Characterization of the phosphorylated intermediate of the K+-translocating Kdp-ATPase from Escherichia coli

During ATP hydrolysis the K+-translocating Kdp-ATPase from Escherichia coli forms a phosphorylated intermediate as part of the catalytic cycle. The influence of effectors (K+, Na+, Mg2+, ATP, ADP) and inhibitors (vanadate, N-ethylmaleimide, bafilomycin A1) on the phosphointermediate level and on the ATPase activity was analyzed in purified wild-type enzyme (apparent Km = 10 microM) and a KdpA mutant ATPase exhibiting a lower affinity for K+ (Km = 6 mM). Based on these data we propose a minimum reaction scheme consisting of (i) a Mg2+-dependent protein kinase, (ii) a Mg2+-dependent and K+-stimulated phosphoprotein phosphatase, and (iii) a K+-independent basal phosphoprotein phosphatase. The findings of a K+-uncoupled basal activity, inhibition by high K+ concentrations, lower ATP saturation values for the phosphorylation than for the overall ATPase reaction, and presumed reversibility of the phosphoprotein formation by excess ADP indicated similarities in fundamental principles of the reaction cycle between the Kdp-ATPase and eukaryotic E1E2-ATPases. The phosphoprotein was tentatively characterized as an acylphosphate on the basis of its alkali-lability and its sensitivity to hydroxylamine. The KdpB polypeptide was identified as the phosphorylated subunit after electrophoretic separation at pH 2.4, 4 degrees C of cytoplasmic membranes or of purified ATPase labeled with [gamma-32P]ATP.     

Rapid, high yield purification and characterization of the K(+)-translocating Kdp-ATPase from Escherichia coli.

The conventional procedure for the purification of the high affinity K+ uptake ATPase (KdpABC) from Escherichia coli involves a tedious three-column protocol (final enzyme purity, approximately 90%; activity yield, 6.5% (Siebers, A., and Altendorf, K. (1988) Eur. J. Biochem. 178, 131-140)). We have now developed a highly effective one-column (Fractogel TSK AF-Red) protocol yielding an enzyme preparation of comparable purity with severalfold higher activity yield. A further increase in enzyme purity up to 98% was achieved by a two-column protocol involving elution over DEAE-Sepharose CL-6B prior to TSK AF-Red affinity chromatography. The reduction of preparation time minimized KdpB protein degradation and led to hitherto unequaled values of specific activity (up to 2000 mumols x g-1 x min-1) and enrichment factors (up to 30-fold). Our results confirm the usefulness of triazine dye matrices for the purification of transport ATPases.     

The Kdp-ATPase of Escherichia coli mediates an ATP-dependent, K+-independent electrogenic partial reaction

Charge transport by the K+ transporting Kdp-ATPase from Escherichia coli was investigated using planar lipid membranes to which liposomes reconstituted with the enzyme were adsorbed. To study reactions in the absence of K+, given some contamination of solutions with K+, we used a mutant of Kdp whose affinity for K+ was 6 mM instead of the wild-type whose affinity is 2 microM. Upon rapid release of ATP from caged ATP, a transient current occurred in the absence of K+. In the presence of K+, a stationary current was seen. On the basis of their structural similarity, we propose a kinetic model for the Kdp-ATPase analogous to that of the Na+K+-ATPase. In this model, the first, K+-independent step is electrogenic and corresponds to the outward transport of a negative charge. The second, K+-translocating step is probably also electrogenic and corresponds to transport of positive charge to the intracellular side of the protein.     

Cytidine deaminase from Escherichia coli B. Purification and enzymatic and molecular properties

