Kinetic studies of L-aspartase from Escherichia coli: substrate activation

The enzyme L-aspartase from Escherichia coli was observed to have a time lag during the production of aspartic acid from fumarate and ammonia. This time lag is pH dependent, with little lag observed below pH 7.0 and a very extensive lag observed above pH 8.0. This time lag was also found to be dependent on both substrate and divalent metal ion concentrations and on the degree of proteolysis of L-aspartase. The observed lag, in the reaction examined in the amination direction, has been found to be correlated with the nonlinear kinetics seen at higher pH in the deamination direction. Both phenomena are consistent with a model in which there is a separate activator site for the substrate, L-aspartic acid, that is distinct from the enzyme active site. Occupation of this site by the substrate, or by various substrate analogues, eliminates both the nonlinearity and the time lag. The D isomer of aspartic acid, which does not bind at the active site, can bind at this newly identified activator site.     

Subsite specificity of divalent metal ions to glucosyltransferase

Glucosyltransferase from oral bacteria Streptococcus mutans is the most significant virulent factor in causing dental caries. The enzyme has two subsites. The binding specificity of divalent metal ions to glucosyl or fructosyl subsite was examined using multiple inhibition kinetics. The interaction factor "alpha" identifies whether the two subsites are exclusive or non-exclusive.     

Kinetic studies of L-aspartase from Escherichia coli: pH-dependent activity changes

The pH dependence of the kinetic parameters of the L-aspartase-catalyzed reaction have been examined in both the amination and the deamination directions. The enzyme isolated from Escherichia coli exists in a pH-dependent equilibrium between a higher pH form that has an absolute requirement for a divalent metal ion and for substrate activation, and a low pH form that does not require activation by either substrate or metal ions. The interconversion between these enzyme forms is observed near neutral pH in the profiles examined for the reaction in either direction. This pH-dependent activation has not been observed for other bacterial aspartases. Loss of activity is observed at high pH with a pK value of 9. The pH profiles of competitive inhibitors such as 3-nitropropionic acid and succinic acid have shown that the enzyme group responsible for this activity loss must be protonated for substrate binding at the active site. An enzymatic group has also been identified that must be protonated in the amination reaction, with a pK value near 6.5, and deprotonated in the deamination reaction. This group, tentatively assigned as a histidyl residue, fulfills the criteria for the acid-base catalyst at the active site of L-aspartase.     

Sequence and substrate specificity of isolated DNA methylases from Escherichia coli C

Two DNA methylase activities of Escherichia coli C, the mec (designates DNA-cytosine-methylase gene, which is also designated dcm) and dam gene products, were physically separated by DEAE-cellulose column chromatography. The sequence and substrate specificity of the two enzymes were studied in vitro. The experiments revealed that both enzymes show their expected sequence specificity under in vitro conditions, methylating symmetrically on both DNA strands. The mec enzyme methylates exclusively the internal cytosine residue of CCATGG sequences, and the dam enzyme methylates adenine residues at GATC sites. Substrate specificity experiments revealed that both enzymes methylate in vitro unmethylated duplex DNA as efficiently as hemimethylated DNA. The results of these experiments suggest that the methylation at a specific site takes place by two independent events. A methyl group in a site on one strand of the DNA does not facilitate the methylation of the same site on the opposite strand. With the dam methylase it was found that the enzyme is incapable of methylating GATC sites located at the ends of DNA molecules.     

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.     

Regulation of the heat-shock response depends on divalent metal ions in an hfIB mutant of Escherichia coli

