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The Biochemical Journal Jun 1991To study the mechanism of re-activation of Zymomonas mobilis pyruvate decarboxylase apoenzyme by its cofactors thiamin diphosphate and Mg2+, cofactor-free enzyme was...
To study the mechanism of re-activation of Zymomonas mobilis pyruvate decarboxylase apoenzyme by its cofactors thiamin diphosphate and Mg2+, cofactor-free enzyme was prepared by dialysis against 1 mM-dipicolinic acid at pH 8.2. This apoenzyme was then used in a series of experiments that included determination of: (a) the affinity towards one cofactor when the other was present at saturating concentrations; (b) cofactor-binding rates by measuring the quenching of tryptophan fluorescence on the apoenzyme; (c) the effect of replacement of cofactors with various analogues; (d) the stoichiometry of bound cofactors in holoenzyme; and (e) the molecular mass of apoenzyme by gel filtration. The results of these experiments form the basis for a proposed model for the re-activation of Z. mobilis pyruvate decarboxylase apoenzyme by its cofactors. In this model there exists two alterative but equivalent pathways for cofactor binding. In each pathway the first step is an independent reversible binding of either thiamin diphosphate (Kd 187 microM) or Mg2+ (Kd 1.31 mM) to free apoenzyme. When both cofactors are present, the second cofactor-binding step to form active holoenzyme is a slow quasi-irreversible step. This second binding step is a co-operative process for both thiamin diphosphate (Kd 0.353 microM) and Mg2+ (Kd 2.47 microM). Both the apo- and the holo-enzyme have a tetrameric subunit structure, with cofactors binding in a 1:1 ratio with each subunit.
Topics: Apoenzymes; Bacteria, Anaerobic; Enzyme Activation; Ethylmaleimide; Gram-Negative Bacteria; Kinetics; Magnesium; Mathematics; Models, Biological; Pyruvate Decarboxylase; Thiamine Pyrophosphate
PubMed: 2049073
DOI: 10.1042/bj2760439 -
Gene Jun 1993In this paper, we describe the gene (nia) coding for the apoenzyme of the nitrate reductase (NR) of petunia. A full-size genomic clone was isolated from a genomic...
In this paper, we describe the gene (nia) coding for the apoenzyme of the nitrate reductase (NR) of petunia. A full-size genomic clone was isolated from a genomic library, using the tobacco nia2 cDNA as a probe, and sequenced. The open reading frame is interrupted by three introns and encodes a protein of 909 amino acids which reveals between 92% and 68% identity to the NADH NR apoenzyme from other higher plants. Southern analyses indicated that the NR apoenzyme is encoded by a single-copy gene, although another region homologous to part of nia was also identified. The analysis of the steady-state level of nia mRNA showed that the petunia nia is regulated by the nitrogen source and is under the control of the circadian rhythm.
Topics: Amino Acid Sequence; Apoenzymes; Base Sequence; Circadian Rhythm; Cloning, Molecular; DNA Mutational Analysis; Gene Expression Regulation, Enzymologic; Genes, Plant; Molecular Sequence Data; Nitrate Reductase; Nitrate Reductases; Nitrogen; Phylogeny; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Messenger; Sequence Homology, Nucleic Acid
PubMed: 8514183
DOI: 10.1016/0378-1119(93)90557-j -
Neuroscience Letters Jan 1987Tryptophan hydroxylase apoenzyme was measured in 21 regions of the rat brain by a competitive enzyme-linked immunoassay (ELISA) technique using a recently developed...
Tryptophan hydroxylase apoenzyme was measured in 21 regions of the rat brain by a competitive enzyme-linked immunoassay (ELISA) technique using a recently developed antiserum from the sheep to this protein. Highest apoenzyme levels were found in the pineal gland and in the dorsal raphé. An insignificant level was observed in the cerebellum. In general, the distribution of tryptophan hydroxylase apoenzyme follows the distribution of serotonin previously detected by immunocytochemistry. A turnover number for tryptophan hydroxylase in a rat brain supernatant fraction of 7.5 s-1 was estimated, a value far higher than that estimated for serotonin turnover in vivo. This result confirms that serotonin biosynthesis is additionally regulated by factors other than tryptophan hydroxylase apoenzyme concentration.
