A study of the dynamic state of histidine residues in tryptophan synthetase subunit by 13 C nuclear magnetic resonance

Tryptophan synthetase α subunit in which the histidine C₂ (ring) positions are enriched in ¹³C and labeled with deuterium was prepared by incorporation of labeled histidine into protein of Escherichia coli. ¹³C nuclear magnetic resonance studies of the specifically labeled enzyme demonstrate that all four histidine residues of α subunit are highly immobilized within the protein matrix.     

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

The heme-based oxygen-sensor enzyme from Escherichia coli (Ec DOS) is a heme-regulated phosphodiesterase with activity on cyclic-di-GMP and is composed of an N-terminal heme-bound sensor domain with the PAS structure and a C-terminal functional domain. The activity of Ec DOS is substantially enhanced by the binding of O₂ to the Fe(II)-protoporphyrin IX complex [Fe(II) complex] in the sensor domain. The binding of O₂ to the Fe(II) complex changes the structure of the sensor domain, and this altered structure becomes a signal that is transduced to the functional domain to trigger catalysis. The first step in intra-molecular signal transduction is the binding of O₂ to the Fe(II) complex, and detailed elucidation of this molecular mechanism is thus worthy of exploration. The X-ray crystal structure reveals that Phe113 is located close to the O₂ molecule bound to the Fe(II) complex in the sensor domain. Here, we found that the O₂ association rate constants (>200 × 10⁻³ μM⁻¹ s⁻¹: F113L; 26 × 10⁻³ μM⁻¹ s⁻¹: F113Y) of the Fe(II) complexes of Phe113 mutants were markedly different from that (51 × 10⁻³ μM⁻¹ s⁻¹) of the wild-type enzyme, and auto-oxidation rates (0.00068 min⁻¹: F113L; 0.039 min⁻¹: F113Y) of the Phe113 mutants also differed greatly from that (0.0062 min⁻¹) of the wild-type enzyme. We thus suggest that Phe113, residing near the O₂ molecule, has a critical role in optimizing the Fe(II)-O₂ complex for effective regulation of catalysis by the oxygen-sensor enzyme. Interactions of CO and cyanide anion with the mutant proteins were also studied.     

Studies on the subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

Both uncomplexed subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium have an absolute requirement for divalent metal ions which can be satisfied by Mg²⁺, Mn²⁺, or Co²⁺. The metal ion kinetics for uncomplexed anthranilate synthetase give biphasic double-reciprocal plots and higher apparent K_m values than those for anthranilate synthetase in the enzyme complex. In contrast, the apparent K_m values for phosphoribosyltransferase are the same whether the enzyme is uncomplexed or complexed with anthranilate synthetase. This suggests that the metal ion sites on anthranilate synthetase, but not those on phosphoribosyltransferase, are altered upon formation of the enzyme complex. These results and the results of studies reported by others, suggest that complex formation between anthranilate synthetase and phosphoribosyltransferase leads to marked alterations at the active site of the former, but not the latter enzyme. Uncomplexed anthranilate synthetase can be stoichiometrically labeled with Co(III) under conditions which lead to inactivation of 75% of its activity. A comparison of the effects of anthranilate and tryptophan on phosphoribosyltransferase activity in the uncomplexed and complexed forms shows that anthranilate, but not tryptophan, inhibits the uncomplexed enzyme. The complexed phosphoribosyltransferase shows substrate inhibition by anthranilate binding to the phosphoribosyltransferase subunits. In contrast, in a tryptophan-hypersensitive variant complex, anthranilate inhibits phosphoribosyltransferase activity by acting on the anthranilate synthetase subunits. The data are interpreted to mean that there are two classes of binding sites for anthranilate, one on each type of subunit, which may participate in the regulation of anthranilate synthetase and phosphoribosyltransferase under different conditions.     