HfIB, also called FtsH, is an essential Escherichia coli protein involved in the proteolysis of the heat-shock regulator σ³² and of the phage regulator λcll. The hfIB1 (Ts) allele (formerly called ftsH1 ) conferring temperature-sensitive growth at 42°C is suppressed by loss of the ferric-uptake repressor Fur and by anaerobic growth. We show here that suppression requires TonB-dependent Fe(III) transport in the hfIB1 (Ts) fur mutant during aerobic growth at 42°C and Feo-dependent Fe(II) transport during anaerobic growth at 42°C. Temperature-resistant growth of hfIB1 (Ts) strains is also observed at 42°C in the presence of a high concentration of Fe(II), Ni(II), Mn(II) or Co(II) salts, but not in the presence of Zn(II), Cd(II), Cu(II), Mg(II), Ca(II) or Cr(III) salts. However, neither Ni(II) nor a fur mutation permits growth in the complete absence of HfIB. The heat-shock response, evaluated by an htpG :: lacZ fusion, is overinduced in hfIB1 (Ts) strains at 42°C because of stabilization of σ³². Growth in the presence of Ni(II) or in the absence of the Fur repressor abolishes this overinduction in the hfIB1 (Ts) strain, and, in the hfIB1 (Ts) fur mutant, σ³² is no longer stabilized at 42°C. These results reinforce the recent observation that HfIB is a metalloprotease active against σ³² in vitro and suggest that it can associate functionally in vivo with Fe(II), Ni(II), Mn(II) and Co(II) ions.     

Examination of polypeptide substrate specificity for Escherichia coli ClpB

Escherichia coli ClpB is a molecular chaperone that belongs to the Clp/Hsp100 family of AAA+ proteins. ClpB is able to form a hexameric ring structure to catalyze protein disaggregation with the assistance of the DnaK chaperone system. Our knowledge of the mechanism of how ClpB recognizes its substrates is still limited. In this study, we have quantitatively investigated ClpB binding to a number of unstructured polypeptides using steady‐state anisotropy titrations. To precisely determine the binding affinity for the interaction between ClpB hexamers and polypeptide substrates the titration data were subjected to global non‐linear least squares analysis incorporating the dynamic equilibrium of ClpB assembly. Our results show that ClpB hexamers bind tightly to unstructured polypeptides with binding affinities in the range of ～3–16 nM. ClpB exhibits a modest preference of binding to Peptide B1 with a binding affinity of (1.7 ± 0.2) nM. Interestingly, we found that ClpB binds to an unstructured polypeptide substrate of 40 and 50 amino acids containing the SsrA sequence at the C‐terminus with an affinity of (12 ± 3) nM and (4 ± 2) nM, respectively. Whereas, ClpB binds the 11‐amino acid SsrA sequence with an affinity of (140 ± 20) nM, which is significantly weaker than other polypeptide substrates that we tested here. We hypothesize that ClpB, like ClpA, requires substrates with a minimum length for optimal binding. Finally, we present evidence showing that multiple ClpB hexamers are involved in binding to polypeptides ≥152 amino acids. Proteins 2015; 83:117–134. © 2014 Wiley Periodicals, Inc.     

Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding.

Gamma-glutamylcysteine synthetase (gamma-GCS, glutamate-cysteine ligase), which catalyzes the first and rate-limiting step in glutathione biosynthesis, is present in many prokaryotes and in virtually all eukaryotes. Although all eukaryotic gamma-GCS isoforms examined to date are rapidly inhibited by buthionine sulfoximine (BSO), most reports indicate that bacterial gamma-GCS is resistant to BSO. We have confirmed the latter finding with Escherichia coli gamma-GCS under standard assay conditions, showing both decreased initial binding affinity for BSO and a reduced rate of BSO-mediated inactivation compared with mammalian isoforms. We also find that substitution of Mn2+ for Mg2+ in assay mixtures increases both the initial binding affinity of BSO and the rate at which BSO causes mechanism-based inactivation. Similarly, the specificity of E. coli gamma-GCS for its amino acid substrates is broadened in the presence of Mn2+, and the rate of reaction for some very poor substrates is improved. These results suggest that divalent metal ions have a role in amino acid binding to E. coli gamma-GCS. Electron paramagnetic resonance (EPR) studies carried out with Mn2+ show that E. coli gamma-GCS binds two divalent metal ions; Kd values for Mn2+ are 1.1 microm and 82 microm, respectively. Binding of l-glutamate or l-BSO to the two Mn2+/gamma-GCS species produces additional upfield and downfield X-band EPR hyperfine lines at 45 G intervals, a result indicating that the two Mn2+ are spin-coupled and thus apparently separated by 5 A or less in the active site. Additional EPR studies in which Cu2+ replaced Mg2+ or Mn2+ suggest that Cu2+ is bound by one N and three O ligands in the gamma-GCS active site. The results are discussed in the context of the catalytic mechanism of gamma-GCS and its relationship to the more fully characterized glutamine synthetase reaction.     

