Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent cation binds to PRPP synthetase and serves as a bridge to the alpha-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site.     

Stereochemical probes of the argininosuccinate synthetase reaction

The stereochemical course of the argininosuccinate synthetase reaction has been determined. The SP isomer of [alpha-17O,alpha-18O,alpha beta-18O]ATP is cleaved to (SP)-[16O,17O,18O]AMP by the action of argininosuccinate synthetase in the presence of citrulline and aspartate. The overall stereochemical transformation is therefore net inversion, and thus the enzyme does not catalyze the formation of an adenylylated enzyme intermediate prior to the synthesis of citrulline adenylate. The RP isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) is a substrate in the presence of Mg2+, but the SP isomer is a substrate when Cd2+ is used as the activating divalent cation. Therefore, the lambda screw sense configuration of the beta,gamma-bidentate metal--ATP complex is preferred by the enzyme as the actual substrate. No significant discrimination could be detected between the RP and SP isomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) when Mg2+ or Mn2+ are used as the divalent cation. Argininosuccinate synthetase has been shown to require a free divalent cation for full activity in addition to the metal ion needed to complex the ATP used in the reaction.     

Characterization of the monovalent cation activator binding site of S-adenosylmethionine synthetase by 205Tl NMR of enzyme-bound Tl+

The structure of the binding site for the monovalent cation activator of S-adenosylmethionine (AdoMet) synthetase from Escherichia coli has been characterized by 205Tl NMR of enzyme-bound Tl+. The chemical shift of the enzyme-Tl+ complex is 176 ppm downfield from aquo Tl+, a shift which is typical only of Tl+ complexes with solely oxygen ligands. The 205Tl resonance shifts upfield to 85 ppm in the enzyme-Mg(II)-Tl+ complex, to 38 ppm in the enzyme-Tl+-AdoMet complex and to 34 ppm in the enzyme-Tl+-AdoMet-Mg(II) complex. The 205Tl chemical shift of enzyme-bound Tl+ was not altered by binding of either methionine, or the Mg(II)-ATP analog Mg(II)-adenyl-5'-yl imidodiphosphate, or Mg(II)-pyrophosphate to the enzyme-Tl+-Mg(II) complex. The NMR data suggest that the substrates or products of the enzyme do not coordinate to the monovalent cation activator and imply that monovalent cation activation results from alterations in protein conformation.     

Kinetic studies of the interaction between MS2 phage and F pilus of Escherichia coli.

The kinetics of the binding reaction of MS2 phage to free F pili, which were highly purified from Escherichia coli, has been studied using a membrane filter assay. The rate of dissociation (kd) of the MS2-phage--F-pilus complex is very slow and follows first-order kinetics with a half-life of 4.2 h at 30 degrees C in the standard buffer. The dissociation rate is rather insensitive to temperature, but becomes more rapid at high ionic strength or at basic pH. In a 0.25 M ionic strength buffer, the half-life of the complex is about 1.0 min. The rate of association is very fast and follows second-order kinetics with the rate constant for association (ka) being 8 x 10(7) M-1 s-1 at 30 degrees C in the standard buffer. The rate of association is almost insensitive to ionic strength but slightly sensitive to pH or temperature. Monovalent cations can also promote the binding reaction as well as divalent cations but the complex formed with monovalent cation is unstable. A study of the kinetics of dissociation suggests that there are two types of interaction between MS2 phage and F pilus; one is a strong interaction formed with divalent cations and the other is a weak one formed with monovalent cations. The physical nature of the bonds involved in the former and the latter seems to be mainly electrostatic and non-electrostatic respectively. The mechanism of the binding reaction is discussed.     

