNAs and the stalling of structured RNAs.     

Allosteric communication between the nucleotide binding domains of caseinolytic peptidase B

ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K(d) = 3-7 μm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K(d) decreases from ≈160-300 to 50-60 μm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.     

Effects of replacement of prolines with alanines on the catalytic activity and thermostability of inorganic pyrophosphatase from thermophilic bacterium PS-3.

Each of the 10 proline residues of the inorganic pyrophosphatase (PPase) subunit of thermophilic bacterium PS-3 (PS-3) was replaced with alanine by the PCR-mutagenesis method. The variants were classified into three groups according to the effects of the replacements on their catalytic activities in 20 mM Tris-HCl, pH 7.8, containing 5 mM MgCl(2): the catalytic activity was (i) slightly affected (P39A and P69A), (ii) considerably reduced (P14A, P43A, P59A, and P116A), and (iii) completely or almost completely abolished (P72A, P100A, P104A, and P146A). HPLC-gel chromatography in the presence of 5 mM MgCl(2) revealed the following subunit assembly of the variants: group (i), a hexamer; group (ii), a hexamer or a mixture of a hexamer and a trimer, although the hexamer was predominant; and group (iii), a trimer or a monomer. The thermostability of the variant PPases depended upon the amount of hexamer remaining in the presence of Mg(2+) at high temperature. The results indicated that the hexamer state formed through protomer-protomer and trimer-trimer interactions is necessary for the PS-3 PPase to retain the correct structure for full catalytic activity and thermostability.     

Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.     

Mixed dimers of insulin-degrading enzyme reveal a cis activation mechanism

Insulin-degrading enzyme (IDE) exists primarily as a dimer being unique among the zinc metalloproteases in that it exhibits allosteric kinetics with small synthetic peptide substrates. In addition the IDE reaction rate is increased by small peptides that bind to a distal site within the substrate binding site. We have generated mixed dimers of IDE in which one or both subunits contain mutations that affect activity. The mutation Y609F in the distal part of the substrate binding site of the active subunit blocks allosteric activation regardless of the activity of the other subunit. This effect shows that substrate or small peptide activation occurs through a cis effect. A mixed dimer composed of one wild-type subunit and the other subunit containing a mutation that neither permits substrate binding nor catalysis (H112Q) exhibits the same turnover number per active subunit as wild-type IDE. In contrast, a mixed dimer in which one subunit contains the wild-type sequence and the other contains a mutation that permits substrate binding, but not catalysis (E111F), exhibits a decrease in turnover number. This indicates a negative trans effect of substrate binding at the active site. On the other hand, activation in trans is observed with extended substrates that occupy both the active and distal sites. Comparison of the binding of an amyloid β peptide analog to wild-type IDE and to the Y609F mutant showed no difference in affinity, indicating that Y609 does not play a significant role in substrate binding at the distal site.     

Substitutions of Glycine Residues Gly100 and Gly147 in Conservative Loops Decrease Rates of Conformational Rearrangements of <Emphasis Type="Italic">Escherichia coli</Emphasis> Inorganic Pyrophosphatase

Escherichia coli inorganic pyrophosphatase (PPase) is a one-domain globular enzyme characterized by its ability to easily undergo minor structure rearrangements involving flexible segments of the polypeptide chain. To elucidate a possible role of these segments in catalysis, catalytic properties of mutant variants of E. coli PPase Gly100Ala and Gly147Val with substitutions in the conservative loops II and III have been studied. The main result of the mutations was a sharp decrease in the rates of conformational changes required for binding of activating Mg2+ ions, whereas affinity of the enzyme for Mg2+ was not affected. The pH-independent parameters of MgPP(i) hydrolysis, kcat and kcat/Km, have been determined for the mutant PPases. The values of kcat for Gly100Ala and Gly147Val variants were 4 and 25%, respectively, of the value for the native enzyme. Parameter kcat/Km for both mutants was two orders of magnitude lower. Mutation Gly147Val increased pH-independent Km value about tenfold. The study of synthesis of pyrophosphate in the active sites of the mutant PPases has shown that the maximal level of synthesized pyrophosphate was in the case of Gly100Ala twofold, and in the case of Gly147Val fivefold, higher than for the native enzyme. The results reported in this paper demonstrate that the flexibility of the loops where the residues Gly100 and Gly147 are located is necessary at the stages of substrate binding and product release. In the case of Gly100Ala PPase, significant impairment of affinity of enzyme effector site for PP(i) was also found.