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Advances in Experimental Medicine and... 2003Enzyme activities involved in tryptophan metabolism along the kynurenine pathway were studied in male New Zealand white rabbits. Activities are expressed both as...
Enzyme activities involved in tryptophan metabolism along the kynurenine pathway were studied in male New Zealand white rabbits. Activities are expressed both as specific activity and per g of fresh tissue. Liver tryptophan 2,3-dioxygenase activity (TDO), when assayed in either the absence (holoenzyme) or presence of added haematin (apoenzyme), did not change. Therefore, in rabbit, TDO was present only in holoenzyme form. Small intestine indole 2,3-dioxygenase was significantly higher than liver TDO. Mitochondrial kynurenine 3-monooxygenase was higher in liver than in kidney. Kynureninase activity was similar in both tissues, whereas kynurenine-oxoglutarate transaminase was markedly higher in kidney than in liver. 3-Hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase activities were higher in kidney than in liver. However, the former enzyme showed much higher activity than the latter. These findings suggest that, in rabbit, tryptophan is mainly metabolised along the kynurenine pathway although the apo-TDO enzyme is lacking, as high indole 2,3-dioxygenase activity can obviate this lack.
Topics: Animals; Apoenzymes; Holoenzymes; Intestine, Small; Kidney; Kynurenine; Kynurenine 3-Monooxygenase; Liver; Male; Mixed Function Oxygenases; Rabbits; Tryptophan; Tryptophan Oxygenase
PubMed: 15206765
DOI: 10.1007/978-1-4615-0135-0_55 -
Applied and Environmental Microbiology Jul 2014Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or...
Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.
Topics: Apoenzymes; Arthrobacter; Computer Simulation; Crystallography, X-Ray; Enzyme Stability; Escherichia coli; Herbicides; Hydrolases; Hydrolysis; Kinetics; Models, Molecular; Mutant Proteins; Mutation, Missense; Protein Conformation; Recombinant Proteins; Solubility; Triazines
PubMed: 24771025
DOI: 10.1128/AEM.00916-14 -
The Journal of Neuroscience : the... Sep 1991The apoenzyme of glutamate decarboxylase [enzyme without bound cofactor, pyridoxal 5'-phosphate (pyridoxal-P)] serves as a reservoir of inactive glutamate decarboxylase...
The apoenzyme of glutamate decarboxylase [enzyme without bound cofactor, pyridoxal 5'-phosphate (pyridoxal-P)] serves as a reservoir of inactive glutamate decarboxylase (GAD) that can be activated when additional GABA synthesis is required. We have investigated which of two molecular forms of GAD is present as apoenzyme in synaptosomes and in cortex, caudate nucleus, hippocampus, and cerebellum of rat brain. Endogenous glutamate apodecarboxylase (apoGAD) was labeled by incubating extracts of synaptosomes or punches of each region with 32P-pyridoxal-P, followed by reduction with NaBH4, to link covalently the 32P-pyridoxal-P to GAD. Proteins were separated by SDS-PAGE. Punches from all four brain regions and forebrain synaptosomes contained two forms of GAD with apparent Mrs of 63 and 65 kDa as identified by immunoblotting with four antiGAD sera. Punches and synaptosomes contained a major 32P-pyridoxal-P-labeled band with an apparent Mr of 63 kDa that was stained on immunoblots by the antiGAD serum 1440 and the monoclonal antibody GAD-6, and a minor labeled band at 65 kDa that was stained by the 1440, 6799, and K2 antisera. Synaptosomes contained remarkably few other strongly labeled proteins, but punches contained several other labeled bands. Three additional lines of evidence indicate that the labeled 63-kDa protein is apoGAD: (1) it was purified by immunoaffinity chromatography with the GAD-1 monoclonal antibody; (2) it yielded one major labeled peptide when digested with chymotrypsin, and that peptide appeared identical in peptide-mapping experiments to the labeled active-site peptide isolated from chromatographically prepared rat brain GAD; and (3) its labeling was selectively blocked by 4-deoxypyridoxine 5'-phosphate, a competitive inhibitor of the binding of pyridoxal-P to GAD.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Apoenzymes; Brain; Caudate Nucleus; Cerebellum; Cerebral Cortex; Chromatography, Affinity; Chymotrypsin; Glutamate Decarboxylase; Hippocampus; Immunoblotting; Kinetics; Molecular Weight; Peptide Mapping; Pyridoxal Phosphate; Rats; Synaptosomes
PubMed: 1880546
DOI: 10.1523/JNEUROSCI.11-09-02725.1991 -
Archives of Biochemistry and Biophysics Jan 1997A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with > or =80% yields. The apoenzyme bound one...
A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with > or =80% yields. The apoenzyme bound one FMN per enzyme monomer with a dissociation constant of 0.2 microM at 23 degrees C. The reconstituted holoenzyme was catalytically as active as the native enzyme. FMN binding resulted in 87 and 92% of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD showed any appreciable binding to the cofactor site of the apoenzyme but both flavins were active substrates for the FMN-containing holoenzyme. 2-ThioFMN bound to the cofactor site of the apoenzyme with an affinity similar to that for FMN binding. The holoenzyme reconstituted with 2-thioFMN showed a 509-nm absorption peak, which represents a 19-nm red shift from the corresponding peak of the free flavin, and was catalytically active in using either FMN or 2-thioFMN as a substrate. The holoenzyme showed a concentration dependence in molecular sieve chromatography corresponding to higher apparent molecular weights at higher concentrations. Both the holoenzyme and the apoenzyme was shown at 4 degrees C by equilibrium ultracentrifugation to undergo dimerization with dissociation constants of 1.8 and 3.3 microM, respectively.
