Phosphoryl Group Transfer Research Articles

How do kinases transfer phosphoryl groups?

Understanding how phosphoryl transfer is accomplished by kinases, a ubiquitous group of enzymes, is central to many biochemical processes. Qualitative analysis of the crystal structures of enzyme-substrate complexes of kinases reveals structural features of these enzymes important to phosphoryl transfer. Recently determined crystal structures which mimic the transition state complex have added new insight into the debate as to whether kinases use associative or dissociative mechanisms of catalysis.

An essential, multicomponent signal transduction pathway required for cell cycle regulation in Caulobacter.

Cell differentiation and division in Caulobacter crescentus are regulated by a signal transduction pathway mediated by the histidine kinase DivJ and the essential response regulator DivK. Here we report genetic and biochemical evidence that the DivJ and DivK proteins function to control the activity of CtrA, a response regulator required for multiple cell cycle events, including flagellum biosynthesis, DNA replication, and cell division. Temperature-sensitive sokA (suppressor of divK) alleles were isolated as extragenic suppressors of a cold-sensitive divK mutation and mapped to the C terminus of the CtrA protein. The sokA alleles also suppress the lethal phenotype of a divK gene disruption and the cold-sensitive cell division phenotype of divJ mutants. The relationship between these signal transduction components and their target was further defined by demonstrating that the purified DivJ kinase phosphorylates CtrA, as well as DivK. Our studies also showed that phospho-CtrA activates transcription in vitro from the class II flagellar genes and that their promoters are recognized by the principal C. crescentus sigma factor sigma73. We propose that an essential signal transduction pathway mediated by DivJ, DivK, and CtrA coordinates cell cycle and developmental events in C. crescentus by regulating the level of CtrA phosphorylation and transcription from sigma73-dependent class II gene promoters. Our results suggest that an unidentified phosphotransfer protein or kinase (X) is responsible for phosphoryl group transfer to CtrA in the proposed DivJ --> DivK --> X --> CtrA phosphorelay pathway.

Phage display selection of peptides against enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system (PTS).

The bacterial phosphoenolpyruvate-sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates and is involved in signal transduction. In response to the availability of carbohydrates it modulates catabolite repression, intermediate metabolism, gene expression and chemotaxis. It is ubiquitous in bacteria but does not occur in animals and plants. Uniqueness and pleiotropic function make the PTS a target for new antibacterial drugs. Enzyme I is the first component of the divergent protein phosphorylation cascade of the PTS. It transfers phosphoryl groups from phosphoenolpyruvate to the general phosphoryl carrier protein HPr. Six 15-mer, nine 10-mer and nine 6-mer peptides that inhibit enzyme I were selected from phage display libraries. Of these, 16 were synthesized and characterized. The majority of the peptides contain a histidine with an adjacent arginine. Two peptides were found to contain cysteines but no histidine. All peptides are rich in basic residues and lack acidic amino acids. The peptides inhibit the phosphotransferase system in vitro with IC50 of between 10 microM and 2 mM. Some, but not all, of the peptides inhibit cell growth in the agar diffusion test by an as yet undefined mechanism. All peptides are phosphorylated by enzyme I, and some are regenerated by slow autocatalytic hydrolysis of the phospho-peptide bond.

Structures of active conformations of UMP kinase from Dictyostelium discoideum suggest phosphoryl transfer is associative.

UMP/CMP kinase from Dictyostelium discoideum (UmpKdicty) catalyzes the specific transfer of the terminal phosphate of ATP to UMP or CMP. Crystal structures of UmpKdicty with substrates and the transition state analogs AlF3 or BeF2 that lock UmpKdicty in active conformations were solved. The positions of the catalytic Mg2+ and the highly conserved lysine of the P loop are virtually invariant in the different structures. In contrast, catalytic arginines move to stabilize charges that develop during this reaction. The location of the arginines indicates formation of negative charges during the reaction at the transferred phosphoryl group, but not at the phosphate bridging oxygen atoms. This is consistent with an associative phosphoryl transfer mechanism but not with a dissociative one.