A nuclear magnetic resonance study of the topography of binding sites of Escherichia coli carbamoyl-phosphate synthetase

Two paramagnetic probes, viz., Mn2+ and Cr3+-ATP, were used to map distances to various loci on carbamoyl-phosphate synthetase by using NMR measurements. The paramagnetic influence of Mn2+ on the 1H of L-glutamate and L-ornithine was measured at 200 and 360 MHz. On the basis of these data, a correlation time for the paramagnetic interaction was determined (2 X 10(-9) s) and used to compute distances. These were in the range 7-9 A. Distances were also calculated from Mn2+ to the 13C-5 atom of glutamate (8.6 A), to the monovalent cation site (approximately 8 A), and to the phosphorus atoms of ATP in the Co(NH3)4ATP complex. For studies of the monovalent cation site relaxation rates of 6Li+, 7Li+, and 15NH4+ were measured. With Cr3+ ATP as a paramagnetic substrate analogue, Cr3+ to 13C distances were measured with the substrates HCO3(-) and [5-13C]glutamate. These NMR data provide the first topographical map of the arrangement of substrates, metal ion activators, and allosteric modifiers on the Escherichia coli carbamoyl-phosphate synthetase dimer.     

Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.     

Structure of the divalent metal ion activator binding site of S-adenosylmethionine synthetase studied by vanadyl(IV) electron paramagnetic resonance

The structure of the divalent metal ion binding site of S-adenosylmethionine synthetase from Escherichia coli has been studied by using the vanadyl(IV) ion (VO2+) as probe. VO2+ binds at a single site per subunit in the presence or absence of substrates. Single turnover experiments measuring S-adenosylmethionine (AdoMet) formation from methionine and the ATP analogue 5'-adenylyl imidodiphosphate show that complexes containing VO2+ and either Mg2+ or Ca2+ as a second metal ion are catalytically active, while a complex containing VO2+ alone is inactive. Electron paramagnetic resonance spectra of the enzyme-VO2+ complex, as well as complexes also containing AdoMet or methionine, indicate the coordination of two water molecules and at least two protein ligands to the VO2+. In complexes with polyphosphate substrates or products (e.g., enzyme-VO2+-ATP-methionine, enzyme-VO2+-PPi-Mg2+), EPR spectral changes reveal ligand substitutions on the VO2+, and 8.5-G isotropic superhyperfine coupling to two 31P nuclei can be resolved. 17O superhyperfine coupling from [17O]pyrophosphate indicates coordination of two oxygen atoms of PPi to the VO2+ ion. Thus the polyphosphate compounds are bidentate ligands to the VO2+, demonstrating that the VO2+ binds at the active site and suggesting a catalytic role for the protein-bound metal ion.     

Spatial proximity of two divalent metal ions at the active site of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is shown to require 2 divalent metal ions/enzyme subunit for maximal enzymatic activity. In the absence of substrate, the tetrameric enzyme binds 1 Mn(II) ion/subunit, whereas in the presence of a nucleotide substrate, adenylylimidodiphosphate, or the product pyrophosphate, there are two Mn(II)-binding sites/subunit. Electron paramagnetic resonance spectra of Mn(II) bound to the enzyme reveal a spin exchange interaction between 2 Mn(II) ions in complexes of enzyme and Mn(II) which also contain adenosylmethionine, K+, and either pyrophosphate or imidotriphosphate. Since a spin exchange interaction requires orbital overlap between the 2 ions, the metal ions must be bound close to one another, and they may share a common ligand.     

A deoxyribonucleic acid ligase from nuclei of rat liver. Purification and properties.

A DNA ligase has been extensively purified from nuclei of rat livers. The ligase seals single strand nicks in DNA with any of the four usual bases on either the 3 or 5 sides. It requires ATP and a divalent cation (Mg-2plus or Mn-2plus) for activity. At low Mg-2plus concentrations the activity is greatly stimulated by a variety of monovalent cations. Relatively small excesses of either monovalent or divalent cation above the amounts which give maximal activity lead to inhibition of activity. Poly(G) and poly(I) inhibit ligase activity; several other polyribonucleotides are not inhibitory. Low concentrations of inorganic pyrophosphate are inhibitory. The molecular weight of the ligase is estimated from gel filtration to be about 10 times 10-4.     