Topics: Apoenzymes; FMN Reductase; Kinetics; Macromolecular Substances; NADH, NADPH Oxidoreductases; Protein Binding; Substrate Specificity; Ultracentrifugation; Vibrio
PubMed: 8990272
DOI: 10.1006/abbi.1996.9746 -
European Journal of Biochemistry Jun 1995Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified...
Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues.
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.
Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Apoenzymes; Electron Spin Resonance Spectroscopy; Gram-Negative Aerobic Bacteria; Molecular Sequence Data; PQQ Cofactor; Quinolones; Spectrophotometry, Ultraviolet
PubMed: 7601151
DOI: 10.1111/j.1432-1033.1995.tb20634.x -
European Journal of Biochemistry Jul 2000Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of...
Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of the coding gene into the multicloning site. E. coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies. Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures. Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.
Topics: Apoenzymes; Catalase; Cell Line; Chromatography, Gel; Enzyme Activation; Escherichia coli; Holoenzymes; Molecular Chaperones; Thermus
PubMed: 10866831
DOI: 10.1046/j.1432-1033.2000.01474.x -
Journal of Nutritional Science and... 1979Transketolase, sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase [EC 2.2.1.1], was extracted from pig liver and purified 96-fold by...
Transketolase, sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase [EC 2.2.1.1], was extracted from pig liver and purified 96-fold by ammonium sulfate fractionation, followed by column chromatography using DEAE-cellulose and a Sephadex G-200. Transketolase from pig liver was stable at pH 6.0 and above, whereas it was unstable at lower pH values. It could be resolved into apoenzyme and thiamine pyrophosphate in an acidic medium, in contrast to baker's or brewer's yeast transketolase which resolved in an alkaline solution. All the activity of pig liver transketolase was lost upon incubation at pH 5.0 for two hours even at 0 degrees C but about 40% of the original activity could be restored by the addition of excess thiamine pyrophosphate and CaCl2. Restoration of the activity was achieved effectively at pH 7.6-8.0.
Topics: Animals; Apoenzymes; Apoproteins; Chromatography, Gel; Coenzymes; Hydrogen-Ion Concentration; Liver; Swine; Transketolase
PubMed: 41031
DOI: 10.3177/jnsv.25.175 -
Clinical Chemistry May 1983To investigate the considerable variation in stimulation of serum aminotransferase activities by pyridoxal-5'-phosphate added in vitro, we determined the...
To investigate the considerable variation in stimulation of serum aminotransferase activities by pyridoxal-5'-phosphate added in vitro, we determined the pyridoxal-5'-phosphate content of plasma, using the tyrosine decarboxylase reaction together with the catalytic activities of alanine aminotransferase and aspartate aminotransferase, with and without pyridoxal-5'-phosphate supplementation, within a group of normal human individuals. We found a very significant inverse linear relationship between plasma pyridoxal-5'-phosphate concentration and stimulation of the activities of these enzymes in serum after supplementation with pyridoxal-5'-phosphate. We conclude that the degree of stimulation of the apoenzyme of the two serum aminotransferases clearly depends on the pyridoxal-5'-phosphate concentration in vivo.
Topics: Apoenzymes; Apoproteins; Pyridoxal Phosphate; Stimulation, Chemical; Transaminases; Tyrosine Decarboxylase
PubMed: 6839455
DOI: No ID Found -
Journal of Neurochemistry Jan 1979
Topics: Animals; Apoenzymes; Apoproteins; Brain; Carboxy-Lyases; Glutamate Decarboxylase; Immune Sera; Immunoassay; Kinetics; Rats
PubMed: 759578
DOI: 10.1111/j.1471-4159.1979.tb04537.x -
The Journal of Biological Chemistry Jun 1985The cleavage of an amino-terminal decapeptide from Escherichia coli alkaline phosphatase has been previously described (Roberts, C. H., and Chlebowski, J. F. (1984) J....
The cleavage of an amino-terminal decapeptide from Escherichia coli alkaline phosphatase has been previously described (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733) by this laboratory. The modest reduction in specific activity of the modified enzyme is paralleled by an apparent alteration in the Zn(II) affinity at one of the three active center metal ion binding sites. In contrast to the behavior of the native enzyme, formation of the metal-free apoprotein results in an irreversible loss of catalytic activity; phosphohydrolase activity is not restored on addition of Zn(II) and Mg(II). Differential scanning calorimetry and velocity sedimentation data indicate that the apo form of the modified enzyme exists as a monomer form which, while capable of binding Zn(II) does not readily reassociate to active dimer. Processive cleavage of the amino termini of the dimer by trypsin results in the transient formation of a hybrid dimer consisting of cleaved and uncleaved subunits. This species can be directly observed and isolated by taking advantage of the differential chromatographic mobility of the native "isozymes" and the resulting products. Coupled with improved procedures for the preparation of the modified protein, these data indicate that the amino-terminal modification results in alterations in the subunit interface domain and provides a species (the hybrid dimer) for the investigation of the propagation of these effects.
Topics: Alkaline Phosphatase; Apoenzymes; Apoproteins; Calorimetry, Differential Scanning; Chlorides; Escherichia coli; Kinetics; Macromolecular Substances; Magnesium; Magnesium Chloride; Molecular Weight; Trypsin; Zinc; Zinc Compounds
PubMed: 3889000
DOI: No ID Found