Identification of a partially rate-determining step in the catalytic mechanism of cAMP-dependent protein kinase: a transient kinetic study using stopped-flow fluorescence spectroscopy.

The kinetics of nucleotide binding and phosphoryl group transfer were measured in the catalytic subunit of cAMP-dependent protein kinase using stopped-flow fluorescence spectroscopy and an acrylodan-labeled derivative of this enzyme, which we have previously shown to have kinetic properties similar to those for the wild-type enzyme (Lew et al., 1996). The fluorescence emission spectrum of this enzyme is quenched differentially by ATP and ADP so that both the binding of ligands and phosphoryl group transfer at the active site can be monitored selectively. The association and dissociation rate constants for both nucleotides were measured using two methods: relaxation and competition binding. The ratio of the observed dissociation and association rate constants by both methods are consistent with Kd measurements (25 microM) determined by equilibrium fluorescence quenching. The dissociation rate constant for ADP (100 s(-1)) is approximately 2.5-fold larger than k(cat) (39 s(-1)). A full viscosity effect was measured for k(cat), suggesting that a diffusive step or steps limit maximum turnover. Pre-steady-state kinetic transients are biphasic and were fitted to observed rate constants of 500 s(-1) and 60 s(-1) at 500 microM Kemptide (LRRASLG). Metal substitution studies (Mg2+ vs Mn2+) indicate that this first phase represents the phosphoryl group transfer step. Phosphopeptide release is faster than this second phase since the substrate is in rapid exchange with the enzyme and phosphorylation reduces the affinity of the peptide. The inability to assign this second phase to the chemical event or to product release implies that it reflects a viscosity-sensitive, protein conformational change that occurs after phosphoryl group transfer and prior to product release. Two conformational steps were detected in the binding of both ATP and ADP by relaxation methods that may be related to this second pre-steady-state kinetic phase. We suggest that this additional step in the kinetic mechanism may also occur in the wild-type enzyme and represents a large structural change in the enzyme during normal catalytic cycling.

Secondary structure of the IIB domain of the Escherichia coli mannose transporter, a new fold in the class of α/β twisted open-sheet structures

The mannose transporter of the Escherichia coli bacterial phosphotransferase system consists of three subunits: IIAB, IIC and IID. IIAB Man transfers phosphoryl groups to the transported substrate via phosphohistidine intermediates. Its IIB domain was overexpressed and isotopically labelled with 13C, 15N and 2H. Heteronuclear 3D triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances. Based on the evaluation of conformationally sensitive parameters, the secondary structure of the IIB Man domain has been determined as an α/β twisted open-sheet structure consisting of a six-stranded parallel β-sheet with the novel strand order 3–2–4–1–5–6, six helices and a short two-stranded antiparallel β-sheet. © 1997 Federation of European Biochemical Societies.

Site-directed mutagenesis of the sodium pump: analysis of mutations to amino acids in the proposed nucleotide binding site by stable oxygen isotope exchange.

A model for the active site of P type ATPases has been tested by site-directed mutagenesis of amino acids in two conserved sequences of Mg(2+)-dependent and Na(+)- and K(+)-stimulated ATPase. The mutants K501R, K501E, D586E, D586N, P587A, and P588A were expressed in yeast cells and compared with wild type. In addition to previously published assays of adenosine 5'-triphosphate binding and hydrolysis, measurements of 18O exchange between Pi and water have been used to identify steps in the E2 half of the reaction cycle affected by the mutations. The study supports the prediction that K501 in the KGAP sequence interacts with adenosine 5'-triphosphate. However, quantitative comparisons of the effect of mutation K501E on the activity with the effects of mutations to an enzyme of known structure that also catalyzes phosphoryl group transfer make a direct role for the positive charge on the side chain of K501 in catalysis by stabilizing the transition state unlikely. No evidence for the predicted interaction between D586 and the hydroxyl groups of ribose was found. However, the data do indicate that the spatial organization of the loop containing the DPPR sequence is critical for phosphorylation of the enzyme. A role for D586 in coordinating the Mg2+ that is required for activity is proposed.