The structure of Escherichia coli ATP-phosphoribosyltransferase: identification of substrate binding sites and mode of AMP inhibition

ATP-phosphoribosyltransferase (ATP-PRT), the first enzyme of the histidine pathway, is a complex allosterically regulated enzyme, which controls the flow of intermediates through this biosynthetic pathway. The crystal structures of Escherichia coli ATP-PRT have been solved in complex with the inhibitor AMP at 2.7A and with product PR-ATP at 2.9A (the ribosyl-triphosphate could not be resolved). On the basis of binding of AMP and PR-ATP and comparison with type I PRTs, the PRPP and parts of the ATP-binding site are identified. These structures clearly identify the AMP as binding in the 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP)-binding site, with the adenosine ring occupying the ATP-binding site. Comparison with the recently solved Mycobacterium tuberculosis ATP-PRT structures indicates that histidine is solely responsible for the large conformational changes observed between the hexameric forms of the enzyme. The role of oligomerisation in inhibition and the structural basis for the synergistic inhibition by histidine and AMP are discussed.     

A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon

Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA^Asp QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA^Asp isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA^Asp anticodon stem and the tRNA^Glu amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA^Asp synthetases, is conserved among prokaryotes.     

Expression, purification and characterization of recombinant (E)-beta-farnesene synthase from Artemisia annua

A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-β-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI = 5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, β-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K_m- and k_cat-values for farnesyl diphosphate, is 2.1 μM and 9.5 × 10⁻³ s⁻¹, respectively resulting in the efficiency 4.5 × 10⁻³ M⁻¹ s⁻¹. The enzyme exhibits substantial activity in the presence of Mg²⁺, Mn²⁺ or Co²⁺ but essentially no activity when Zn²⁺, Ni²⁺ or Cu²⁺ is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 μM for Mg²⁺, Co²⁺ or Mn²⁺, respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme.     

Comparative study of catalase-peroxidases (KatGs)

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900 s⁻¹ and 8-25 s⁻¹, respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC₅₀ values of 0.2-20 μM and 50-170 μM, respectively. The K_Ms of the enzymes for H₂O₂ in the catalase reaction were relatively invariant between 3 and 5 mM at pH 7.0, but increased to values ranging from 20 to 225 mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H₂O₂ binding to Cpd I. The catalatic k_cat was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002 s⁻¹, respectively. Only the NADH oxidase reaction varied more widely between 10⁻⁴ and 10⁻² s⁻¹ with the fastest rate being exhibited by the enzyme from B. pseudomallei.     

Zinc uptake across the apical membrane of freshwater rainbow trout intestine is mediated by high affinity, low affinity, and histidine-facilitated pathways

Zinc is both a vital nutrient and an important toxicant to aquatic biota. In order to understand the interplay between nutrition and toxicity, it will be important to determine the mechanisms and the factors that regulate zinc uptake. The mechanism of apical intestinal Zn(II) uptake in freshwater rainbow trout and its potential modification by the complexing amino acid histidine was investigated using brush-border membrane vesicles (BBMVs). Following characterisation of the BBMV preparation, zinc uptake in the absence of histidine was both time- and concentration-dependent and consisted of two components. A saturable phase of uptake was described by an affinity constant of 57±17 μM and a transport capacity of 1867±296 nmol mg membrane protein⁻¹ min⁻¹. At higher zinc levels (>500 μM) a linear, diffusive component of uptake was evident. Zinc transport was also temperature-dependent, with Q₁₀ values suggesting zinc uptake was a carrier-mediated process. Zinc uptake by vesicles in the presence of histidine was correlated to a mono-histidine species (Zn(His)⁺) at all Zn(II) concentrations examined.     

Reversible adenylylation of glutamine synthetase is dynamically counterbalanced during steady-state growth of Escherichia coli

