Pro-tRNA synthetase from Phaseolus aureus and Delonix regia

Pro-tRNA synthetase from Phaseolus aureus was photoinactivated in the presence of methylene blue or rose bengal. Pro and several imino acid analogues protected the enzyme against dye-mediated photoinactivation but ATP was ineffective. Together with kinetic data, this evidence suggested that a His-residue near the Pro-binding site was involved in the enzyme reaction. In the absence of methylene blue, Phaseolus enzyme was stable to light whilst that from Delonix was rapidly and reversibly photoinactivated. ATP as well as Pro, protected the Delonix enzyme against dye-independent photoinactivation. In the presence of methylene blue, the Delonix enzyme was more rapidly photoinactivated than in the absence of the dye. p-Chloromercuribenzoate (pCMB)-inhibited enzyme from both Phaseolus and Delonix was reactivated by sulphydryl reducing reagents. Reactivation of Delonix enzyme was markedly temperature-dependent whilst Phaseolus enzyme was reactivated equally efficiently at all temperatures tested. ATP, tRNA, Pro and several analogues of Pro, protected both the Phaseolus and Delonix enzymes against pCMB inhibition. The possible roles of the His-residue and SH group are discussed in relation to the known differences in substrate specificity between the Phaseolus and Delonix enzymes.     

The activation of amino acid analogues by phenylalanyl- and tyrosyl-tRNA synthetases from plants

Partially purified preparations of Phe- and Tyr-tRNA synthetases were obtained from seed or seedlings of Phaseolus aureus, Delonix regia and Caesalpinia tinctoria, and the ability of a variety of structural analogues of Phe or Tyr to act as alternative substrates or inhibitors was tested. 3-Hydroxymethylphenylalanine, a natural product of C. tinctoria, formed a particularly effective substrate for the Tyr-tRNA synthetase from P. aureus. The structural features commensurate with substrate activity in an analogue molecule are discussed.     

Cold-lability of prolyl-tRNA synthetase from higher plants

Pro-tRNA synthetase from D. regia and P. aureus lost enzymic activity more rapidly at 0° than at room temperature. The enzyme from a number of higher plants that produce azetidine-2-carboxylic acid (A2C) was more rapidly inactivated in the cold than the enzyme from plants which do not contain A2C. The rate of cold inactivation was dependent on temperature and on the concentration of glycerol, protein and sulphydryl-reducing reagents. Substrates of Pro-tRNA synthetase also stabilized the enzyme against cold inactivation. Enzyme which had been completely inactivated by storage in the cold, could be reactivated by warming in the presence of a sulphydryl-reducing reagent. The rate of reactivation was dependent on temperature, pH and the concentration of sulphydryl-reducing reagent. Kinetic analysis indicated the existence of more than one molecular form of the enzyme. It is suggested that the cold-lability of Pro-tRNA synthetase may be due to dissociation of the active enzyme molecule into inactive subunits.     

Investigations of Amino Acids in the ATP Binding Site of 5,10-Methenyltetrahydrofolate Synthetase

5-Formyltetrahydrofolate is a compound that is administered as a rescue agent in methotrexate chemotherapy and in 5-fluorouracil chemotherapy for synergistic effects. It has also recently been suggested to play a role in bacterial resistance to antifolate therapy. 5,10-methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme known to catalyze the conversion of this compound to 5,10-methenyltetrahydrofolate along with the hydrolysis of ATP to ADP. To better understand the roles of specific amino acids in the ATP binding pocket of this enzyme, we used site-directed mutagenesis to create 10 modified forms of the Mycoplasma pneumoniae ortholog. The Michaelis constant (K_m) for each substrate and the turnover number (k_cat) was determined for each mutant to help elucidate the role of individual amino acids. Data were compared to crystal structures of human and M. pneumoniae orthologs of MTHFS. Results were largely consistent with a simple coulombic and proximity model; the larger the predicted charges of an interaction and the closer those interactions were to the phosphate transferred between the substrates, the greater the reduction in ATP binding and catalytic activity of the enzyme.     