The fructose transporter of Bacillus subtilis encoded by the lev operon: backbone assignment and secondary structure of the IIB(Lev) subunit.

The fructose transporter of the Bacillus subtilis phosphotransferase system consists of two membrane associated (IIA and IIB) and two transmembrane (IIC and IID) subunits [Martin-Verstraete, I., Débarbouille, M., Klier, A. & Rapoport, G. (1990) J. Mol. Biol. 214, 657-671] . It mediates uptake by a mechanism which couples translocation to phosphorylation of the transported solute. The 18-kDa IIBLev subunit transfers phosphoryl groups from His9 of the IIA subunit to the sugar. The three-dimensional structure of IIBLev or similar proteins is not known. IIBLev was overexpressed in Escherichia coli and isotopically labelled with 13C/15N in H2O as well as in 70% D2O. 15N-edited NOESY, 13C-edited NOESY and 13C,15N triple-resonance experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances. Based on qualitative interpretation of NOE, scalar couplings, chemical shift values and amide exchange data, the secondary structure and topology of IIBLev was determined. IIBLev comprises six parallel beta-strands, one antiparallel beta-strand and 5 alpha-helices. The order of the major secondary-structure elements is (beta alpha)5beta (strand order 7651423). Assuming that the (beta alpha beta)-motives form right-handed turn structures, helices alphaA and alphaB are packed to one face and helices alphaC, alphaD and alphaE to the opposite face of the parallel beta-sheet. His15 which is transiently phosphorylated during catalysis is located in the loop beta1/alphaA of the topological switch point. The amino terminal (beta/alpha)4 part of IIBLev has the same topology as phosphoglyceromutase (PGM; PDB entry 3pgm). Both proteins catalyze phosphoryltransfer reactions which proceed through phosphohistidine intermediates and they show a similar distribution of invariant residues in the topologically equivalent positions of their active sites. The protein fold of IIBLev has no similarity to any of the known structures of other phosphoenolpyruvate-dependent-carbohydrate-phosphotransferase-system proteins.

Basal phosphorylation of the PEST domain in the I(kappa)B(beta) regulates its functional interaction with the c-rel proto-oncogene product.

The product of the c-rel proto-oncogene (c-Rel) belongs to the NF-kappaB/Rel family of polypeptides and has been implicated in the transcriptional control of cell proliferation and immune function. In human T lymphocytes, c-Rel is sequestered in the cytoplasmic compartment by constitutively phosphorylated inhibitors, including I(kappa)B(alpha) and I(kappa)B(beta). Studies with bacterially expressed forms of these inhibitory proteins revealed that unphosphorylated I(kappa)B(alpha) but not I(kappa)B(beta) assembles with c-Rel and inhibits its DNA binding activity. Furthermore, latent I(kappa)B(beta)-c-Rel complexes derived from mammalian cells were sensitive to phosphatase treatment, whereas I(kappa)B(alpha)-c-Rel complexes were resistant. We have identified a constitutive protein kinase in unstimulated T cells that associates with and phosphorylates I(kappa)B(beta) in vitro. The substrate specificity, electrophoretic mobility, and antigenic properties of this I(kappa)B(beta)-associated kinase (BAK) suggest identity with casein kinase II (CKII), an enzyme known to mediate basal phosphorylation of I(kappa)B(alpha). Phosphorylation of recombinant I(kappa)B(beta) by either BAK or CKII restored the capacity of this inhibitor to antagonize the DNA binding activity of c-Rel. Peptide mapping and mutational analyses localized the bulk of the basal phosphorylation sites in I(kappa)B(beta) to the C-terminal PEST domain, which contains two potential acceptors for CKII-mediated phosphoryl group transfer (Ser-313 and Ser-315). Point mutations introduced into the full-length inhibitor at Ser-313 and Ser-315 led to a significant reduction in the phosphorylation of I(kappa)B(beta) and severely impaired its c-Rel inhibitory function in vivo. Taken together, these findings strongly suggest that basal phosphorylation of the PEST domain of I(kappa)B(beta) at consensus CKII sites is required for the efficient formation of latent I(kappa)B(beta)-c-Rel complexes.

