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| 2. |
- Bushnell, Eric A. C., et al.
(författare)
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The first branching point in porphyrin biosynthesis : a systematic docking, molecular dynamics and quantum mechanical/molecular mechanical study of substrate binding and mechanism of uroporphyrinogen-III decarboxylase
- 2011
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Ingår i: Journal of Computational Chemistry. - New York : John Wiley & Sons. - 0192-8651 .- 1096-987X. ; 32:5, s. 822-834
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Tidskriftsartikel (refereegranskat)abstract
- In humans, uroporphyrinogen decarboxylase is intimately involved in the synthesis of heme, where the decarboxylation of the uroporphyrinogen-III occurs in a single catalytic site. Several variants of the mechanistic proposal exist; however, the exact mechanism is still debated. Thus, using an ONIOM quantum mechanical/molecular mechanical approach, the mechanism by which uroporphyrinogen decarboxylase decarboxylates ring D of uroporphyrinogen-III has been investigated. From the study performed, it was found that both Arg37 and Arg50 are essential in the decarboxylation of ring D, where experimentally both have been shown to be critical to the catalytic behavior of the enzyme. Overall, the reaction was found to have a barrier of 10.3 kcal mol−1 at 298.15 K. The rate-limiting step was found to be the initial protontransfer from Arg37 to the substrate before the decarboxylation. In addition, it has been found that several key interactions exist between the substrate carboxylate groups and backbone amides of various activesite residues as well as several other functional groups.
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| 3. |
- Erdtman, Edvin, et al.
(författare)
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Computational insights into the mechanism of porphobilinogen synthase
- 2010
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Ingår i: Journal of Physical Chemistry B. - Washington : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 114:50, s. 16860-16870
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Tidskriftsartikel (refereegranskat)abstract
- Porphobilinogen synthase (PBGS) is a key enzyme in heme biosynthesis that catalyzes the formation of porphobilinogen (PBG) from two 5-aminolevulinic acid (5-ALA) molecules via formation of intersubstrateC-N and C-C bonds. The active site consists of several invariant residues, including two lysyl residues (Lys210 and Lys263; yeast numbering) that bind the two substrate moieties as Schiff bases. Based on experimental studies, various reaction mechanisms have been proposed for this enzyme that generally can be classified according to whether the intersubstrate C-C or C-N bond is formed first. However, the detailed catalytic mechanism of PBGS remains unclear. In the present study, we have employed density functional theory methods in combination with chemical models of the two key lysyl residues and two substrate moieties in order to investigate various proposed reaction steps and gain insight into the mechanism of PBGS. Importantly, it is found that mechanisms in which the intersubstrate C-N bond is formed first have a ratelimiting barrier (17.5 kcal/mol) that is lower than those in which the intersubstrate C-C bond is formed first (22.8 kcal/mol).
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| 5. |
- Erdtman, Edvin, et al.
(författare)
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Computational studies on Schiff-base formation : Implications for the catalytic mechanism of porphobilinogen synthase
- 2011
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Ingår i: Computational and Theoretical Chemistry. - Amsterdam : Elsevier. - 2210-271X .- 2210-2728. ; 963:2-3, s. 479-489
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Tidskriftsartikel (refereegranskat)abstract
- Schiff bases are common and important intermediates in many bioenzymatic systems. The mechanism by which they are formed, however,is dependent on the solvent, pH and other factors. In the present study we have used density functional theory methods in combination with appropriate chemical models to get a better understanding of the inherent chemistry of the formation of two Schiff bases that have been proposed to be involved in the catalytic mechanism of porphobilinogensynthase (PBGS), a key enzyme in the biosynthesis of porphyrins. More specifically, we have investigated the uncatalysed reaction of its substrate 5-aminolevulinic acid (5-ALA) with a lysine residue for theformation of the P-site Schiff base, and as possibly catalysed by the second active site lysine, water or the 5-ALA itself. It is found that cooperatively both the second lysine and the amino group of the initial 5-ALA itself are capable of reducing the rate-limiting energy barrier to14.0 kcal mol-1. We therefore propose these to be likely routes involved in the P-site Schiff-base formation in PBGS.
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| 7. |
- Sekhar, Sreeja C., et al.
(författare)
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A H2AX-CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage
- 2019
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Ingår i: Cancers. - : MDPI. - 2072-6694. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- Cell Cycle and Apoptosis Regulatory Protein (CARP-1/CCAR1) is a peri-nuclear phosphoprotein that regulates apoptosis via chemotherapeutic Adriamycin (doxorubicin) and a novel class of CARP-1 functional mimetic (CFM) compounds. Although Adriamycin causes DNA damage, data from Comet assays revealed that CFM-4.16 also induced DNA damage. Phosphorylation of histone 2AX (gamma H2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Adriamycin or CFM-4.16 treatments inhibited cell growth and caused elevated CARP-1 and gamma H2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and gamma H2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished gamma H2AX, their co-localization, and apoptosis in CFM-4.16- or Adriamycin-treated cells. We found that CARP-1 directly binds with H2AX, and H2AX interacted with CARP-1, but not CARP-1 (Delta 600-652) mutant. Moreover, cells expressing CARP-1 (Delta 600-652) mutant were resistant to apoptosis, and had diminished levels of gamma H2AX, when compared with cells expressing wild-type CARP-1. Mutagenesis studies revealed that H2AX residues 1-35 harbored a CARP-1-binding epitope, while CARP-1 amino acids 636-650 contained an H2AX-interacting epitope. Surface plasmon resonance studies revealed that CARP-1 (636-650) peptide bound with H2AX (1-35) peptide with a dissociation constant (K-d) of 127 nM. Cells expressing enhanced GFP (EGFP)-tagged H2AX (1-35) peptide or EGFP-tagged CARP-1 (636-650) peptide were resistant to inhibition by Adriamycin or CFM-4.16. Treatment of cells with transactivator of transcription (TAT)-tagged CARP-1 (636-650) peptide resulted in a moderate, statistically significant abrogation of Adriamycin-induced growth inhibition of cancer cells. Our studies provide evidence for requirement of CARP-1 interaction with H2AX in apoptosis signaling by Adriamycin and CFM compounds.
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