Structural and kinetic properties of Bacillus subtilis S-adenosylmethionine synthetase expressed in Escherichia coli

S-adenosylmethionine (SAM) synthetase (EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionine using l-methionine and ATP as substrates. SAM synthetase gene (metE) from Bacillus subtilis was cloned and over-expressed, for the first time, in the heterologus host Escherichia coli as an active enzyme. Size-exclusion chromatography (SEC) revealed a molecular weight of ~180 kDa, suggesting that the enzyme is a homotetramer stabilized by non-covalent interactions. SAM synthetase exhibited optimal activity at pH 8.0 and 45 degrees C with the requirement of divalent cation Mg(2+), and stimulated by the monovalent cation K(+). The enzyme followed sequential mechanism with a V(max) of 0.362 micromol/min/mg, and a K(m) of 920 microM and 260 microM for ATP and l-methionine, respectively. The urea-induced unfolding equilibrium of the recombinant enzyme revealed a multistate process, comprising partially unfolded tetramer, structural dimer, structural monomer and completely unfolded monomer, as evidenced by intrinsic and extrinsic fluorescence, circular dichroism (CD) and SEC. Absence of trimer in the SEC implicates that the enzyme is a dimer of dimer. Concordance between results of SEC and enzyme activity in the presence of urea amply establishes that tetramer alone with intersubunit active site(s) exhibits enzyme activity.     

Structures of the mono- and divalent metal nucleotide complexes in the myosin ATPase

The structure of both the mono- and the divalent metal nucleotide complexes active in the myosin subfragment 1 ATPase has been determined using the phosphorothioate analogs of ATP in the presence of various cations. Both the Sp and the Rp diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) were substrates in the presence of Mg2+, Ca2+, Mn2+, Co2+, Zn2+, and Cd2+ as well as with NH4+ and T1+. The Sp/Rp activity ratios obtained were largely independent of the cation. The simplest explanation of these results is that both mono- and divalent cations do not coordinate to the alpha-phosphate group. With adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), essentially only the Sp diastereomer was active with Mg2+ with Sp/Rp ratio of greater 3000. As the divalent metal ion was varied in the series given above, this ratio was progressively lowered to the value of 0.2 found with Cd2+. Similar changes in stereoselectivity were seen with monovalent cations. Thus, with NH4+, an Sp/Rp ratio of 8 was observed, whereas with T1+, this figure was reduced to 0.04. These data indicate that both mono- and divalent cations coordinate to the beta-phosphate group of the nucleoside triphosphate substrate. These results obtained with ATP alpha S and ATP beta S suggest that myosin uses the mono- or divalent cation delta, beta, gamma-bidentate nucleotide chelate as substrate.     

Metal ion activation of S-adenosylmethionine decarboxylase reflects cation charge density

S-Adenosylmethionine decarboxylase from Escherichia coli is a pyruvoyl cofactor-containing enzyme that requires a metal cation for activity. We have found that the enzyme is activated by cations of varying charge and ionic radius, such as Li+, A13+, Tb3+, and Eu3+, as well as the divalent cations Mg2+, Mn2+, and Ca2+. All of the activating cations provide kcat values within 30-fold of one another, showing that the charge of the cation does not greatly influence the rate-limiting step for decarboxylase turnover. Cation concentrations for half-maximal activation decrease by >100-fold with each increment of increase in the cation charge, ranging from approximately 300 mM with Li+ to approximately 2 microM with trivalent lanthanide ions. The cation affinity is related to the charge/radius ratio of the ion for those ions with exchangeable first coordination sphere ligands. The exchange-inert cation Co(NH3)63+ activates in the presence of excess EDTA (and NH4+ does not activate), indicating that direct metal coordination to the protein or substrate is not required for activation. The binding of metal ions (monitored by changes in the protein tryptophan fluorescence) and enzyme activation are both cooperative with Hill coefficients as large as 4, the active site stoichiometry of this (alphabeta)4 enzyme. The Hill coefficients for Mg2+ binding and activation increase from 1 to approximately 4 as the KCl concentration increases, which is also observed with NaCl or KNO3; neither Na+ nor K+ activates the enzyme. The single tryptophan in the protein is located 16 residues from the carboxyl terminus of the pyruvoyl-containing alpha chain, in a 70-residue segment that is not present in metal ion independent AdoMet decarboxylases from other organisms. The results are consistent with allosteric metal ion activation of the enzyme, congruent with the role of the putrescine activator of the mammalian AdoMet decarboxylase.     