Insight into tyrosine phosphorylation in v-Fps using proton inventory techniques.

The phosphoryl group transfer step in the kinase domain of v-Fps, a nonreceptor tyrosine protein kinase, was analyzed using proton inventory and viscosometric techniques. The latter studies show that two nine-residue peptide substrates for v-Fps, peptides I (EAEAYEAIE) and II (EAEIYEAIE), are in rapid equilibrium with the active site and bind with similar affinities (Ks = 2.2 and 1.7 mM for peptides I and II). While phosphoryl group transfer is the rate-limiting step in kcat for peptide I (5 s-1) at neutral pH, peptide II is converted to product by a kinetic mechanism in which phosphoryl group transfer (45 s-1) and product release (20 s-1) partially control this parameter. Significant solvent isotope effects on kcat (k0/kn approximately 1.6) are observed for the phosphorylation of both peptides in 95% D2O. Proton inventories on kcat for peptide I are linear, indicating that the phosphoryl group transfer step is associated with a single proton transfer. Conversely, proton inventories on kcat for peptide II are "bowed" up, consistent with a "virtual" transition state in which phosphoryl group transfer and product release steps partially control this parameter. The lack of solvent isotope effects on kcat/Kpeptide for both peptides can be explained by an equilibrium isotope effect on substrate binding that offsets the kinetic isotope effect for phosphoryl group transfer. In keeping with this proposal, the KI for the inhibitor peptide, EAEIFEAIE, is 11 +/- 0.80 and 6.5 +/- 0.82 mM in 0 and 60% D2O, respectively. Fitting of the proton inventory plots using modified forms of the Gross-Butler equation provide intrinsic isotope effects of 1.7 and 3.6 for peptides I and II. The combined data are consistent with a mechanism involving either an acid-base catalyst or a conformational change preceding the release of products that is accompanied by the disruption of a single hydrogen donor-acceptor pair.

Rate-determining steps for tyrosine phosphorylation by the kinase domain of v-fps.

The rate-determining steps in the phosphorylation of four tyrosine-containing peptides by the kinase domain of the nonreceptor tyrosine protein kinase v-fps were measured using viscosometric methods. The peptides were phosphorylated by a fusion protein of glutathione-S-transferase and the kinase domain of v-fps (GST-kin) and the initial velocities were determined by a coupled enzyme assay. Peptides I (EEEIYEEIE), II (EAEIYEAIE), and III (DADIYDAID) were phosphorylated by GST-kin with similar kinetic constants. The viscosogens, glycerol and sucrose, were found to have intermediate effects on kcat and no effect on kcat/Kpeptide for the phosphorylation of these three peptides. The data are interpreted according to the Stokes-Einstein equation and a simple three-step mechanism involving substrate binding, phosphoryl group transfer, and net product release. Two competitive inhibitors (EAEIFEAIE and DADIFDAID) exhibited K1 values that are 6-10-fold higher than the Kpeptide values for their analogous peptide substrates. The data imply that peptides I-III are in rapid equilibrium with the enzyme and that kcat is partially limited by both phosphoryl group transfer (40-100 s-1) and product release (17-22 s-1). GST-kin phosphorylates peptide IV (R5AENLEYamide) with a low Km (100 microM) and a kcat that is 40-fold lower than that for peptide I. No effect of solvent viscosity was observed for the phosphorylation of this peptide on either kcat or kcat/Kpeptide. This suggests that highly viscous solutions do not perturb structure and that the rate-determining step for this poor substrate is phosphoryl group transfer. The data indicate that the kinase domain of v-fps phosphorylates its best substrate with a chemical rate constant that is at least 5-fold lower than that for the serine-specific cAMP-dependent protein kinase and its best substrate LRRASLG (Adams & Taylor, 1992). Interestingly, both enzymes exhibit a similar affinity for their substrates and both enzymes release their products at a similar rate. This implies that the differences in catalytic efficiency between serine- and tyrosine-specific protein kinases lie exclusively in the rate constants for phosphoryl group transfer and not in substrate absorption or product desorption.

