The biological sulphation of L-tyrosyl peptides.
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ABSTRACT:
SUBMITTER: Jones JG
PROVIDER: S-EPMC1264805 | biostudies-other | 1966 Jan
REPOSITORIES: biostudies-other
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Project description:1. The kinetics of the enzymic transfer of sulphate from adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate to derivatives of l-tyrosine were investigated with a partially purified enzyme preparation from rat liver. 2. At pH7.5 and 37 degrees C the K(m) values for l-tyrosine methyl ester and adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate are 0.3mm and 8nm respectively. The K(m) value for either substrate is independent of the concentration of the other. The available data are consistent with the sulphation reaction proceeding according to a rapid-equilibrium random Bi Bi mechanism. 3. From the effect of pH on the K(m) and V(max.) values for l-tyrosine methyl ester, tyramine and N-acetyl-l-tyrosine ethyl ester it is concluded that the enzyme is specific for substrate molecules with a free and unprotonated amino group and an un-ionized hydroxyl group. 4. The only ionizing group that can be positively attributed to the enzyme appears to influence the binding of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate and has an apparent pK value of approx. 9.5. It is suggested that this group may be an essential thiol. 5. The enzyme is inhibited by iodoacetamide at pH7.5 and 30 degrees C and this inhibition is prevented by the presence of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate but not by l-tyrosine methyl ester.
| S-EPMC1185502 | biostudies-other
Project description:Tyrosyl radicals are important in long-range electron transfer in several enzymes, but the protein environmental factors that control midpoint potential and electron transfer rate are not well understood. To develop a more detailed understanding of the effect of protein sequence, we have performed 14N and 15N electron spin echo envelope modulation (ESEEM) measurements on tyrosyl radical, generated either in polycrystalline tyrosinate or in its 15N-labeled isotopomer, by UV photolysis. 14N-ESEEM was also performed on tyrosyl radical generated in tyrosine-containing pentapeptide samples. Simulation of the 14N- and 15N-tyrosyl radical ESEEM measurements yielded no significant isotropic hyperfine splitting to the amine or amide nitrogen; the amplitude of the anisotropic, nitrogen hyperfine coupling (0.21 MHz) was consistent with a dipole-dipole distance of 3.0 A. Density functional theory was used to calculate the isotropic and anisotropic hyperfine couplings to the amino nitrogen in four different tyrosyl radical conformers. Comparison with the simulated data suggested that the lowest energy radical conformer, generated in tyrosine at pH 11, has a 76 degrees Calpha-Cbeta-C1'-C2' ring and a -73 degrees C-Calpha-Cbeta-C1' backbone dihedral angle. In addition, the magnitude, orientation, and asymmetry of the nuclear quadrupole coupling tensor were derived from analysis of the tyrosyl radical 14N-ESEEM. The simulations showed differences in the coupling and orientation of the nuclear quadrupole tensor, when the tyrosinate and pentapeptide samples were compared. These results suggest sequence- or conformation-induced changes in the ionic character of the NH bond in different tyrosine-containing peptides.
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Project description:1. An enzyme that catalyses the transfer of sulphate from adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate to l-tyrosine methyl ester and tyramine was purified approx. 70-fold from female rat livers. 2. The partially purified preparation is still contaminated with adenosine 3'-phosphate 5'-sulphatophosphate-phenol sulphotransferase (EC 2.8.2.1), but a partial separation of the two enzymes can be achieved by chromatography on columns of Sephadex G-200 and DEAE-Sephadex. 3. The enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is activated by dithiothreitol, 2-mercaptoethanol and GSH, the degree of activation being more marked with preparations previously stored at 0 or -10 degrees C. In contrast, the enzymic sulphation of p-nitrophenol is inhibited by all three thiols. Again, there is a quantitative difference in the degree of inhibition of the two enzymes by o-iodosobenzoate, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetate. 4. Mixed-substrate experiments support the hypothesis that the enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is separate from that responsible for the sulphation of p-nitrophenol. However, p-nitrophenol is a potent inhibitor of the sulphation of both tyrosyl derivatives whereas these latter compounds have no effect on the sulphation of p-nitrophenol.