Incorporation of glutamic acid into protein by a soluble system

A heat-labile, non-dialyzable factor(s) in soluble fractions from Escherichia coli strains and Bacillus subtilis was found to incorporate the radioactivity of [14C]glutamic acid into 95 degrees C CCl3COOH-insoluble fraction. Incorporation catalyzed by a partially purified factor from E. coli B required ATP, Mg2+, tRNA, casein, carbonate, and 2-mercaptoethanol. A mixture of nineteen amino acids other than glutamic acid had no effect on the incorporation. Heparin, spermine and monovalent cations were inhibitory. Incorporation proceeded via glutamyl-tRNA. The incorporation from [14C]glutamyl-tRNA required Mg2+, casein, carbonate, and 2-mercaptoethanol, and there was no incorporation from [14C]aspartyl-tRNA. The reaction product was identified as protein. The incorporated moiety was the glutamyl moiety of glutamic acid and it retained a free alpha-amino group in the product protein. The incorporating factor of E. coli B was demonstrated to be glutamyl-tRNA synthetase.     

Enzymatic properties of S-adenosylmethionine synthetase from the archaeon Methanococcus jannaschii

S-Adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, MAT) catalyzes a unique enzymatic reaction that leads to formation of the primary biological alkylating agent. MAT from the hyperthermophilic archaeon Methanococcus jannaschii (MjMAT) is a prototype of the newly discovered archaeal class of MAT proteins that are nearly unrecognizable in sequence when compared with the class that encompasses both the eucaryal and bacterial enzymes. In this study the functional properties of purified recombinant MjMAT have been evaluated. The products of the reaction are AdoMet, PP(i), and P(i); >90% of the P(i) originates from the gamma-phosphoryl group of ATP. The circular dichroism spectrum of the dimeric MjMAT indicates that the secondary structure is more helical than the Escherichia coli counterpart (EcMAT), suggesting a different protein topology. The steady state kinetic mechanism is sequential, with random addition of ATP and methionine; AdoMet is the first product released, followed by release of PP(i) and P(i). The substrate specificity differs remarkably from the previously characterized MATs; the nucleotide binding site has a very broad tolerance of alterations in the adenosine moiety. MjMAT has activity at 70 degrees C comparable with that of EcMAT at 37 degrees C, consistent with the higher temperature habitat of M. jannaschii. The activation energy for AdoMet formation is larger than that for the E. coli MAT-catalyzed reaction, in accord with the notion that enzymes from thermophilic organisms are often more rigid than their mesophilic counterparts. The broad substrate tolerance of this enzyme proffers routes to preparation of novel AdoMet analogs.     

林肯链霉菌谷氨酰胺合成酶的酶学性质

在分离纯化的基础上,报道了pH、温度和金属离子对林肯链霉菌(Streptomyceslincolnensis)Z-512谷氨酸胺合成酶(GS)活力的影响及GS底物专一性的研究结果.在动力学性质的研究中,发现林肯链霉菌GS在生物合成反应系统中,对底物NH_4CI的饱和曲线不遵守米氏方程.Hill作图呈两相曲线.在NH_4CI浓度低的情况下,Hill系数大于1,具有正协同效应;当NH_4CI浓度增加到一定程度时,Hill系数小于1,具有负协同效应.这说明NH_4CI不仅作为林肯链霉菌GS的底物,而且作为一种效应物调节GS的活性.林肯链霉菌GS对底物Glu及ATP的饱和曲线遵守米氏方程.在不同的激活离子存在下,GS对Glu、ATP的Km值也不同.     