Comparison of inline and non-inline associative and dissociative reaction pathways for model reactions of phosphate monoester hydrolysis

The energies, geometries and transition state orders for several different potential phosphate ester group hydrolysis mechanisms are determined at different levels of phosphate group and nucleophile prnotonation to define factors which might favour one phosphoryl group transfer mechanism over another. With respect to the phosphate ester group, protonation is considered as a generic model for phosphate anion charge neutralisation by, for example, alkylation or metal ion chelation. The results indicate that the protonation state of either the phosphate group and/or the nucleophile can produce dramatic changes in the relative energies of intermediates and transition states for any particular hydrolytic mechanism and that the magnitude of the changes can be sufficiently large to cause a change in favoured mechanism. For example, the results of the calculations predict that the favoured mechanism should change from a dissociative one at low levels of phosphate group protonation, to an associative one at higher levels of phosphate group protonation. Furthermore, the results indicate that under conditions in which a highly stable pentacoordinate intermediate is formed, the favoured reaction pathway involves a non-inline displacement of the leaving group by the nucleophile rather than an inline displacement. The relevance of these findings to understanding enzyme-catalysed phosphoryl group transfer is discussed.

Relation between the oligomerization state and the transport and phosphorylation function of the Escherichia coli mannitol transport protein: interaction between mannitol-specific enzyme II monomers studied by complementation of inactive site-directed mutants.

Previous experiments with the mannitol-specific enzyme II of Escherichia coli, EIImtl, have demonstrated that (1) the enzyme is a dimer, (2) the dimer is necessary for maximum activity, and (3) phosphoryl groups could be transferred between EIImtl subunits [van Weeghel et al. (1991) Biochemistry 30, 1768-1773; Weng et al. (1992) J. Biol. Chem. 267, 19529-19535; Weng & Jacobson (1993) Biochemistry 32, 11211-11216; Stolz et al. (1993) J. Biol. Chem. 268, 27094-27099]. The experiments in this article address the mechanistic role of the dimer. They indicate that the A, B, and C domains of EIImtl preferentially interact within the same subunit. Site-directed mutants in each of the three domains of EIImtl were used to study phosphoryl group transfer by the EIImtl dimer in vitro and mannitol transport in vivo. The C domain mutant, EIImtl-G196D, which was unable to bind mannitol, and the separated C domain, IICmtl, which was unable to phosphorylate mannitol, formed a heterodimer which was capable of mannitol phosphorylation in vitro and mannitol transport in vivo. The rates of phosphorylation were approximately 10-fold lower in heterodimers containing two inactive subunits relative to the rates in heterodimers containing one inactive and one wild type subunit; phosphoryl group transfer through one subunit is kinetically preferred to intersubunit transfer. Heterodimers formed in vivo between one wild type EIImtl subunit and the CB domain double mutant, EIImtl-G196D/C384S, transported mannitol as rapidly as wild type EIImtl alone; the presence of the inactive double mutant subunit did not reduce the transport rate. Thus, only one active A, B, and C domain in the dimer is sufficient for transport and phosphorylation activity, and if all three domains are situated on the same subunit, maximum rates are achieved.

The structure of nucleotidylated histidine-166 of galactose-1-phosphate uridylyltransferase provides insight into phosphoryl group transfer.

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate to form UDP-galactose and glucose 1-phosphate during normal cellular metabolism. The reaction proceeds through a double displacement mechanism characterized by the formation of a stable nucleotidylated histidine intermediate. This paper describes the preparation of the uridylyl-enzyme complex on the crystalline enzyme from Escherichia coli and its subsequent structure determination by X-ray crystallography. The refined structure has an R-factor of 19.6% (data between 65 and 1.86 A resolution) and reveals modest conformational changes at the active site compared to the inactive UMP/UDP-enzyme complex reported previously [Wedekind, J.E., Frey, P.A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. In particular, positions of the respective UMP alpha-phosphoryl groups differ by approximately 4 A. Well-defined electron density for the nucleotidylated imidazole supports the existence of a covalent bond between N epsilon 2 of the nucleophile and the alpha-phosphorus of UMP. A hydrogen bond that is conserved in both complexes between His 166 N delta 1 and the carbonyl O of His 164 serves to properly orient the nucleophile and electrostatically stabilize the positively charged imidazolium that results from nucleotidylation. Hydrogen bonds from side-chain Gln 168 to the nonbridging phosphoryl oxygens of the nucleotidyl intermediate appear crucial for the formation and reaction of the uridylyl-enzyme complex as well. The significance of the latter interaction is underscored by the fact that the predominant cause of the metabolic disease galactosemia is the mutation of the corresponding Gln (Gln 188 in humans) to Arg. A comparison to other phosphohistidyl enzymes is described, as well as a revised model for the mechanism of the uridylyltransferase.