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Project description:Tdp1 and Tdp2 are two tyrosyl-DNA phosphodiesterases that can repair damaged DNA resulting from topoisomerase inhibitors and a variety of other DNA-damaging agents. Both Tdp1 and Tdp2 inhibition could hypothetically potentiate the cytotoxicities of topoisomerase inhibitors. This study reports the successful structure-based design and synthesis of new 7-azaindenoisoquinolines that act as triple inhibitors of Top1, Tdp1, and Tdp2. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures establish that modification of the lactam side chain of the 7-azaindenoisoquinolines can modulate their inhibitory potencies and selectivities vs Top1, Tdp1, and Tdp2. Molecular modeling of selected target compounds bound to Top1, Tdp1, and Tdp2 was used to design the inhibitors and facilitate the structure-activity relationship analysis. The monitoring of DNA damage by γ-H2AX foci formation in human PBMCs (lymphocytes) and acute lymphoblastic leukemia CCRF-CEM cells documented significantly more DNA damage in the cancer cells vs normal cells.
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Project description:Antimicrobial peptides are diverse group of biologically active molecules with multidimensional properties. In recent past, a wide variety of AMPs with diverse structures have been reported from different sources such as plants, animals, mammals, and microorganisms. The presence of unusual amino acids and structural motifs in AMPs confers unique structural properties to the peptide that attribute for their specific mode of action. The ability of these active AMPs to act as multifunctional effector molecules such as signalling molecule, immune modulators, mitogen, antitumor, and contraceptive agent makes it an interesting candidate to study every aspect of their structural and biological properties for prophylactic and therapeutic applications. In addition, easy cloning and recombinant expression of AMPs in heterologous plant host systems provided a pipeline for production of disease resistant transgenic plants. Besides these properties, AMPs were also used as drug delivery vectors to deliver cell impermeable drugs to cell interior. The present review focuses on the diversity and broad spectrum antimicrobial activity of AMPs along with its multidimensional properties that could be exploited for the application of these bioactive peptides as a potential and promising drug candidate in pharmaceutical industries.
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Project description:Substances with dual tyrosyl-DNA phosphodiesterase I-topoisomerase I inhibitory activity in one low molecular weight compound would constitute a unique class of anticancer agents that could potentially have significant advantages over drugs that work against the individual enzymes. The present study demonstrates the successful synthesis and evaluation of the first dual Top1-Tdp1 inhibitors, which are based on the indenoisoquinoline chemotype. One bis(indenoisoquinoline) had significant activity against human Tdp1 (IC(50) = 1.52 ± 0.05 μM), and it was also equipotent to camptothecin as a Top1 inhibitor. Significant insights into enzyme-drug interactions were gained via structure-activity relationship studies of the series. The present results also document the failure of the previously reported sulfonyl ester pharmacophore to confer Tdp1 inhibition in this indenoisoquinoline class of inhibitors even though it was demonstrated to work well for the steroid NSC 88915 (7). The current study will facilitate future efforts to optimize dual Top1-Tdp1 inhibitors.
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Project description:Peptides are small biological molecules that are attractive in drug delivery and materials engineering for applications including therapeutics, molecular building blocks and cell-targeting ligands. Peptides are small but can possess complexity and functionality as larger proteins. Due to their intrinsic properties, peptides are able to overcome the physiological and transport barriers presented by diseases. In this review, we discuss the progress of identifying and using peptides to shuttle across biological barriers and facilitate transport of drugs and drug delivery systems for improved therapy. Here, the focus of this review is on rationally designed, phage display peptides, and even endogenous peptides as carriers to penetrate biological barriers, specifically the blood-brain barrier(BBB), the gastrointestinal tract (GI), and the solid tumor microenvironment (T). We will discuss recent advances of peptides as drug carriers in these biological environments. From these findings, challenges and potential opportunities to iterate and improve peptide-based approaches will be discussed to translate their promise towards the clinic to deliver drugs for therapeutic efficacy.
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