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The Biochemical Journal Mar 1986Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of...
Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase'. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.
Topics: Alcohol Oxidoreductases; Apoenzymes; Apoproteins; Calcium; Enzyme Induction; Kinetics; Pseudomonas; Spectrophotometry; Substrate Specificity
PubMed: 3521592
DOI: 10.1042/bj2340611 -
Journal of Biochemistry May 1976The interaction of hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2, 2'-disulfonate (MBAS), with pig heart lipoamide...
The interaction of hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2, 2'-disulfonate (MBAS), with pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3] was investigated. When ANS or MBAS was mixed with the apoenzyme of lipoamide dehydrogenase, the fluorescence quantum yield, of each dye was enhancedd markedly and the emission maxima concurrently shifted to the blue. The quantum yield, 0.038, of ANS bound to the apoenzyme, calculated from the corrected emission spectrum, was eight times higher than that in buffer solution, and the value, 0.0090, for bound MBAS was eighteen times higher than that in buffer solution. Moreover, the absortion bands of both ANS and MBAS shifted to the red upon binding with the apoenzyme. A general feature of the absorption spectra of these dyes observed on changing the solvent from polar to apolar was a red shift of the absorption bands. These results indicate that ANS or MBAS bound to the apoenzyme of lipoamide dehydrogenase is situated in a hydrophobic region of the apoenzyme molecule. It was found that 2 moles of each dye was bound per mole of the apoenzyme, which contains two polypeptide chains. The dissociation constants for the ANS- and MBAS-apoenzyme complexes were estimated to be 1.03X10(-5) and 1.54X10(-5) M, respectively. The enhanced fluorescence of both dyes bound to the apoenzyme decreased linearly upon adding FAD and disappeared at about 2 moles of FAD per mole of the apoenzyme. This suggests that both ANS and MBAS were displaced from their binding sites on the apoenzyme by FAD. The protein fluorescence spectrum of the apoenzyme had a maximum at 352 nm, which was blue-shifted by 6 nm from that of tryptophan in the buffer. Upon binding ANS or MBAS, the maximum of the protein fluorescence of the apoenzyme returned to 350 nm for the holoenzyme, and the fluorescence intensity decreased. Thus, the conformation around some tryptophan residues was affected by the binding of the dyes. When guanidine hydrochloride (GuHCl) was added to the ANS-apoenzyme complex solution, the enhanced fluorescence due to the bound ANS decreased and the emission maximum concurrently shifted to the red. Further, the maximum of the protein fluorescence of the apoenzyme shifted to the red, indicating the exposure of some tryptophan residues buried in an apolar region of the apoenzyme. Thus the binding of ANS to the apoenzyme was inhibited by protein denaturation due to GuHCL. In contrast, the holoenzyme of lipoamide dehydrogenase did not bind ANS or MBAS at all.
Topics: Anilino Naphthalenesulfonates; Animals; Apoenzymes; Apoproteins; Binding Sites; Dihydrolipoamide Dehydrogenase; Guanidines; Myocardium; Protein Binding; Protein Conformation; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Swine
PubMed: 956145
DOI: 10.1093/oxfordjournals.jbchem.a131164 -
Current Computer-aided Drug Design Sep 2011The design of biological active compounds from the apoenzyme is still a challenging task. Herein a simple yet efficient technique is reported to generate a receptor... (Comparative Study)
Comparative Study
The design of biological active compounds from the apoenzyme is still a challenging task. Herein a simple yet efficient technique is reported to generate a receptor based pharmacophore solely using a ligand-free protein crystal structure. Human leukocyte antigen-related phosphatase (PTP-LAR) is an apoenzyme and a receptor like transmembrane phosphatase that has emerged as a drug target for diabetes, obesity and cancer. The prior knowledge of the active residues responsible for the mechanism of action of the protein was used to generate the LUDI interaction map. Then, the complement negative image of the binding site was used to generate the pharmacophore features. A unique strategy was followed to design a pharmacophore query maintaining crucial interactions with all the active residues, essential for the enzyme inhibition. The same query was used to screen several databases consisting of the Specs, IBS, MiniMaybridge, NCI and an in-house PTP inhibitor databases. In order to overcome the common bioavailability problem associated with phosphatases, the hits obtained were filtered by Lipinski's Rule of Five, SADMET properties and validated by docking studies in Glide and GOLD. These docking studies not only suggest the essential ligand binding interactions but also the binding patterns necessary for the LAR inhibition. The ligand pharmacophore mapping studies further validated the screened protocol and supported that the final screened molecules, presumably, showed potent inhibitory activity. Subsequently, these molecules were subjected to Derek toxicity predictions and nine new molecules with different scaffold were obtained as non-toxic PTP-LAR inhibitors. The present prospective strategy is a powerful technique to identify potent inhibitors using the protein 3D structure alone and is a valid alternative to other structure-based and random docking approaches.