Glutamine synthetase (GS) is the central enzyme for nitrogen assimilation in Escherichia coli and is subject to reversible adenylylation (inactivation) by a bifunctional GS adenylyltransferase/adenylyl-removing enzyme (ATase). In vitro, both of the opposing activities of ATase are regulated by small effectors, most notably glutamine and 2-oxoglutarate. In vivo, adenylyltransferase (AT) activity is critical for growth adaptation when cells are shifted from nitrogen-limiting to nitrogen-excess conditions and a rapid decrease of GS activity by adenylylation is needed. Here, we show that the adenylyl-removing (AR) activity of ATase is required to counterbalance its AT activity during steady-state growth under both nitrogen-excess and nitrogen-limiting conditions. This conclusion was established by studying AR⁻/AT⁺ mutants, which surprisingly displayed steady-state growth defects in nitrogen-excess conditions due to excessive GS adenylylation. Moreover, GS was abnormally adenylylated in the AR⁻ mutants even under nitrogen-limiting conditions, whereas there was little GS adenylylation in wild-type strains. Despite the importance of AR activity, we establish that AT activity is significantly regulated in vivo, mainly by the cellular glutamine concentration. There is good general agreement between quantitative estimates of AT regulation in vivo and results derived from previous in vitro studies except at very low AT activities. We propose additional mechanisms for the low AT activities in vivo. The results suggest that dynamic counterbalance by reversible covalent modification may be a general strategy for controlling the activity of enzymes such as GS, whose physiological output allows adaptation to environmental fluctuations.     

Investigation of the enzymatic activity of the Na,K-ATPase via isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) is shown here to be a sensitive and accurate method for assaying the steady-state enzyme activity of the Na⁺,K⁺-ATPase. Single ATP injection experiments yield an apparent enthalpy change for the ATP hydrolysis reaction catalyzed by the enzyme of −51 (± 1) kJ mol⁻¹. This value is independent of the amount of ADP accumulated in the sample cell, which indicates that under the experimental conditions studied here (saturating Na⁺ and K⁺ concentrations) ADP does not inhibit enzyme activity by reversal of the phosphorylation reaction and resynthesizing ATP. Multiple ATP injection titration experiments in which varying concentrations of ADP were initially included in the sample cell could be adequately explained by a Michaelis-Menten kinetic model incorporating noncompetitive inhibition. This suggests that ADP inhibits the enzyme by binding to more than one enzyme intermediate and inhibiting forward reactions of the enzyme. Values of K_m and K_I obtained for the fits agree with literature values obtained by other methods. Because ITC is a direct method of continually monitoring enzyme activity, it is a valuable supplement to less direct or noncontinuous methods such as colorimetric, enzyme-coupled or radioactivity-based assays.     

Diffusion Coefficients of Endogenous Cytosolic Proteins from Rabbit Skinned Muscle Fibers

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10⁻¹⁰ cm² s⁻¹) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10⁻⁷ cm² s⁻¹ (parvalbumin) to 0.20 × 10⁻⁷ cm² s⁻¹ (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Triose phosphate isomerase from the blood fluke Schistosoma mansoni: Biochemical characterisation of a potential drug and vaccine target

The glycolytic enzyme triose phosphate isomerase from Schistosoma mansoni is a potential target for drugs and vaccines. Molecular modelling of the enzyme predicted that a Ser-Ala-Asp motif which is believed to be a helminth-specific epitope is exposed. The enzyme is dimeric (as judged by gel filtration and cross-linking), resistant to proteolysis and highly stable to thermal denaturation (melting temperature of 82.0 °C). The steady-state kinetic parameters are high (K_m for dihydroxyacetone phosphate is 0.51 mM; K_m for glyceraldehyde 3-phosphate is 1.1 mM; k_cat for dihydroxyacetone phosphate is 7800 s⁻¹ and k_cat for glyceraldehyde 3-phosphate is 6.9 s⁻¹).     

Substrate-inhibitory analysis of the cholinesterase in the freshwater oligochaete Lumbriculus variegatus (O.F. Műller, 1773; Lumbriculidae,Oligochaeta)

The substrate-inhibitory analysis has shown that single “atypical” cholinesterase (ChE) presents in tissues of freshwater oligochaete Lumbriculus variegatus (O.F. M?ller). This enzyme differs both from “typical” acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Specific activity of oligochaete ChE ranges 55–100 μmol ATCh g⁻¹ tissue min⁻¹ or 0.7–1 μmol ATCh mg⁻¹ protein min⁻¹, ratio of maximal rates (V) of substrate hydrolysises is 100:72:71:83 for acetyl-, propionyl-, butyryl- and acetyl-β-metylthiocholine respectively. Values of Michaelis constant (K_m) for these substrates are (1.9–2.5) × 10⁻⁴ M. The bimolecular enzyme inhibition rate constants (k_II) for organophosphorus inhibitors paraoxon, DDVP, and iso-OMPA are 10⁷, 10⁶ и 10³ mol⁻¹ | min⁻¹. ATCh and BuTCh exhibit the effect of substrate inhibition of ChE activity, while PrTCh and MeTCh do not.     

The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2

Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K_M of 2.4 μM for human thioredoxin and a very low K_M for H₂O₂ (?0.7 μM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H₂O₂ or peroxynitrite, were determined by competition kinetics, k₂ = 1.0 × 10⁸ and 1.4 × 10⁷ M⁻¹ s⁻¹ at 25 °C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K_d < 10⁻²³ M⁴ which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.     

Kinetic and thermodynamic properties of the folding and assembly of formate dehydrogenase

The folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10⁻¹³ M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate (k₁) of about 2 × 10⁻³ s⁻¹ (by deduction k₋₁ is about10⁻⁴ s⁻¹) and assembles into the active dimeric state with a bimolecular rate constant (k₂) of about 2 × 10⁴ M⁻¹ s⁻¹. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k₋₂ ∼ 3 × 10⁻⁷ s⁻¹).     

Characterization of Ni transport into brush border membrane vesicles (BBMVs) isolated from the kidney of the freshwater rainbow trout (Oncorhynchus mykiss)

The transport of nickel (Ni) across the renal brush border membrane of the rainbow trout was examined in vitro using brush border membrane vesicles (BBMVs). Both transmembrane transport of Ni into an osmotically active intravesicular space, and binding of Ni to the brush border membrane itself, were confirmed. Nickel (Ni) uptake fitted a two component kinetic model. Saturable, temperature-dependent transport dominated at lower Ni concentrations, with a moderate linear diffusive component of Ni transport apparent at higher Ni concentrations. An affinity constant (K_m) for Ni transport within the specifically described vesicular media was calculated as 17.9 ± 1.9 μM, the maximal rate of transport (J_max) was calculated as 108.3 ± 3.7 nmol mg protein⁻¹ min⁻¹, and the slope of the linear diffusive component was calculated as 0.049 ± 0.005 nmol mg protein⁻¹ min⁻¹ per μM of Ni. Efflux of Ni from BBMVs was fitted to an exponential decay curve with a half-time (T_1/2) of 125.2 ± 7.3 s. Ni uptake into renal BBMVs was inhibited by magnesium at a 100:1 Mg to Ni molar ratio, and by magnesium and calcium at a 1000:1 molar ratio. In the presence of histidine at a 100:1 histidine to Ni ratio, Ni uptake was almost completely abolished. At a 1:1 molar ratio, histidine inhibited Ni uptake by approximately 50%. Ni-histidine complexation was rapid, with a T_1/2 of 12.2 s describing the Ni-histidine equilibration time needed to inhibit Ni uptake into renal BBMVs by 50%. Characterization of Ni transport across cellular membranes is an important step in understanding both the processes underlying homeostatic regulation of Ni, and the toxicological implications of excessive Ni exposure in aquatic ecosystems.     

Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N⁶-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164 ± 5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (k_cat/K_m 2.1 × 10⁶ s^− 1 M^− 1), followed by 2′-deoxyadenosine (k_cat/K_m 4.2 × 10⁵ s^− 1 M^− 1). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.     

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10⁵-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k_cat′ for desulfination of l-cysteine sulfinate increased to 0.5 s^− 1 (from 0.05 s^− 1 in wild-type enzyme), whereas k_cat′ for transamination of the same substrate was reduced from 510 s^− 1 to 0.05 s^− 1. Similarly, k_cat′ for β-decarboxylation of l-aspartate increased from < 0.0001 s^− 1 to 0.07 s^− 1, whereas k_cat′ for transamination was reduced from 530 s^− 1 to 0.13 s^− 1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.