Faculty Opinions recommendation of Coordination environment of a site-bound metal ion in the hammerhead ribozyme determined by 15N and 2H ESEEM spectroscopy.

2007 ◽  
Author(s):  
Thorsten Dieckmann
Keyword(s):  
Metal Ion ◽  
Hammerhead Ribozyme ◽  
Coordination Environment ◽  
A Site

scholarly journals Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by15N and2H ESEEM Spectroscopy

2006 ◽  
Vol 128 (51) ◽  
pp. 16764-16770 ◽  
Author(s):  
Matthew Vogt ◽  
Simanti Lahiri ◽  
Charles G. Hoogstraten ◽  
R. David Britt ◽  
Victoria J. DeRose
Keyword(s):  
Metal Ion ◽  
Hammerhead Ribozyme ◽  
Coordination Environment ◽  
A Site

scholarly journals Characterization of the two 5-aminolaevulinic acid binding sites, the A- and P-sites, of 5-aminolaevulinic acid dehydratase from Escherichia coli

1995 ◽  
Vol 305 (1) ◽  
pp. 151-158 ◽  
Author(s):  
P Spencer ◽  
P M Jordan
Keyword(s):  
Schiff Base ◽  
Binding Sites ◽  
Metal Ion ◽  
Substrate Binding ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Carboxylate Groups ◽  
Catalytic Metal ◽  
A Site ◽  
Aminolaevulinic Acid Dehydratase

Experiments are described in which the individual properties of the two 5-aminolaevulinic acid (ALA) binding sites, the A-site and the P-site, of 5-aminolaevulinic acid dehydratase (ALAD) have been investigated. The ALA binding affinity at the A-site is greatly enhanced (at least 10-fold) on the binding of the catalytic metal ion (bound at the alpha-site). The nature of the catalytic metal ion, Mg2+ or Zn2+, also gave major variations in the substrate Km, P-site affinity for ALA, the effect of potassium and phosphate ions and the pH-dependence of substrate binding. Modification of the P-site by reaction of the enzyme-substrate Schiff base with NaBH4 and analysis of the reduced adduct by electro-spray mass spectrometry indicated a maximum of 1 mol of substrate incorporated/mol of subunit, correlating with a linear loss of enzyme activity. The reduced Schiff-base adduct was used to investigate substrate binding at the A-site by using rate-of-dialysis analysis. The affinity for ALA at the A-site of Mg alpha Zn beta ALAD was found to determine the Km for the reaction and was pH-dependent, with its affinity increasing from 1 mM at pH 6 to 70 microM at pH 8.5. The affinity of ALA at the P-site of Zn alpha An beta ALAD is proposed to limit the Km at pH values above 7, since the measured Kd for ALA at the A-site in 45 microM Tris, pH 8, was well below the observed Km (600 microM) under the same conditions. The amino group of the ALA molecule bound at the P-site was identified as a critical binding component for the A-site, explaining why ALA binding to ALAD is ordered, with the P-site ALA binding first. Structural requirements for ALA binding at the A- and P-sites have been identified: the P-site requires the carbonyl and carboxylate groups, whereas the A-site requires the amino, carbonyl and carboxylate groups of the substrate.

scholarly journals Functional Determinants of Metal Ion Transport and Selectivity in Paralogous Cation Diffusion Facilitator Transporters CzcD and MntE in Streptococcus pneumoniae

2016 ◽  
Vol 198 (7) ◽  
pp. 1066-1076 ◽  
Author(s):  
Julia E. Martin ◽  
David P. Giedroc
Keyword(s):  
Streptococcus Pneumoniae ◽  
Metal Ion ◽  
Transmembrane Domain ◽  
Divalent Metal ◽  
Cation Diffusion ◽  
Metal Specificity ◽  
Cation Diffusion Facilitator ◽  
Content Type ◽  
Metal Selectivity ◽  
A Site

ABSTRACTCation diffusion facilitators (CDFs) are a large family of divalent metal transporters that collectively possess broad metal specificity and contribute to intracellular metal homeostasis and virulence in bacterial pathogens.Streptococcus pneumoniaeexpresses two homologous CDF efflux transporters, MntE and CzcD. Cells lackingmntEorczcDare sensitive to manganese (Mn) or zinc (Zn) toxicity, respectively, and specifically accumulate Mn or Zn, respectively, thus suggesting that MntE selectively transports Mn, while CzcD transports Zn. Here, we probe the origin of this metal specificity using a phenotypic growth analysis of pneumococcal variants. Structural homology toEscherichia coliYiiP predicts that both MntE and CzcD are dimeric and each protomer harbors four pairs of conserved metal-binding sites, termed the A site, the B site, and the C1/C2 binuclear site. We find that single amino acid mutations within both the transmembrane domain A site and the B site in both CDFs result in a cellular metal sensitivity similar to that of the corresponding null mutants. However, multiple mutations in the predicted cytoplasmic C1/C2 cluster of MntE have no impact on cellular Mn resistance, in contrast to the analogous substitutions in CzcD, which do have on impact on cellular Zn resistance. Deletion of the MntE-specific C-terminal tail, present only in Mn-specific bacterial CDFs, resulted in only a modest growth phenotype. Further analysis of MntE-CzcD functional chimeric transporters showed that Asn and Asp in theND-DD A-site motif of MntE and the most N-terminal His in theHD-HD site A of CzcD (the specified amino acids are underlined) play key roles in transporter metal selectivity.IMPORTANCECation diffusion facilitator (CDF) proteins are divalent metal ion transporters that are conserved in organisms ranging from bacteria to humans and that play important roles in cellular physiology, from metal homeostasis and resistance to type I diabetes in vertebrates. The respiratory pathogenStreptococcus pneumoniaeexpresses two metal CDF transporters, CzcD and MntE. How CDFs achieve metal selectivity is unclear. We show here that CzcD and MntE are true paralogs, as CzcD transports zinc, while MntE selectively transports manganese. Through the use of an extensive collection of pneumococcal variants, we show that a primary determinant for metal selectivity is the A site within the transmembrane domain. This extends our understanding of how CDFs discriminate among transition metals.