Topics: Animals; Apoenzymes; Apoproteins; Brain; Enzyme-Linked Immunosorbent Assay; Male; Rats; Rats, Inbred Strains; Tryptophan Hydroxylase
PubMed: 3561858
DOI: 10.1016/0304-3940(87)90033-4 -
Archives of Biochemistry and Biophysics Dec 1996Soluble, periplasmic quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus (sGDH; EC 1.1.99.17) was produced in good yield in the apoenzyme form (without the...
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 Ca2+ 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 Ca2+. 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 PQQH2-like spectrum when glucose was added. Full restoration of activity was achieved upon addition of Ca2+ 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 Ca2+ 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.
Topics: Acinetobacter calcoaceticus; Apoenzymes; Glucose 1-Dehydrogenase; Glucose Dehydrogenases; Protein Conformation; Protein Processing, Post-Translational; Recombinant Proteins; Solubility
PubMed: 8951033
DOI: 10.1006/abbi.1996.0530 -
Journal of Molecular Biology Aug 1992The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A. Molecular... (Comparative Study)
Comparative Study
The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A. Molecular replacement, using the partially refined structure of porcine mitochondrial malate dehydrogenase as a probe, provided initial phases. The structure of this prokaryotic enzyme is closely homologous with the mitochondrial enzyme but somewhat less similar to cytosolic malate dehydrogenase from eukaryotes. However, all three enzymes are dimeric and form the subunit-subunit interface through similar surface regions. A citrate ion, found in the active site, helps define the residues involved in substrate binding and catalysis. Two arginine residues, R81 and R153, interacting with the citrate are believed to confer substrate specificity. The hydroxyl of the citrate is hydrogen-bonded to a histidine, H177, and similar interactions could be assigned to a bound malate or oxaloacetate. Histidine 177 is also hydrogen-bonded to an aspartate, D150, to form a classic His.Asp pair. Studies of the active site cavity indicate that the bound citrate would occupy part of the site needed for the coenzyme. In a model building study, the cofactor, NAD, was placed into the coenzyme site which exists when the citrate was converted to malate and crystallographic water molecules removed. This hypothetical model of a ternary complex was energy minimized for comparison with the structure of the binary complex of porcine cytosolic malate dehydrogenase. Many residues involved in cofactor binding in the minimized E. coli malate dehydrogenase structure are homologous to coenzyme binding residues in cytosolic malate dehydrogenase. In the energy minimized structure of the ternary complex, the C-4 atom of NAD is in van der Waals' contact with the C-3 atom of the malate. A catalytic cycle involves hydride transfer between these two atoms.
Topics: Amino Acid Sequence; Animals; Apoenzymes; Binding Sites; Citrates; Escherichia coli; Malate Dehydrogenase; Models, Molecular; Molecular Sequence Data; Myocardium; Protein Conformation; Sequence Homology, Nucleic Acid; Swine; Thermodynamics; X-Ray Diffraction
PubMed: 1507230
DOI: 10.1016/0022-2836(92)90637-y -
Biochemistry. Biokhimiia Oct 2002Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5'-phosphate dependent transformation of amino acid as an... (Review)
Review
Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5'-phosphate dependent transformation of amino acid as an example. A new type of antigens analogous to enzyme--substrate compounds is suggested for the production of such antibodies. Approaches for the development of pyridoxal antiidiotypic antibody using analogs of coenzyme--substrate compounds and corresponding apoenzyme complexes are reviewed.
Topics: Antibodies, Anti-Idiotypic; Antibodies, Catalytic; Antigens; Apoenzymes; Protein Conformation; Protein Engineering; Pyridoxal Phosphate
PubMed: 12460106
DOI: 10.1023/a:1020955005503 -
Signal Transduction and Targeted Therapy Jun 2021
Topics: Apoenzymes; Crystallography, X-Ray; Humans; Ligands; Protein Conformation; Receptors, Serotonin; Serotonin; Serotonin Receptor Agonists
PubMed: 34145221
DOI: 10.1038/s41392-021-00668-3 -
Biochemistry Dec 2006The Vibrio harveyi NADPH-specific flavin reductase FRP follows a ping-pong mechanism but switches to a sequential mechanism in the luciferase-coupled reaction. The bound...