Topics: Apoenzymes; Binding Sites; Chemistry, Pharmaceutical; Crystallography, X-Ray; Drug Design; HLA Antigens; Humans; Models, Molecular; Predictive Value of Tests; Prospective Studies; Quantitative Structure-Activity Relationship; Random Allocation; Receptor-Like Protein Tyrosine Phosphatases, Class 2
PubMed: 21726194
DOI: 10.2174/157340911796504288 -
The American Journal of Clinical... Feb 1978The erythrocyte apoenzyme activities of transketolase, glutathione reductase, and glutamic-pyruvic transaminase were determined in 236 pregnant women during the first or...
The erythrocyte apoenzyme activities of transketolase, glutathione reductase, and glutamic-pyruvic transaminase were determined in 236 pregnant women during the first or second trimester and again during the third trimester. There were no differences in erythrocyte glutathione reductase and erythrocyte glutamic-pyruvic transaminase activities during these two periods. In contrast, erythrocyte transketolase decreased significantly in the third trimester. No statistically significant correlations were found between levels of activity for the various enzymes and dietary intakes of protein, vitamins or calories. The percent of subjects with low erythrocyte transketolase activity (a value one standard deviation or more below the mean initial value) increased significantly during the third trimester. The percent of subjects with low erythrocyte glutamic-pyruvic transaminase activity was significantly reduced during the third trimester although the mean apoenzyme level did not change. Vitamin deficiencies as measured by enzyme stimulation tests tended to occur less frequently among subjects with low enzyme activities but in no instance was there a statistically significant difference. Hence, no association could be found between apoenzyme activity and the incidence of vitamin deficiencies.
Topics: Alanine Transaminase; Apoenzymes; Erythrocytes; Female; Glutathione Reductase; Humans; Pregnancy; Pregnancy Trimester, First; Pregnancy Trimester, Second; Pregnancy Trimester, Third; Transketolase; Vitamin B Deficiency
PubMed: 623040
DOI: 10.1093/ajcn/31.2.202 -
Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme.Journal of Bacteriology Aug 1990Urease was purified from recombinant Klebsiella aerogenes which was grown in the absence of nickel. The protein was inactive and contained no transition metals, yet it...
Urease was purified from recombinant Klebsiella aerogenes which was grown in the absence of nickel. The protein was inactive and contained no transition metals, yet it possessed the same heteropolymeric structure as native enzyme, demonstrating that Ni is not required for intersubunit association. Ni did, however, substantially increase the stability of the intact metalloprotein (Tm = 79 degrees C) compared with apoenzyme (Tm = 62 degrees C), as revealed by differential scanning calorimetric analysis. An increased number of histidine residues were accessible to diethyl pyrocarbonate in apourease compared with holoenzyme, consistent with possible Ni ligation by histidinyl residues. Addition of Ni to purified apourease did not yield active enzyme; however, urease apoenzyme was very slowly activated in vivo by addition of Ni ions to Ni-free cell cultures, even after treatment of the cells with spectinomycin to inhibit protein synthesis. In contrast, sonicated cells and cells treated with dinitrophenol or dicyclohexylcarbodiimide were incapable of activating apourease. These results indicate that apourease activation is an energy-dependent process that is destroyed by cell disruption.
Topics: Apoenzymes; Apoproteins; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Kinetics; Klebsiella pneumoniae; Spectinomycin; Thermodynamics; Urease
PubMed: 2142939
DOI: 10.1128/jb.172.8.4427-4431.1990 -
Journal of Molecular Biology Jul 2011Caspase-6 has been identified as a key component in the pathway of neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. It has been the focus...