Spin-labelled Ligands

1974 ◽  
Vol 52 (13) ◽  
pp. 2454-2462 ◽  
Author(s):  
C. T. Cazianis ◽  
D. R. Eaton
Keyword(s):  
Coordination Chemistry ◽  
Unpaired Electron ◽  
Ligand Exchange ◽  
Metal Ion ◽  
Functional Group ◽  
Lewis Base ◽  
Exchange Reactions ◽  
Base Site ◽  
Relative Electron ◽  
A Site

A spin-labelled ligand is defined as a molecule with two functional groups, one of which provides a Lewis base site suitable for complexing to a metal ion. The second functional group comprises a site bearing an unpaired electron. The possible utility of such compounds in coordination chemistry has been explored. The spin density distribution in 4-pyridyl-t-butyl nitroxide provides information on the relative electron accepting abilities of different metal ions in various ligand environments. A method for measuring the rates of very rapid ligand exchange reactions is also suggested.

Comparison of Metal-Ion-Dependent Cleavages of RNA by a DNA Enzyme and a Hammerhead Ribozyme

2002 ◽  
Vol 3 (1) ◽  
pp. 69-83 ◽  
Author(s):  
Qiu-Chen He ◽  
Jing-Min Zhou ◽  
De-Min Zhou ◽  
Yuka Nakamatsu ◽  
Tadashi Baba ◽  
...  
Keyword(s):  
Metal Ion ◽  
Hammerhead Ribozyme

Interaction of neutrophil elastase with hydrophobic polyanionic chelators

1991 ◽  
Vol 69 (9) ◽  
pp. 624-629 ◽  
Author(s):  
Suresh C. Tyagi ◽  
Sanford R. Simon
Keyword(s):  
Calcium Ions ◽  
Neutrophil Elastase ◽  
Metal Ion ◽  
Electrostatic Interactions ◽  
Enzyme Inhibitor ◽  
Human Neutrophil Elastase ◽  
Tetraacetic Acid ◽  
Fura 2 ◽  
A Site ◽  
Substrate Binding Domain

The polyanionic calcium chelators, ethylenediamine-tetraacetic acid (EDTA), ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA), [bis-(O-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 1-[2-amino-5-(6-carboxy-indol-2-yl)phenoxyl]-2-(2′-amino-5′-methylphenoxy)ethane- N,N,N′,N′-tetraacetic acid (INDO-1), 1-[2-(5-carboxyoxazol-2yl)-6-phenoxyl]-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid (FURA-2), and 2-{[2-bis-(carboxymethyl)-amino-5-methylphenoxy]-methyl}-6-methyl-8-bis-(bis-(carboxymethyl)-aminoquinoline (QUIN-2), are all inhibitors of amidolytic activity of human neutrophil elastase (HNE). With MeOSuc-Ala-Ala-Pro-Val-pNA as substrate, these chelators all display mixed partial competitive and partial noncompetitive inhibition, but with the smaller substrate, pGlu-Pro-Val-pNA, only the noncompetitive component persists. The most effective inhibitor is FURA-2, with an apparent Ki of 0.5–0.7 mM. QUIN-2 is somewhat less effective, with a Ki of 2 mM, while EDTA is much less effective, with a Ki of 7 mM. In general, the more hydrophobic chelators are the best inhibitors, although INDO-1, which is about the same size as FURA-2, is surprisingly ineffective as an inhibitor. The chelators no longer function as effective inhibitors if their carboxyl groups are blocked by esterification with acetoxymethyl groups or by complexation with calcium ions, indicating that their binding to HNE is mediated in part through electrostatic interactions with a center of positive charge on the protein. The excitation spectrum of the complex of FURA-2 with HNE differs from that of the chelator with calcium ions, indicating that the structure of the enzyme-inhibitor complex is not like that of the coordination complex of the chelator with the metal ion. The inhibitory capacity of FURA-2 apparently arises from binding to a site that is in the vicinity of the S4 and S5 subsites of the extended substrate binding domain on HNE through a combination of hydrophobic and electrostatic interactions with the enzyme.Key words: elastase, protease inhibitors, chelators, poly anions, calcium.

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