The Vibrio harveyi NADPH-specific flavin reductase FRP follows a ping-pong mechanism but switches to a sequential mechanism in the luciferase-coupled reaction. The bound FMN co-isolated with FRP, while acting as a genuine cofactor in the single-enzyme reaction, functions in the luciferase-coupled reaction as a prebound substrate and is directly transferred to luciferase once it is reduced [Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623-14629]. With the aim of better understanding the functions of FMN in the FRP holoenzyme, this study was undertaken to quantify and compare the thermodynamic properties of the binding of oxidized and reduced FMN by the FRP apoenzyme. By isothermal titration calorimetry (ITC) measurements in various buffers at pH 7.0 and 15-30 degrees C, the binding of FMN by apo-FRP was found to be noncooperative, exothermic, and primarily enthalpy driven. The binding free energy change (hence, the association constant) was nearly invariant over this temperature range. Significant conformational changes in FRP upon binding of FMN were indicated. Equilibrium bindings of reduced flavins by flavin-dependent proteins have rarely been studied. In this work, the thermodynamic properties of binding of reduced FMN by apo-FRP were found to closely resemble those of FMN binding under three sets of experimental conditions via ITC measurements and, in one case, fluorescence quenching. The kinetically deduced ping-pong mechanism of FRP is now supported by direct measurements of binding affinities of the oxidized and reduced FMN cofactors. These findings are also discussed in relation to the function of FRP as a reduced flavin donor in the FRP-luciferase couple.
Topics: Apoenzymes; Bacterial Proteins; FMN Reductase; Flavin Mononucleotide; Kinetics; Models, Biological; Oxidation-Reduction; Spectrometry, Fluorescence; Thermodynamics; Vibrio
PubMed: 17144671
DOI: 10.1021/bi0610956 -
Biochemistry Feb 1993The structure of the His-94-->Cys variant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods to a resolution of 2.3 A with a...
The structure of the His-94-->Cys variant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods to a resolution of 2.3 A with a final crystallographic R factor of 0.155. This variant of CAII crystallizes in orthorhombic space group P2(1)2(1)2(1) which is the first example of a new crystal form for this important zinc hydrase (the wild-type enzyme crystallizes in monoclinic space group P21 under similar crystallization conditions). Although the overall structure of the enzyme in the orthorhombic crystal form is similar to that of the wild-type protein in the monoclinic crystal form, the rms deviation of C alpha atoms between the two structures is 0.5 A. Larger structural deviations occur in regions of the protein molecule involved in crystal lattice contacts, and significant structural changes are found in the polypeptide strand containing Cys-94. Surprisingly, no electron density corresponding to a zinc ion is found in the active site of crystalline His-94-->Cys CAII, even though the stoichiometry of zinc binding to this variant in solution is confirmed by atomic absorption spectroscopy. However, the KD for zinc dissociation from the variant is increased 10(4)-fold compared with wild-type enzyme; furthermore, under the crystallization conditions of high ionic strength (1.75-2.5 M ammonium sulfate), the observed KD is increased further, which leads to zinc dissociation. Spectroscopic analysis of Co(2+)-substituted His-94-->Cys CAII indicates that the metal binds in a tetrahedral geometry with a new thiolate bond.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Amino Acid Sequence; Apoenzymes; Binding Sites; Carbonic Anhydrases; Cloning, Molecular; Cysteine; Escherichia coli; Histidine; Humans; Hydrogen Bonding; Isoenzymes; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Engineering; Recombinant Proteins; Spectrophotometry; X-Ray Diffraction; Zinc
PubMed: 8431430
DOI: 10.1021/bi00057a015 -
The Biochemical Journal Feb 1989Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed...
Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.
Topics: Apoenzymes; Apoproteins; Enzyme Activation; Enzyme Stability; Escherichia coli; Flavin-Adenine Dinucleotide; Oxidoreductases; Phosphoenolpyruvate; Protease Inhibitors; Temperature
PubMed: 2649085
DOI: 10.1042/bj2580187