Signal transduction in bacteria: phospho-neural network(s) inEscherichia coli?

The molecular basis of many forms of signal transfer in living organisms is provided via the transient phosphorylation of regulatory proteins by transfer of phosphoryl groups between these proteins. The dominant form of signal transduction in prokaryotic microorganisms proceeds via so-called two-component regulatory systems. These systems constitute phosphoryl transfer pathways, consisting of two or more components. Most of these pathways are linear, but some converge and some are divergent. The molecular properties of some of the well-characterised representatives of two-component systems comply with the requirements to be put upon the elements of a neural network: they function as logical operators and show the phenomenon of autoamplification. Because there are many phosphoryl transfer pathways in parallel and because there also appears to be cross-talk between these pathways, the total of all two-component regulatory systems in a single prokaryotic cell may show the typical characteristics of a 'phospho-neural network'. This may well lead to signal amplification, associative responses and memory effects, characteristics which are typical for neural networks. One of the main challenges in molecular microbial physiology is to determine the extent of the connectivity of the constituting elements of this presumed 'phospho-neural network', and to outline the extent of intelligence-like behaviour this network can generate. Escherichia coli is the organism of choice for this characterization.

High-resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer.

The structure of adenylate kinase from yeast ligated with the two-substrate-mimicking inhibitor Ap5A and Mg2+ has been refined to 1.96 A resolution. In addition, the refined structure of the same complex with a bound imidazole molecule replacing Mg2+ has been determined at 1.63 A. These structures indicate that replacing Mg2+ by imidazole disturbs the water structure and thus the complex. A comparison with the G-proteins shows that Mg2+ is exactly at the same position with respect to the phosphates. However, although the Mg2+ ligand sphere of the G-proteins is a regular octahedron containing peptide ligands, the reported adenylate kinase has no such ligands and an open octahedron leaving space for the Mg2+ to accompany the transferred phosphoryl group. A superposition of the known crystalline and therefore perturbed phosphoryl transfer geometries in the adenylate kinases demonstrates that all of them are close to the start of the forward reaction with bound ATP and AMP. Averaging all observed perturbed structures gives rise to a close approximation of the transition state, indicating in general how to establish an elusive transition state geometry. The average shows that the in-line phosphoryl transfer is associative, because there is no space for a dissociative metaphosphate intermediate. As a side result, the secondary dipole interaction in the alpha-helices of both protein structures has been quantified.

Separate site catalysis by pyruvate phosphate dikinase as revealed by deletion mutants.

Previous studies had indicated that pyruvate phosphate dikinase (PPDK), an enzyme which catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP), is made up of 25, 13, 18, and 35 kDa domains [Carroll, L. J., Xu, Y., Thrall, S. H., Martin, B. M. & Dunaway-Mariano, D. (1994) Biochemistry 33, 1134]. The catalytic histidine (which mediates the phosphoryl group transfers from ATP to P(i) and pyruvate) is located on the 18 kDa domain while the 25 and 13 kDa domains appear to contain the ATP binding site and the 35 kDa domain appears to contain the pyruvate binding site, respectively. The goal of this investigation was to examine functional interdependency of the putative ATP and pyruvate binding domains. Two truncated forms of PPDK were created by using recombinant DNA techniques. The 35 kDa (C-terminal) deletion mutant was found to catalyze the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction but not the E-P+pyruvate<-->E+PEP partial reaction. The 25 kDa (N-terminal) deletion mutant was found to catalyze the E-P+pyruvate<-->E+PEP partial reaction but not the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction. Neither mutant catalyzes the full ATP+P(i)+pyruvate<-->AMP+PP(i)+PEP reaction. These results are interpreted to mean that the ATP and pyruvate binding domains in PPDK are functionally independent, thus providing evidence for separate active sites for catalysis of the two partial reactions.