Caspase-6 has been identified as a key component in the pathway of neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. It has been the focus of drug development for some time, but only recently have structural data become available. The first study identified a novel noncanonical conformation of apo-caspase-6 contrasting with the typical caspase conformation. Then, the structures of both caspase-6 zymogen and the Ac-VEID-CHO peptide inhibitor complex described caspase-6 in the canonical conformation, raising the question of why the intermediate between these two structures (mature apo-caspase-6) would adopt the noncanonical conformation. In this study, we present a new crystal form of the apoenzyme in the canonical conformation by identifying the previous apostructure as a pH-inactivated form of caspase-6. Our new apostructure is further compared to the Ac-VEID-CHO caspase-6 inhibitor complex. The structural comparison allows us to visualize the organization of loops L2, L3, and L4 upon ligand binding and how the catalytic groove forms to accommodate the inhibitor.
Topics: Apoenzymes; Caspase 6; Caspase Inhibitors; Catalytic Domain; Crystallization; Crystallography, X-Ray; Enzyme Activation; Enzyme Inhibitors; Humans; Ligands; Protein Binding; Protein Conformation
PubMed: 21621544
DOI: 10.1016/j.jmb.2011.05.020 -
Biochemistry Jul 1997Porphobilinogen deaminase (PBG-D), an early enzyme of the tetrapyrrole biosynthetic pathway, catalyzes the formation of a tetrapyrrole chain, preuroporphyrinogen, from... (Comparative Study)
Comparative Study
Reconstitution of the holoenzyme form of Escherichia coli porphobilinogen deaminase from apoenzyme with porphobilinogen and preuroporphyrinogen: a study using circular dichroism spectroscopy.
Porphobilinogen deaminase (PBG-D), an early enzyme of the tetrapyrrole biosynthetic pathway, catalyzes the formation of a tetrapyrrole chain, preuroporphyrinogen, from four molecules of porphobilinogen (PBG). The PBG-D apoenzyme is responsible for the autocatalytic synthesis and covalent attachment of a dipyrromethane cofactor at its active site. In this paper an efficient method for the purification of Escherichia coli PBG-D apoenzyme using an affinity chromatography resin is reported. Circular dichroism (CD) spectra of apoenzyme and holoenzyme were recorded and significant differences in both the backbone and aromatic region of the spectra were observed. The differences in the spectra allowed the reconstitution of holoenzyme from purified apoenzyme with PBG and preuroporphyrinogen in solution to be monitored separately by CD. Apoenzyme incubated with preuroporhyrinogen gave a CD spectrum that was much more like the CD spectrum of holoenzyme than apoenzyme incubated with PBG. The results showed clearly that the cofactor was generated much more rapidly from preuroporphyrinogen than from PBG. Changes in the CD spectrum associated with the aromatic side-chain region, in particular the contribution assigned to phenylalanine-62, were found to correlate well with the activity of the reconstituted enzyme. Phenylalanine-62 is located in close proximity to the cofactor and acts as a sensitive probe to active-site changes. The stability of the holoenzyme and apoenzyme were compared with respect to both heat and susceptibility to proteolysis. The results were consistent with a model for the apoenzyme in which, in the absence of the cofactor, the three domains of the protein are held less rigidly together, thereby making the protein more susceptible to heat denaturation and proteolysis. The CD spectrum of the holoenzyme was found to be similar at both pH 5.1 and 7.4, suggesting that the crystal structure, determined at pH 5.1, is likely to be similar at physiological pH values.
Topics: Apoenzymes; Circular Dichroism; Escherichia coli; Humans; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylbilane Synthase; Porphobilinogen; Trypsin; Urobilinogen
PubMed: 9230062
DOI: 10.1021/bi9702602 -
The Journal of Biological Chemistry Jan 1962
Topics: Apoenzymes; Humans; Iodide Peroxidase; Iodides; Oxidoreductases; Thyroid Gland
PubMed: 13860353
DOI: No ID Found -
Scientific Reports May 2016The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the...
The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain.
Topics: Apoenzymes; Calcium-Calmodulin-Dependent Protein Kinases; Dictyostelium; Protein Domains; Protozoan Proteins
PubMed: 27211275
DOI: 10.1038/srep26634 -
Journal of Molecular Biology Mar 2003To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of...
To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.
Topics: Adenosine; Adenosine Triphosphate; Amino Acid Sequence; Animals; Apoenzymes; Binding Sites; Crystallography, X-Ray; Cyclic AMP-Dependent Protein Kinases; Hydrophobic and Hydrophilic Interactions; Mice; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Subunits; Ribose; Static Electricity; Substrate Specificity
PubMed: 12614615
DOI: 10.1016/s0022-2836(02)01446-8