Crystal structure of lactaldehyde dehydrogenase from Escherichia coli and inferences regarding substrate and cofactor specificity

Aldehyde dehydrogenases catalyze the oxidation of aldehyde substrates to the corresponding carboxylic acids. Lactaldehyde dehydrogenase from Escherichia coli (aldA gene product, P25553) is an NAD(+)-dependent enzyme implicated in the metabolism of l-fucose and l-rhamnose. During the heterologous expression and purification of taxadiene synthase from the Pacific yew, lactaldehyde dehydrogenase from E. coli was identified as a minor (相似文献   

Col V plasmids and the resistance of Escherichia coli to divalent copper ions

Free organisms of both plasmid-free and plasmid-carrying strains of Escherichia coli were killed by incubation in water containing low levels of cupric ions. Sensitivity was temperature-dependent with killing being more marked at 20° or 25°C than at 10° or 15°C. In contrast to the effects of other inhibitors from natural waters (which affect free Col V⁺organisms more than Col^-ones), free Col^-and Col V⁺organisms were equally sensitive to kill by Cu²⁺. Attachment to glass beads essentially abolished sensitivity to cupric ions with full survival after exposure to 15 μ g/ml. This applied to both p⁺and p^-strains but attachment would have more effect on the survival of p⁺organisms in natural waters because some plasmids markedly enhance attachment.     

Quantifying Escherichia coli glutaredoxin-3 substrate specificity using ligand-induced stability

Traditionally, quantification of protein-ligand affinity is performed using kinetic or equilibrium measurements. However, if the binding reaction proceeds via a stable covalent complex, these approaches are often limited. By exploiting the fact that the conformational stabilization of a protein is altered upon ligand binding due to specific interactions, and using an array of selectively chosen ligand analogs, one can quantify the contribution individual interactions have on specificity. We have used ligand-induced stability as a basis to dissect the interaction between glutaredoxin-3 (Grx3) and one of its native substrates, the tripeptide glutathione. Taking advantage of the fact that Grx3 can be trapped in a covalent mixed disulfide to glutathione or to selected synthetic glutathione analogs as part of the natural catalytic cycle, individual contributions to binding of specific molecular groups can be quantified by changes in ligand-induced stability. These changes in conformational stability are interpreted in terms of interaction energies (i.e. specificity) of the particular groups present on the ligand analog. Our results illustrate that although Grx3 recognizes glutathione predominantly through independent and additive ionic interactions at the N- and C-terminal of glutathione, van der Waals interactions from the unique gamma-glutamate moiety of glutathione also play an important role. This study places us closer to understanding the complex task of accommodating multiple substrate specificities in proteins of the thioredoxin superfamily and underscores the general applicability of ligand-induced stability to probe substrate specificity.     

The metal specificity and selectivity of ZntA from Escherichia coli using the acylphosphate intermediate

ZntA from Escherichia coli is a P-type ATPase that confers resistance to Pb(II), Zn(II), and Cd(II) in vivo. We had previously shown that purified ZntA shows ATP hydrolysis activity with the metal ions Pb(II), Zn(II), and Cd(II). In this study, we utilized the acylphosphate formation activity of ZntA to further investigate the substrate specificity of ZntA. The site of phosphorylation was Asp-436, as expected from sequence alignments. We show that in addition to Pb(II), Zn(II), and Cd(II), ZntA is active with Ni(II), Co(II), and Cu(II), but not with Cu(I) and Ag(I). Thus, ZntA is specific for a broad range of divalent soft metal ions. The activities with Ni(II), Co(II), and Cu(II) are extremely low; the activities with these non-physiological substrates are 10-20-fold lower compared with the values obtained with Pb(II), Zn(II), and Cd(II). Similar results were obtained with DeltaN-ZntA, a ZntA derivative lacking the amino-terminal metal binding domain. By characterizing the acylphosphate formation reaction in ZntA in detail, we show that a step prior to enzyme phosphorylation, most likely the metal ion binding step, is the slow step in the reaction mechanism in ZntA. The low activities with Ni(II), Co(II), and Cu(II) are because of a further decrease in the rate of binding of these metal ions. Thus, metal ion selectivity in ZntA and possibly other P1-type ATPases is based on the charge and the ligand preference of particular metal ions but not on their size.