Purification and properties of the synthetase catalyzing the biotination of the aposubunit of transcarboxylase from Propionibacterium shermanii

The synthetase that attaches biotin to the aposubunit of transcarboxylase (biotin-[methylmalonyl-CoA-carboxyltransferase]ligase) (EC 6.3.4.9) was purified to homogeneity by ion-exchange chromatography on cellulose DE-52 and CM-cellulose. The synthetase is a monomer of molecular weight 30,000. The pH and temperature optima for the synthetase are 6.0 and 37 degrees C, respectively. The apparent Km for the substrates ATP, biotin, and apo 1.3 S subunit of apotranscarboxylase are 38, 2.0, and 0.9 microM, respectively. Ni2+, Co2+, Zn2+, or Mn2+ could replace Mg2+ in the reaction. The affinity of synthetase toward metals is as follows: Zn2+ greater than Ni2+ greater than Mn2+ greater than Co2+ greater than Mg2+, and the activity with Zn2+ was much greater than that with the other divalent metals. EDTA completely inactivates the enzyme. The metals are necessary not only for the catalytic activity but also for the storage stability of the enzyme. The synthetase shows absolute specificity toward ATP.     

Catalytic cycle of the biosynthetic reaction catalyzed by adenylylated glutamine synthetase from Escherichia coli

The covalently attached AMP moiety of adenylylated glutamine synthetase from Escherichia coli has been replaced by its fluorescent analog, 2-aza-1,N6-etheno-AMP (aza-epsilon-AMP). The modified glutamine synthetase (aza-epsilon-GS) exhibits divalent cation requirement (Mn2+, rather than Mg2+), pH profile, Vmax, and Km similar to those of naturally adenylylated glutamine synthetase. Whereas naturally adenylylated glutamine synthetase exhibits only negligible fluorescence changes upon the binding of substrates, aza-epsilon-GS exhibits large fluorescence changes. The fluorescence changes have been used by means of a stopped flow technique to reveal the involvement of five fluorometrically distinct intermediates in the catalytic cycle for the biosynthesis of glutamine catalyzed by the adenylylated glutamine synthetase. The mechanism is very similar to that previously established for the unadenylylated enzyme, using intrinsic tryptophan fluorescence. Substrates bind via a rapid equilibrium random mechanism, but the reaction proceeds in a stepwise manner. The formation of an enzyme-bound intermediate (probably gamma-glutamyl phosphate + ADP) from ATP and L-glutamate is the rate-limiting step, with the subsequent reaction of the enzyme-bound intermediate occurring very rapidly. The success in elucidating this complex mechanism is due largely to the vastly different amplitudes of the fluorescence changes at the two excitation maxima (300 nm and 360 nm) of the aza-epsilon-AMP moiety which accompany the formation of the various intermediates.     

Identification of ribonuclease P activity from chick embryos

RNAase P (EC 3.1.26.5) activity has been identified in chick embryo thigh tissue on the basis of specific cleavage of Escherichia coli 129 nucleotide tRNATyr precursor and has been partially purified by the procedure used for human tissue culture KB cell RNAase P. RNAase P from chick resembles the KB cell RNAase P in substrate specificity, requirement for a divalent cation (Mg2+) and a monovalent cation (K+, Na+ or NH4+) for activity, inhibition by bulk tRNA, ready inactivation by proteases, and increasing instability; with purification. RNAase P activity is also present in whole chick embryos, as well as in liver and heart tissues. Furthermore, crude preparations of RNAase P from chick embryo heart tissue are relatively free of contaminating nucleases.     

Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg2+ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca2+ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg2+ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca2+. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mm Mg2+, other free divalent ions are not needed. Reactions containing low Mg2+ concentrations are strongly inhibited by the rapid Ca2+ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca2+ or Mg2+, suggesting that Ca2+ does not perform a unique signaling function. When the need for Mg2+, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion.