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Ikuo Satoh - One of the best experts on this subject based on the ideXlab platform.
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Amperometric biosensing of heavy metal ions using a hybrid type of Apoenzyme membrane in flow streams
Sensors and Actuators B: Chemical, 1993Co-Authors: Ikuo SatohAbstract:Abstract Flow-amperometric microdetermination of heavy metal ions based on an Apoenzyme reactivation method is proposed. A hybrid type of enxyme membrane was prepared by co-immobilizing alkaline phosphatase (zinc enzyme) and ascorbate oxidase (copper enzyme) onto a porous polymer membrane and thereby used as the recognition element for zinc(II) and copper(II) ions. The biosensing system was assembled with the hybrid-enzyme membrane attached to a sensing part of a flow-through oxygen electrode for monitoring the enzymic activity. Zinc(II) ions in 2 to 200 μM levels and also copper(II) ions in 2 to 100 μM levels were amperometrically determined through the activation of each immobilized metal-free enzyme (Apoenzyme). The Apoenzyme membrane was regenerated by pumping chelating agents to remove each of the heavy metal ions from the catalytic site of alkaline phosphatase or ascorbate oxidase.
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Flow-injection amperometric biosensing of copper(II) ions using a contact-type of an Apoenzyme sensor
Biosensors '92 Proceedings, 1992Co-Authors: Ikuo Satoh, H. Itoh, H. AnzaiAbstract:Flow-injection amperometric determination of copper(II) ions based on an Apoenzyme reactivation method is proposed. Ascorbate oxidase immobilized onto a porous polymer membrane is used as the recognition element for copper(II) ions. The biosensing system is assembled with the enzyme-membrane attached onto a flow-through type of an oxygen electrode for monitoring the enzymic activity. Copper(II) ions are amperometrically determined in 5.0-50 μM through its activation of the immobilized metal-free enzyme(Apoenzyme). The activity is assessed by injecting a 0.1 ml of 10 mM L-ascorbate solution as the substrate and thereby, the change in oxygen uptake is measured. The membrane is regenerated by pumping a 0.8 mM N,N- diethyldithiocarbamate solution(pH 8.0) between successive samples.
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An Apoenzyme thermistor microanalysis for zinc(II) ions with use of an immobilized alkaline phosphatase reactor in a flow system
Biosensors and Bioelectronics, 1991Co-Authors: Ikuo SatohAbstract:Abstract Calorimetric microdetermination of zinc(II) ions with use of an Apoenzyme thermistor in a flow stream is proposed. Alkaline phosphatase as the selective recognition element was immobilized onto oxirane-acrylic beads (Eupergit-C) and packed into a small polymer column. The flow-injection biosensing system was assembled with the immobilized enzyme reactor and a thermistor device for monitoring the enzyme activity. Zinc(II) ions were calorimetrically determined in the range 0·01–1·0 m m for 0·5 ml samples through their activation of the immobilized metal-free alkaline phosphatase (Apoenzyme) reactor. The activity of the reactor was assessed by injecting 0·1 ml of 100 m m p -nitrophenyl phosphate solution. Regeneration of the reactor was performed by pumping 20 m m 2,6-pyridine dicarboxylate (pH 6·0) between successive samples. The system could be repeatedly used at least 120 times during 2 months of operation.
Alfred A Antson - One of the best experts on this subject based on the ideXlab platform.
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structures of apo and holo tyrosine phenol lyase reveal a catalytically critical closed conformation and suggest a mechanism for activation by k ions
Biochemistry, 2006Co-Authors: Dalibor Milic, Vitalia V Kulikova, T Y Demidkina, Dubravka Matkoviccalogovic, Nina I Sinitzina, Alfred A AntsonAbstract:Tyrosine phenol-lyase, a tetrameric pyridoxal 5‘-phosphate dependent enzyme, catalyzes the reversible hydrolytic cleavage of l-tyrosine to phenol and ammonium pyruvate. Here we describe the crystal structure of the Citrobacter freundii holoenzyme at 1.9 A resolution. The structure reveals a network of protein interactions with the cofactor, pyridoxal 5‘-phosphate, and details of coordination of the catalytically important K+ ion. We also present the structure of the Apoenzyme at 1.85 A resolution. Both structures were determined using crystals grown at pH 8.0, which is close to the pH of the maximal enzymatic activity (8.2). Comparison of the Apoenzyme structure with the one previously determined at pH 6.0 reveals significant differences. The data suggest that the decrease of the enzymatic activity at pH 6.0 may be caused by conformational changes in the active site residues Tyr71, Tyr291, and Arg381 and in the monovalent cation binding residue Glu69. Moreover, at pH 8.0 we observe two different active si...
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crystallographic studies of tyrosine phenol lyase
1994Co-Authors: Alfred A Antson, G G Dodson, Keith S Wilson, S V Pletnev, E G Harutyunyan, T Y DemidkinaAbstract:Monovalent cation binding site of the enzyme was determined using X-ray data obtained from Apoenzyme crystals soaked in K+ and Cs+ containing solutions. Glu 69 is involved in formation of that site and is conserved in all other tyrosine phenol-lyases and tryptophan indol-lyases with known sequence. Three dimensional structures of holoenzyme and its complex with 3-(4-hydroxyphenyl)propionic acid were solved and refined at 2.7A and 2.5 A respectively. The structures explicitly reveal the cofactor and substrate binding pockets.
Johannis A. Duine - One of the best experts on this subject based on the ideXlab platform.
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Reconstitution of Membrane-Integrated Quinoprotein Glucose Dehydrogenase Apoenzyme with PQQ and the Holoenzyme's Mechanism of Action
Biochemistry, 1998Co-Authors: Asteriani R. Dewanti, Johannis A. DuineAbstract:Membrane-integrated quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus was produced by heterologous expression of the gene for it in an Escherichia coli recombinant strain. The Apoenzyme (lacking the cofactor pyrroloquinoline quinone, PQQ) was solubilized with Triton X-100 and purified to homogeneity. Reconstitution of the Apoenzyme to full activity in the assay was achieved with a stoichiometric amount of PQQ in the presence of Mg2+. Just as for other PQQ-containing dehydrogenases where Ca2+ fulfills this role, Mg2+ anchors PQQ to the mGDH protein and activates the bound cofactor. This occurs in a precise way since high anomer specificity was found for the enzyme toward the sugars tested. Although the steady-state-type kinetics were as expected for a dye-linked dehydrogenase (ping-pong) and the PQQ in it was present in oxidized form, addition of glucose to the holoenzyme resulted in a very slow but continuous production of gluconolactone; i.e., the reaction did not stop after one turnover, with O2 apparently acting as an (albeit poor) electron acceptor by reoxidizing PQQH2 in the enzyme. The surprisingly low reactivity with glucose, in the absence of dye, as compared to the activity observed in the steady-state assay appeared to be due to formation of an anomalous enzyme form, mGDH. Formation of normal holoenzyme, mGDH, reducing added glucose immediately to gluconolactone (in one turnover), was achieved by treating mGDH with sulfite, by reconstituting Apoenzyme with PQQ in the presence of sulfite, or by applying assay conditions to mGDH (addition of PMS/DCPIP). As compared to other quinoprotein dehydrogenases, mGDH appears to be unique with respect to the mode of PQQ-binding, as expressed by the special conditions for reconstitution and the absorption spectra of the bound cofactor, and the reactivity of the reduced enzyme toward O2. The primary cause for this seems not to be related to a different preference for the activating bivalent metal ion but to the special way of binding of PQQ to mGDH.
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Production, Characterization, and Reconstitution of Recombinant Quinoprotein Glucose Dehydrogenase (Soluble Type; EC 1.1.99.17) Apoenzyme ofAcinetobacter calcoaceticus
Archives of biochemistry and biophysics, 1996Co-Authors: Arjen J. J. Olsthoorn, Johannis A. DuineAbstract:Abstract Soluble, periplasmic quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus (sGDH; EC 1.1.99.17) was produced in good yield in the Apoenzyme form (without the cofactor pyrroloquinoline quinone, PQQ) by an Escherichia coli recombinant strain provided with a plasmid containing the gene under control of a lac promoter. Structural analysis of the purified Apoenzyme revealed that the E. coli strain used produces the correct mature protein. Titration of the Apoenzyme with PQQ in the presence of Ca 2+ showed that a linear relation exists between the amount of added PQQ and activity observed, and that the subunit and PQQ associate in a molar ratio of 1:1. Based on spectral and enzymatic criteria, it is concluded that the present holoenzyme preparation has a better quality than the previously described preparations of authentic holoenzyme. As isolated here, the recombinant Apoenzyme was in the dimeric form. Partial monomerization occurred upon gel filtration in a buffer with chelator and the process could be reversed with Ca 2+ . PQQ binds to the dimer in the presence of chelator, not to the monomer. However, the PQQ-containing dimer was not active and showed an unusual absorption spectrum which was slowly converted into a PQQH 2 -like spectrum when glucose was added. Full restoration of activity was achieved upon addition of Ca 2+ and the spectra were immediately converted into those of normal holoenzyme in the oxidized and reduced form, respectively. Addition of chelator to holoenzyme did not lead to inactivation or monomerization. It is concluded, therefore, that Ca 2+ has a dual role in this enzyme, being required for dimerization of the subunits as well as for functionalization of the bound PQQ, and that it is more firmly attached to the holoenzyme than to the Apoenzyme.
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Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the Apoenzyme with pyrroloquinoline quinone analogues.
European journal of biochemistry, 1995Co-Authors: G. A. H. De Jong, J.a. Jongejan, Arie Geerlof, Joke Stoorvogel, S. De Vries, Johannis A. DuineAbstract:Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) Apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified Apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to Apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of Apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the Apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.
Rainer Jaenicke - One of the best experts on this subject based on the ideXlab platform.
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stability and reconstitution of pyruvate oxidase from lactobacillus plantarum dissection of the stabilizing effects of coenzyme binding and subunit interaction
Protein Science, 1992Co-Authors: Bernhard Risse, Gunter Stempfer, Rainer Rudolph, Hans Mollering, Rainer JaenickeAbstract:Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable Apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the Apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the Apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size-exclusion high-performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 micrograms/mL, whereas the Apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of < 20 micrograms/mL. In the presence of divalent cations, the coenzymes FAD and TPP bind to the Apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect. Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of Ka = 2 x 10(7) M-1. The Apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 microM FAD. This association step obeys second-order kinetics with an association rate constant k = 7.4 x 10(3) M-1 s-1 at 20 degrees C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.
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characterization of the stabilizing effect of point mutations of pyruvate oxidase from lactobacillus plantarum protection of the native state by modulating coenzyme binding and subunit interaction
Protein Science, 1992Co-Authors: Bernhard Risse, Gunter Stempfer, Rainer Rudolph, Gunter Schumacher, Rainer JaenickeAbstract:Point mutations in the gene of pyruvate oxidase from Lactobacillus plantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions, or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the Apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased. The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 degrees C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant Apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.
P A Karplus - One of the best experts on this subject based on the ideXlab platform.
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structures of the klebsiella aerogenes urease Apoenzyme and two active site mutants
Biochemistry, 1996Co-Authors: E Jabri, P A KarplusAbstract:Urease from Klebsiella aerogenes [Jabri et al. (1995) Science 268, 998−1004] is an (αβγ)3 trimer with each α-subunit having an (αβ)8-barrel domain containing a binickel active center. Here we examine structure−function relations for urease in more detail through structural analysis of the urease Apoenzyme at 2.3 A resolution and mutants of two key catalytic residues (H219A and H320A) at 2.5 A resolution. With the exception of the active site, in which a water molecule takes the place of the missing carbamate and nickel atoms, the structure of the Apoenzyme is nearly identical to that of the holoenzyme, suggesting a high degree of preorganization which helps explain the tight binding of nickel. In the structure of H219A, the major change involves a conformational shift and ordering of the active site flap, but a small shift in the side chain of Aspα221 could contribute to the lower activity of H219A. In the H320A structure, the catalytic water, primarily a Ni-2 ligand in the holoenzyme, shifts into a bridg...