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from Escherichia coli has been purified to homogeneity through a rapid and efficient two-step procedure consisting of anion-exchange chromatography followed by preparative electrophoresis. The final preparation is homogeneous, as judged by a single band obtained by disc gel electrophoresis performed in the absence and presence of denaturing agents. The native protein molecular weight determined by gel filtration is 56 000. Sodium dodecyl sulfate disc gel electrophoresis experiments conducted upon previous incubation of the enzyme with dimethyl suberimidate suggest an oligomeric structure of two identical subunits of 33 000 molecular weight. The absorption spectrum of the protein reveals a maximum at 277 nm and a minimum at 255 nm. The isoelectric point is at pH 4.35. Amino acid analysis indicates an excess of acidic amino acid residues as well as six half-cystine residues. No interchain disulfide groups have been evidenced. According to Cleland's nomenclature, kinetic analysis shows a rapid-equilibrium random Uni-Bi mechanism. Cytidine deaminase is competitively inhibited by various nucleosides. Km values for cytidine, deoxycytidine, and 5-methylcytidine are 1.8 X 10(-4), 0.9 X 10(-4), and 12.5 X 10(-4) M, respectively.     

Purification and properties of nitrite reductase from Escherichia coli K12.

NADH-nitrite oxidoreductase (EC 1.6.4) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4 mol of flavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20 micron-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by 1 micron-sulphite and 10mM-arsenite, but insensitive to 1 mM-2,2'-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.     

The K+-translocating KdpFABC complex from Escherichia coli: A P-type ATPase with unique features

The prokaryotic KdpFABC complex from the enterobacterium Escherichia coli represents a unique type of P-type ATPase composed of four different subunits, in which a catalytically active P-type ATPase has evolutionary recruited a potassium channel module in order to facilitate ATP-driven potassium transport into the bacterial cell against steep concentration gradients. This unusual composition entails special features with respect to other P-type ATPases, for example the spatial separation of the sites of ATP hydrolysis and substrate transport on two different polypeptides within this multisubunit enzyme complex, which, in turn, leads to an interesting coupling mechanism. As all other P-type ATPases, also the KdpFABC complex cycles between the so-called E1 and E2 states during catalysis, each of which comprises different structural properties together with different binding affinities for both ATP and the transport substrate. Distinct configurations of this transport cycle have recently been visualized in the working enzyme. All typical features of P-type ATPases are attributed to the KdpB subunit, which also comprises strong structural homologies to other P-type ATPase family members. However, the translocation of the transport substrate, potassium, is mediated by the KdpA subunit, which comprises structural as well as functional homologies to MPM-type potassium channels like KcsA from Streptomyces lividans. Subunit KdpC has long been thought to exhibit an FXYD protein-like function in the regulation of KdpFABC activity. However, our latest results are in favor of the notion that KdpC might act as a catalytical chaperone, which cooperatively interacts with the nucleotide to be hydrolyzed and, thus, increases the rather untypical weak nucleotide binding affinity of the KdpB nucleotide binding domain.     

K+-translocating KdpFABC P-type ATPase from Escherichia coli acts as a functional and structural dimer

The membrane-embedded K (+)-translocating KdpFABC complex from Escherichia coli belongs to the superfamily of P-type ATPases, which share common structural features as well as a well-studied catalytic mechanism. However, little is known about the oligomeric state of this class of enzymes. For many P-type ATPases, such as the Na (+)/K (+)-ATPase, Ca (2+)-ATPase, or H (+)-ATPase, an oligomeric state has been shown or is at least discussed but has not yet been characterized in detail. In the KdpFABC complex, kinetic analyses already indicated the presence of two cooperative ATP-binding sides within the functional enzyme and, thus, also point in the direction of a functional oligomer. However, the nature of this oligomeric state has not yet been fully elucidated. In the present work, a close vicinity of two KdpB subunits within the functional KdpFABC complex could be demonstrated by chemical cross-linking of native cysteine residues using copper 1,10-phenanthroline. The cysteines responsible for cross-link formation were identified by mutagenesis. Cross-linked and non-cross-linked KdpFABC complexes eluted with the same apparent molecular weight during gel filtration, which corresponded to the molecular weight of a homodimer, thereby clearly indicating that the KdpFABC complex was purified as a dimer. Isolated KdpFABC complexes were analyzed by transmission electron microscopy and exhibited an approximately 1:1 distribution of mono- and dimeric particles. Finally, reconstituted functional KdpFABC complexes were site-directedly labeled with flourescent dyes, and intermolecular single-molecule FRET analysis was carried out, from which a dissociation constant for a monomer/dimer equilibrium between 30 and 50 nM could be derived.     