Substrate discrimination by prolyl-tRNA synthetase from various higher plants

Prolyl-tRNA synthetase from plants (e.g. Delonix regia) containing azetidine-2-carboxylic acid (A2C), activated imino acid analogues larger than proline (Pro) more efficiently than did the enzyme from plants lacking A2C. The reverse situation was observed for analogues, including A2C itself, that are smaller than Pro. The enzyme from A2C-producing species was quite labile and salt-sensitive, with a high pH optima for the ATP-³²PPi exchange reaction, whereas the enzyme from non-producer species was stable and insensitive to salts, with a lower pH optimum. Certain analogues of Pro, which failed to stimulate ATP-³²PPi in the presence of a particular type of Pro-tRNA synthetase, nevertheless could bind to the enzyme and inhibit the esterification of tRNA by Pro. In the absence of tRNA, no significant ATP-³²PPi exchange was catalyzed by the Delonix enzyme on addition of A2C; the addition of tRNA resulted in a low but real level of activation of the analogue relative to Pro. These findings are discussed in relation to the ability of the enzyme from A2C-producing plants to discriminate against the analogue.     

Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA^Gln and Asn-tRNA^Asn by conversion of the misacylated Glu-tRNA^Gln and Asp-tRNA^Asn species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction.A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn²⁺ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn²⁺ binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA^Gln or Asn-tRNA^Asn.     

S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10–250 μM the K_m for methionine was 20 μM, and this changed to 200 μM for the range 0.5–5.0 mM. In the range 10–250 μM the K_m for ATP was 53 μM, and this changed to 1.75 mM for the range 0.5–5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [¹⁴C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (K_i 240 μM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-α-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration ≈ 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.     

Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase to inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II–IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site in subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase.     

Amino acid substrate specificity of asparaginyl-,aspartyl- and glutaminyl-tRNA synthetase isolated from higher plants

AspNH₂-, Asp- and GluNH₂-tRNA synthetases were purified from Phaseolus aureus; their optimum assay conditions, substrate specificities and salt sensitivities were investigated. AspNH₂-tRNA synthetase from β-cyanoalanine-producing (Vicia sativa), and non-producing (P. aureus and V. faba) species was able to utilize the analogue as a substrate irrespective of the source of the enzyme. Asp-tRNA synthetase from P. aureus was able to utilize α-aminomalonate and threo-β-hydroxy Asp as a substrate. The transfer of ¹⁴C-GluNH₂ to tRNA, catalyzed by GluNH₂-tRNA synthetase, was only inhibited by high concentrations of those analogues tested; albizziine was the most efficient, but no difference could be demonstrated between the substrate specificities of the enzyme isolated from an albizziine-producer (A. julibrissin and a non-producer (P. aureus) species.     

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His₆-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted l-Ala, l-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for l-Ala. S. aureus MurE was very specific for l-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and l-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (l-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and l-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.     

Regulation of the activity of mung bean (Phaseolus aureus) glutamine synthetase by amino acids and nucleotides.

The activity of glutamine synthetase isolated from the germinated seedlings of Phaseolus aureus was regulated by feedback inhibition by alanine, glycine, histidine, AMP, and ADP. When glutamate was the varied substrate, alanine, histidine, and glycine were partial noncompetitive, competitive, and mixed-type inhibitors, respectively. The type of inhibition by these amino acids was confirmed by fractional inhibition analysis. The adenine nucleotides, AMP and ADP, completely inhibited the enzyme activity and were competitive with respect to ATP. Multiple inhibition analyses revealed the presence of separate and nonexclusive binding sites for the amino acids and mutually exclusive sites for adenine nucleotides. Cumulative inhibition was observed with these end products.     

Glutamyl transfer ribonucleic acid synthetase of Escherichia coli. Study of the interactions with its substrates.

The binding of the various substrates to Escherichia coli glutamyl-tRNA synthetase has been investigated by using as experimental approaches the binding study under equilibrium conditions and the substrate-induced protection of the enzyme against its thermal inactivation. The results show that ATP and tRNAGlu bind to the free enzyme, whereas glutamate binds only to an enzyme form to which glutamate-accepting tRNAGlu is associated. By use of modified E. coli tRNAsGlu and heterologous tRNAsGlu, a correlation could be established between the ability of tRNAGlu to be aminoacylated by glutamyl-tRNA synthetase and its abilities to promote the [32P]PPi-ATP isotope exchange and the binding of glutamate to the synthetase. These results give a possible explanation for the inability of blutamyl-tRNA synthetase to catalyze the isotope exchange in the absence of amino acid accepting tRNAGlu and for the failure to detect an enzyme-adenylate complex for this synthetase by using the usual approaches. One binding site was detected for each substrate. The specificity of the interaction of the various substrates has been further investigated. Concerning ATP, inhibition studies of the aminoacylation reaction by various analogues showed the existence of a synergistic effect between the adenine and the ribose residues for the interaction of adenosine. The primary recognition of ATP involves the N-1 and the 6-amino group of adenine as well as the 2'-OH group of ribose. This first interaction is then strengthened by the phosphate groups- Inhibition studies by various analogues of glutamate showed a strong decrease in the affinity of this substrate for the synthetase after substitution of the alpha- or gamma-carboxyl groups. The enzyme exhibits a marked tendency to complex tRNAs of other specificities even in the presence of tRNAGlu. MgCl2 and spermidine favor the specific interactions. The influence of monovalent ions and of pH on the interaction between glutamyl-tRNA synthetase and tRNAGlu is similar to those reported for other synthetases not requiring their cognate tRNA to bind the amino acid. Finally, contrary to that reported for other monomeric synthetases, no dimerization of glutamyl-tRNA synthetase occurs during the catalytic process.     