Probing the catalytic roles of n2‐site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS.

The glucose transporter of Escherichia coli. Overexpression, purification, and characterization of functional domains.

The glucose transporter of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar. It consists of a transmembrane subunit (IICBGlc) and a hydrophilic subunit (IIAGlc). The IICBGlc subunit consists of two domains. The NH2-terminal IIC domain (residues 1-386) spans the membrane eight times and contains the substrate binding site. The COOH-terminal hydrophilic IIB domain (residues 391-476) is accessible from the cytoplasmic side of the membrane. It contains the phosphorylation site (Cys421) and together with the IIC domain catalyzes the transfer of phosphoryl groups from the IIAGlc subunit to the transported solute. Starting from a plasmid vector containing ptsG under an inducible promoter, the IIB and the IIC domains have been subcloned separately, overexpressed in Escherichia coli, and purified by Ni2+ chelate affinity chromatography. Approximately 40 mg of IIBGlc-6H and 4 mg of IICGlc-6H could be purified from 1 liter of culture. Cells expressing IIBGlc-6H and IICGlc-6H separately have a three times longer generation time on glucose minimal medium than cells expressing wild-type IICBGlc. The rate of IIBGlc-6H phosphorylation determined in a nitrocellulose filter binding assay is indistinguishable from wild-type IICBGlc. The in vitro specific activity of IICGlc-6H in the presence of excess IIBGlc-6H is 2% of the control. IIBGlc-6H also complements the activity of a IICBGlc mutant with an inactive IIB domain (C421S) indicating that IIC and IIB are flexibly linked such that a free IIB domain can displace an inactive IIB domain from its contact site on the IIC domain. Based on this work, the secondary structure of the IIBGlc domain has been determined by isotope-edited NMR spectroscopy (Golic Grdadolnik, S., Eberstadt, M., Gemmecker, G., Kessler, H., Buhr, A., and Erni, B. (1994) Eur. J. Biochem. 219, 945-952).

Effects of nitrate respiration on expression of the Arc-controlled operons encoding succinate dehydrogenase and flavin-linked L-lactate dehydrogenase.

Expression of sdhCDAB (encoding succinate dehydrogenase) and lctD (encoding the flavin-linked L-lactate dehydrogenase) is elevated aerobically and repressed anaerobically in Escherichia coli. The repression is initiated by autophosphorylation of the sensor protein ArcB, followed by phosphoryl group transfer to the regulator ArcA. ArcA-P, a global transcriptional regulator, then prevents sdh and lct expression. The stimulus for ArcB is not O2 deficiency per se. In vitro experiments showed that ArcB phosphorylation is enhanced by pyruvate, D-lactate, acetate, and NADH, the concentrations of which are likely to increase with the lack of an effective exogenous electron sink. In addition to their aerobic function, the two primary dehydrogenases also have roles in anaerobic nitrate respiration. Results presented here indicate that the increase of sdh and lct expression by nitrate depended on its chemical reduction, which in turn diminished the ArcA-P pool. Unexpectedly, a mutation in the fnr gene (encoding a global regulator involved in anaerobic metabolism) also alleviated the anaerobic repressions. Mutations in arcB or arcA were epistatic over that of fnr. Moreover, since this relief was counteracted by pyruvate in the growth medium, Fnr appears to affect formation of stimuli for ArcB. It is possible that Fnr also indirectly affects some of the other members of the arcA modulon, e.g., cyoABCDE (encoding the cytochrome o complex), cydAB (encoding the cytochrome d complex), and sodA (encoding the manganese-dependent superoxide dismutase).