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Inorganic Chemistry Oct 2012Carbonic anhydrase (CA, general abbreviation for human carbonic anhydrase II) is a well-studied, zinc-dependent metalloenzyme that catalyzes hydrolysis of carbon dioxide...
Carbonic anhydrase (CA, general abbreviation for human carbonic anhydrase II) is a well-studied, zinc-dependent metalloenzyme that catalyzes hydrolysis of carbon dioxide to the bicarbonate ion. The apo-form of CA (apoCA, CA where Zn(2+) ion has been removed) is relatively easy to generate, and reconstitution of the human erythrocyte CA has been initially investigated. In the past, these studies have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (K(a) ≈ 1.2 × 10(12)) for Zn(2+). However, new reactivity data and isothermal titration calorimetry (ITC) data reported herein call that number into question. As shown in the ITC experiments, the catalytic site binds a stoichiometric quantity of Zn(2+) with a strong equilibrium constant (K(a) ≈ 2 × 10(9)) that is 3 orders of magnitude lower than the previously established value. Thermodynamic parameters associated with Zn(2+) binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with zinc binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the zinc sites in both the as-isolated and the reconstituted ZnCA (active CA containing a mononuclear Zn(2+) center) were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the zinc center in the reconstituted carbonic anhydrase is nearly identical to that of the as-isolated protein and confirm the notion that the metal binding data reported herein is the reconstitution of the zinc active site of human CA II.
Topics: Apoenzymes; Carbonic Anhydrase II; Catalytic Domain; Humans; Models, Molecular; Temperature; Thermodynamics; Zinc
PubMed: 23030313
DOI: 10.1021/ic301645j -
Bulletin de La Societe de Chimie... 1962
Topics: Amino Acids; Apoenzymes; Coenzymes; Oxidoreductases
PubMed: 14008703
DOI: No ID Found -
The Journal of Vitaminology Sep 1960
Topics: Animals; Apoenzymes; Metabolism; Oxidative Phosphorylation; Rats; Thiamine; Thiamine Deficiency; Vitamin B Deficiency; Vitamins
PubMed: 13731420
DOI: 10.5925/jnsv1954.6.202 -
The Journal of Biological Chemistry Jul 1975The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon...
The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.
Topics: Animals; Apoenzymes; Aspartate Aminotransferases; Binding Sites; Cattle; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Electrophoresis, Polyacrylamide Gel; Ligands; Mathematics; Mitochondria, Liver; Pyridoxal Phosphate; Spectrometry, Fluorescence
PubMed: 1141243
DOI: No ID Found -
Nature Chemical Biology Oct 2013Hydrogenases catalyze the formation of hydrogen. The cofactor ('H-cluster') of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] subcluster...
Hydrogenases catalyze the formation of hydrogen. The cofactor ('H-cluster') of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe] subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H₂-producing catalysts.
Topics: Apoenzymes; Biocatalysis; Catalytic Domain; Coenzymes; Enzyme Activation; Hydrogen; Hydrogenase; Iron; Iron-Sulfur Proteins; Models, Molecular
PubMed: 23934246
DOI: 10.1038/nchembio.1311 -
Plant Physiology Mar 2011Prenyltransferases (PTSs) are involved in the biosynthesis of terpenes with diverse functions. Here, a novel PTS from Arabidopsis (Arabidopsis thaliana) is identified as...
Prenyltransferases (PTSs) are involved in the biosynthesis of terpenes with diverse functions. Here, a novel PTS from Arabidopsis (Arabidopsis thaliana) is identified as a trans-type polyprenyl pyrophosphate synthase (AtPPPS), which forms a trans-double bond during each homoallylic substrate condensation, rather than a homomeric C10-geranyl pyrophosphate synthase as originally proposed. Biochemical and genetic complementation analyses indicate that AtPPPS synthesizes C25 to C45 medium/long-chain products. Its close relationship to other long-chain PTSs is also uncovered by phylogenetic analysis. A mutant of contiguous surface polar residues was produced by replacing four charged surface amino acids with alanines to facilitate the crystallization of the enzyme. The crystal structures of AtPPPS determined here in apo and ligand-bound forms further reveal an active-site cavity sufficient to accommodate the medium/long-chain products. The two monomers in each dimer adopt different conformations at the entrance of the active site depending on the binding of substrates. Taken together, these results suggest that AtPPPS is endowed with a unique functionality among the known PTSs.
Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Apoenzymes; Arabidopsis; Arabidopsis Proteins; Biocatalysis; Biosynthetic Pathways; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Molecular Sequence Data; Phylogeny; Polyisoprenyl Phosphates; Protein Structure, Secondary; Sequence Alignment; Substrate Specificity
PubMed: 21220764
DOI: 10.1104/pp.110.168799 -
Clinica Chimica Acta; International... Mar 1976Apoenzyme of aspartate aminotransferase in serum can be reactivated conveniently by addition of 100 mumoles/l pyridoxal phosphate to the reaction mixture, without...
Apoenzyme of aspartate aminotransferase in serum can be reactivated conveniently by addition of 100 mumoles/l pyridoxal phosphate to the reaction mixture, without extending the usual minimum pre-incubation period in the operation of the LKB 8600 reaction rate analyzer. Normal sera contain some apoenzyme, the amount of which, as well as that of holoenzyme, is greatly increased by damage to skeletal muscle. This may be due to direct injury or to the indirect effects of anoxia; e.g., following surgery with extracorporeal circulation. Myocardial infarction also increases the levels of both apo- and holoenzymes, but changes in the two levels follow similar time courses and apo- and holo-aminotransferases disappear from the circulation at similar rates.
Topics: Apoenzymes; Aspartate Aminotransferases; Cardiac Surgical Procedures; Enzyme Activation; Humans; Hypoxia; Kinetics; Muscles; Myocardial Infarction; Pyridoxal Phosphate; Wounds and Injuries
PubMed: 1248153
DOI: 10.1016/0009-8981(76)90256-4 -
European Journal of Biochemistry Mar 19761. Glyceraldehyde-3-phosphate dehydrogenase from bacillus stearothermophilus can be extensively succinylated in the presence of substrates and coenzyme without...
1. Glyceraldehyde-3-phosphate dehydrogenase from bacillus stearothermophilus can be extensively succinylated in the presence of substrates and coenzyme without appreciable loss of activity. 2. The apoenzyme in the absence of substrates is rapidly inhibited by small amounts of succinic anhydride. NAD+, glyceraldehyde-3-phosphate and inorganic phosphate all afford protection from inhibition, and inhibition is slowly reversed in the presence of pyrophosphate at pH 8.5. 3. Kinetic and spectral studies have shown that the specific inhibition is associated with the succinylation of the aliphatic hydroxyl group of a serine or threonine residue. 4. The residue specifically succinylated has been identified as one of the two threonine residues, most probably Thr-150, adjacent to the activ-site cysteine residue in the primary structure. Its unusual reactivity is discussed in relation to the three-dimensional structure of the enzyme. 5. A second residue, a lysine homologous with Lys-212 in the pig muscle enzyme, can be succinylated in both holoenzyme and apoenzyme with no detectable effect upon the enzymic activity.
Topics: Amino Acid Sequence; Apoenzymes; Binding Sites; Geobacillus stearothermophilus; Glyceraldehyde-3-Phosphate Dehydrogenases; Hydrogen-Ion Concentration; Kinetics; NAD; Peptide Fragments; Phosphates; Protein Binding; Succinates; Threonine
PubMed: 4305
DOI: 10.1111/j.1432-1033.1976.tb10195.x -
Nutrition Reviews Apr 1964
Topics: Apoenzymes; Avitaminosis; Biological Clocks; Coenzymes; Humans; Research
PubMed: 14152888
DOI: 10.1111/j.1753-4887.1964.tb04856.x -
Biochemical Society Transactions Feb 1994
Topics: Ammonium Sulfate; Apoenzymes; Apoproteins; Clostridium; Dihydrolipoamide Dehydrogenase; Flavin Mononucleotide; Flavodoxin; Kinetics
PubMed: 8206286
DOI: 10.1042/bst022057s