5-Aminolaevulinic Acid Dehydratase: Characterization of the α and β Metal-Binding Sites of the Escherichia Coli Enzyme

2007 ◽  
pp. 50-69 ◽  
Author(s):  
P. Spencer ◽  
P. M. Jordan
Keyword(s):  
Escherichia Coli ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Binding Sites ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Aminolaevulinic Acid Dehydratase

scholarly journals Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli

1994 ◽  
Vol 300 (2) ◽  
pp. 373-381 ◽  
Author(s):  
P Spencer ◽  
P M Jordan
Keyword(s):  
Escherichia Coli ◽  
Metal Ions ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Protein Fluorescence ◽  
Metal Binding Sites ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Aminolaevulinic Acid Dehydratase

Two distinct metal-binding sites, termed alpha and beta, have been characterized in 5-aminolaevulinic acid dehydratase from Escherichia coli. The alpha-site binds a Zn2+ ion that is essential for catalytic activity. This site can also utilize other metal ions able to function as a Lewis acid in the reaction mechanism, such as Mg2+ or Co2+. The beta-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation, although a metal bound at this site only appears to be essential for activity if Mg2+ is to be bound at the alpha-site. The alpha- and beta-sites may be distinguished from one another by their different abilities to bind divalent-metal ions at different pH values. The occupancy of the beta-site with Zn2+ results in a decrease of protein fluorescence at pH 6. Occupancy of the alpha- and beta-sites with Co2+ results in u.v.-visible spectral changes. Spectroscopic studies with Co2+ have tentatively identified three cysteine residues at the beta-site and one at the alpha-site. Reaction with N-ethyl[14C]maleimide preferentially labels cysteine-130 at the alpha-site when Co2+ occupies the beta-site.

scholarly journals Purification and characterization of 5-aminolaevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain

1993 ◽  
Vol 290 (1) ◽  
pp. 279-287 ◽  
Author(s):  
P Spencer ◽  
P M Jordan
Keyword(s):  
Escherichia Coli ◽  
Metal Binding ◽  
Sequence Data ◽  
Protein Chemistry ◽  
Second Molar ◽  
Aminolaevulinic Acid ◽  
Disulphide Bonds ◽  
Acid Dehydratase ◽  
Metal Binding Domain ◽  
Aminolaevulinic Acid Dehydratase

5-Aminolaevulinic acid dehydratase (ALAD) from a recombinant strain of Escherichia coli was purified to homogeneity. The enzyme is a homo-octamer of subunit M(r) 36554 +/- 17. Enzyme activity was dependent on the presence of Zn2+ ions and an exogenous thiol. Two molar equivalents of Zn2+ are bound/mol of subunit under reducing conditions. On exposure to the metal chelator EDTA, the two Zn2+ ions are removed, giving an inactive metal-depleted apo-ALAD. On oxidation of holo-ALAD, two disulphide bonds are formed with the loss of 1 mol of Zn2+/mol of subunit. The formation of the first disulphide led to the loss of catalytic activity. Replacement of the two bound Zn2+ ions with Co2+ resulted in the formation of a green protein with a spectrum indicative of the presence of charge-transfer bands from one or more cysteine-Co2+ ligands. While Mg2+ could not activate apo-ALAD alone, it was able to substitute for the second molar equivalent of bound Zn2+, leading to a further 4-fold stimulation in activity. The four cysteine residues involved in the formation of the two disulphide bonds were identified by protein-chemistry studies and were all located in a region of the protein extending from amino acid residues 120-134. Protein sequence data obtained in the present study has permitted the resolution of several differences between the published gene-derived protein sequences for ALAD from E. coli.

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.

Principles and methods used to grow and optimize crystals of protein–metallodrug adducts, to determine metal binding sites and to assign metal ligands

Metallomics ◽  
2017 ◽  
Vol 9 (11) ◽  
pp. 1534-1547 ◽  
Author(s):  
Irene Russo Krauss ◽  
Giarita Ferraro ◽  
Andrea Pica ◽  
José A. Márquez ◽  
John R. Helliwell ◽  
...  
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Biological Macromolecules ◽  
Metal Ligands ◽  
Metal Binding Sites

Examples from the literature and experience in our own laboratory on the characterization of the interactions between biological macromolecules and metal-based drugs are provided.

Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system

2015 ◽  
Vol 472 (2) ◽  
pp. 205-216 ◽  
Author(s):  
Isabelle Petit-Härtlein ◽  
Kevin Rome ◽  
Eve de Rosny ◽  
Florian Molton ◽  
Carole Duboc ◽  
...  
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Zinc Binding ◽  
Accessory Protein ◽  
Two Component System ◽  
Physiological Characterization ◽  
Metal Binding Sites ◽  
Two Component ◽  
Zinc Resistance

ZraP is an octamer containing four interfacial metal-binding sites contributing to dimer stability. Zinc binding enhances its chaperone properties and zinc-bound ZraP represses the expression of the zraPSR operon. None of the Zra proteins are involved in zinc resistance.

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