The structures of Thermoplasma volcanium phosphoribosyl pyrophosphate synthetase bound to ribose-5-phosphate and ATP analogs

Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P2₁. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP-Mg²⁺ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively.     

Mutational and Structural Analysis of Conserved Residues in Ribose-5-Phosphate Isomerase B from Leishmania donovani: Role in Substrate Recognition and Conformational Stability

Ribose-5-phosphate isomerase B from Leishmania donovani (LdRpiB) is one of the potential drug targets against visceral leishmaniasis. In the present study, we have targeted several conserved amino acids for mutational analysis (i.e. Cys69, His11, His102, His138, Asp45, Tyr46, Pro47 and Glu149) to gain crucial insights into their role in substrate binding, catalysis and conformational stability of the enzyme. All the eight LdRpiB variants were cloned, sequenced, expressed and purified. C69S, H102N, D45N and E149A mutants exhibited complete loss of enzyme activity indicating that they are indispensable for the enzyme activity. Kinetic parameters were altered in case of H138N, H11N and P47A variants; however Y46F exhibited similar kinetic behaviour as wild type. All the mutants except H138N exhibited altered protein structure as determined by CD and fluorescence spectral analysis. This data was supported by the atomic level details of the conformational changes and substrate binding using molecular dynamic simulations. LdRpiB also exhibited activity with D-form of various aldose substrates in the order of D-ribose > D-talose > D-allose > D-arabinose. Our study provides insights for better understanding of substrate enzyme interactions which can rationalize the process of drug design against parasite RpiB.     

Kinetics of cytochrome c oxidase from yeast. Membrane-facilitated electrostatic binding of cytochrome c showing a specific interaction with cytochrome c oxidase and inhibition by ATP (With an appendix on the occurrence of two-dimensional diffusion of cytochrome c on the membrane surface)

The steady-state kinetics of purified yeast cytochrome c oxidase were investigated at low ionic strength where the electrostatic interaction with cytochrome c is maximized. In 10 mM cacodylate/Tris (pH 6.5) the oxidation kinetics of yeast iso-1-cytochrome c were sigmoidal with a Hill coefficient of 2.35, suggesting cooperative binding. The half-saturation point was 1.14 μM. Horse cytochrome c exhibited Michaelis-Menten kinetics with a higher affinity (K_m = 0.35 μM) and a 100% higher maximal velocity.In 67 mM phosphate the Hill coefficient for yeast cytochrome c decreased to 1.42, and the species differences in Hill coefficients were lessened. Under the latter conditions, a yeast enzyme preparation partially depleted of phospholipids was activated on addition of diphosphatidylglycerol liposomes. When the enzyme was incorporated into sonicated yeast promitochondrial particles the apparent K_m for horse cytochrome c was considerably lower than the value for the isolated enzyme.ATP was found to inhibit both the isolated oxidase and the membrane-bound enzyme. With the isolated enzyme in 10 mM cacodylate/Tris, 3 mM ATP increased the half-saturation point with yeast cytochrome c 3-fold, without altering the maximal velocity or the Hill coefficient. 67 mM phosphate abolished the inhibition of the isolated oxidase by ATP.The increase in affinity for cytochrome c produced by binding the oxidase to the membrane was not observed in the presence of 3 mM ATP, with the result that the membrane-bound enzyme was more sensitive to inhibition by ATP. ADP was a less effective inhibitor than ATP, and did not prevent the inhibition by ATP.It is proposed that non-specific electrostatic binding of cytochrome c to phospholipid membranes, followed by rapid lateral diffusion, is responsible for the dependence of the affinity on the amount and nature of the phospholipids and on the ionic strength.ATP may interfere with the membrane-facilitated binding of cytochrome c by a specific electrostatic interaction with the membrane or by binding to cytochrome c.     