Arginyl-tRNA synthetase from Escherichia coli K12. Purification, properties, and sequence of substrate addition.

Arginyl-tRNA synthetase from Escherichia coli K12 has been purified more than 1000-fold with a recovery of 17%. The enzyme consists of a single polypeptide chain of about 60 000 molecular weight and has only one cysteine residue which is essential for enzymatic activity. Transfer ribonucleic acid completely protects the enzyme against inactivation by p-hydroxymercuriben zoate. The enzyme catalyzes the esterification of 5000 nmol of arginine to transfer ribonucleic acid in 1 min/mg of protein at 37 degrees C and pH 7.4. One mole of ATP is consumed for each mole of arginyl-tRNA formed. The sequence of substrate binding has been investigated by using initial velocity experiments and dead-end and product inhibition studies. The kinetic patterns are consistent with a random addition of substrates with all steps in rapid equilibrium except for the interconversion of the cental quaternary complexes. The dissociation constants of the different enzyme-substrate complexes and of the complexes with the dead-end inhibitors homoarginine and 8-azido-ATP have been calculated on this basis. Binding of ATP to the enzyme is influenced by tRNA and vice versa.     

Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli.

beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase.     

Purification and some properties of uracil phosphoribosyltransferase from Escherichia coli K12

Uracil phosphoribosyltransferase from Escherichia coli K12 was purified to homogeneity as determined by polyacrylamide gel electrophoresis. For this purpose a pyrimidine-requiring strain harboring the upp gene on a ColE1 plasmid was used, which showed 15-times higher uracil phosphoribosyltransferase activity in a crude extract. When this strain was grown under conditions of uracil starvation, an additional 10-times elevation of the enzyme activity was obtained. The molecular weight of uracil phosphoribosyltransferase was determined to be 75000; the enzyme consists of three subunits with a molecular weight of 23500. Uracil phosphoribosyltransferase is specific for uracil and some uracil analogues. The apparent Km values for uracil and PRib-PP were 7 microM and 300 microM, respectively. As an effector of enzyme activity, GTP lowered the Km for PRib-PP to 90 microM and increased the Vmax value 2-fold, but had no effect on the Km for uracil. The effect of GTP was found to be pH-dependent. The enzymatic characterization of uracil phosphoribosyltransferase and the observed regulation of its synthesis emphasizes the role of the enzyme in pyrimidine salvage.     

Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties.

Glutamine 5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase (amidophosphoribosyl-transferase) has been purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was 194,000 by sedimentation equilibrium centrifugation and 224,000 by gel filtration. A subunit Mr = 57,000 was estimated by gel electrophoresis in sodium dodecyl sulfate. Cross-linking experiments gave species of Mr = 57,000, 117,000, and 177,000. A trimer or tetramer of identical subunits is indicated for the native enzyme. Highly active E. coli amidophosphoribosyl-transferase lacks significant nonheme iron. Enzyme activity was not enhanced by addition of iron salts and sulfide. Amidophosphoribosyltransferase exhibited both NH3- and glutamine-dependent activities. Glutaminase activity was detected in the absence of other substrates. Both glutamine- and NH3-dependent activities were subject to end product inhibition by purine 5'-ribonucleotides. AMP and GMP, in combination, gave synergistic inhibition. AMP and GMP exhibited positive cooperativity. In addition, GMP promoted cooperativity for saturation by 5-phosphoribosyl-1-pyrophosphate. Glutamine utilization was inhibited by NH3, suggesting that the amide of glutamine is transferred to the NH3 site prior to amination of 5-phosphoribosyl-1-pyrophosphate. The glutamine-dependent activity was selectively inactivated by the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo L-norleucine (DON) and by iodoacetamide. Incorporation of 1 eq of DON/subunit (Mr = 57,000) caused complete inactivation of the glutamine-dependent activity, thus providing evidence for one glutamine site per monomer and for the functional identity of the subunits. Following alkylation with iodoacetamide, carboxymethylcysteine was the only modified amino acid isolated from an acid hydrolysate. The glutamine-dependent activity was sensitive to oxidation. Inactivation by exposure to air was reversed by incubation with high concentrations of dithiothreitol.