Investigation of the role of conserved residues Ser13, Asn48 and Pro49 in the catalytic mechanism of the tau class glutathione transferase from Glycine max

Plant glutathione transferases (GSTs) play a key role in the metabolism of various xenobiotics. In this report, the catalytic mechanism of the tau class GSTU4-4 isoenzyme from Glycine max (GmGSTU4-4) was investigated by site-directed mutagenesis and steady-state kinetic analysis. The catalytic properties of the wild-type enzyme and three mutants of strictly conserved residues (Ser13Ala, Asn48Ala and Pro49Ala) were studied in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction. The results showed that the mutations significantly affect substrate binding and specificity. The effect of Ser13Ala mutation on the catalytic efficiency of the enzyme could be explained by assuming the direct involvement of Ser13 to the reaction chemistry and the correct positioning of GSH and CDNB in the ternary catalytic complex. Asn48 and Pro49 were found to have a direct role on the structural integrity of the GSH-binding site (G-site). Moreover, mutation of Asn48 and Pro49 residues may bring about secondary effects altering the thermal stability and the catalytic activity (k_cat) of the enzyme without affecting the nature of the rate-limiting step of the catalytic reaction.     

The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus)

In a previous study, a mutant of tyrosyl-tRNA synthetase in which a threonine residue (Thr51) was converted to proline dramatically improved the affinity of the enzyme for its ATP substrate. How does Pro51 improve the enzyme's affinity for ATP? A priori, Pro51 might interact directly with the ATP, or it might distort the polypeptide backbone and thereby force new or improved contacts elsewhere from the enzyme to ATP. By making mutants of the Pro51 enzyme at two residues that make hydrogen bonds to the ATP substrate, we show that Pro51 greatly improves the strength of one of these contacts. Thus the propagation of a structural change in an enzyme induced by mutation may be detected by the introduction of further mutations.     

Purification and properties of asparagine synthetase from soybean root nodules

Asparagine synthetase was purified 240-fold from soybean (Glycine max (L.) Merr.) root nodules with a final recovery of 5% using Reactive Blue 2-crossed linked Agarose affinity gel chromatography. High levels of sulfhydryl protectants were required and the inclusion to glycerol and substrates in the extraction buffer helped to stabilize the enzyme. The final preparation had a specific activity of 3.77 mkat/kg protein when assayed at 30°C and was free of contaminating asparaginase activity. The enzyme had a broad pH maximum around pH 8.0 and apparent K_m values for the substrates aspartate, Mg · ATP, and glutamine were 1.24 mM, 0.076 mM and 0.16 mM, respectively. Ammonium ion could partially replace glutamine as the nitrogen donor. Initial velocity patterns yielded parallel inverse plots with all substrate pairs suggesting an overall ping-pong reaction mechanism. Product inhibition patterns provided evidence that glutamine was the first substrate to bind to the enzyme and asparagine was the last product released.     

Purification and characterization of glutamine synthetase from the unicellular cynabacterium Anacystis nidulans

Glutamine synthetase from the unicellular cynabacterium Anacystis nidulans was found associated with the membrane fraction of cell-free extracts. The enzyme could be solubilized by treatment of the cell membranes with the detergent alkyltrimethylammoniun and was purified to electrophoretical homogeneity by using affinity chromatography on 2′,5′-ADP-Sepharose. The molecular weight of the native enzyme was approx. 575000 but only a single protein band of 47 kDa was detected after sodium dodecyl sulphate gel electrophoresis, which implies a native enzyme complex with twelve identically sized subunits. Values for apparent Michaelis constant of the purified enzyme for ammonium, glutamate and ATP were 20, 5000 and 700 μM, respectively. Alanine behaved as an inhibitor of both activities (transferase and biosynthetic) of glutamine synthetase, whereas aspartate, leucine and lysine inhibited the biosynthetic activity of the enzyme, and glycine and serine only inhibited the transferase activity. Glutamate analogs, such as hydroxylysine, methionine sulfone, methionine sulfoximine and phosphinothricin, which inhibited ammonium uptake in vivo, behaved as potent inhibitors of glutamine synthetase in vitro. A. nidulans glutamine synthetase was inhibited by p-hydroxymercuribenzoate, the effect being reversed by treatment with dithioerythritol, dithiothreitol or mercaptoethanol.