Biomaterial Database

5-Aminolaevulinic acid dehydratase: characterization of the alpha and beta metal-binding sites of the Escherichia coli enzyme. 1994

P Spencer, and P M Jordan

School of Biological Sciences, Queen Mary and Westfield College, University of London, UK.

The alpha and beta metal-binding sites of 5-aminolaevulinic acid dehydratase (ALAD) (porphobilinogen synthase, EC 4.2.1.24) from Escherichia coli were investigated to determine the function of each metal ion and the role of the reactive cysteines in metal binding. Occupancy of the alpha site by Zn2+ restored virtually all catalytic activity to the inactive metal-depleted ALAD (apoALAD). Occupancy of the alpha site by Co2+ also yielded an active enzyme and resulted in a charge-transfer band indicative of a single cysteine amongst the metal ligands. Subsequent labelling of this cysteine residue with 14C-labelled N-ethylmaleimide, followed by peptide analysis, indicated the involvement of Cys-130. The metal ion at the alpha site is thought to be essential for binding of the second molecule of substrate at the A substrate-binding site that forms the acetic acid side of the product, porphobilinogen. Binding of Zn2+ to the beta site restored little activity if the alpha site was unfilled. Metal ion binding to the beta site could be monitored by following the change in protein fluorescence with Zn2+ titration of apoALAD at pH 6. A conformational change upon beta site occupancy may explain why binding of Mg2+ at the alpha site can occur only if Zn2+ is bound at the beta site. The binding of Co2+ at the beta site produced an inactive enzyme that exhibited a charge-transfer band indicative of at least three cysteine ligands.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D003035	Cobalt	A trace element that is a component of vitamin B12. It has the atomic symbol Co, atomic number 27, and atomic weight 58.93. It is used in nuclear weapons, alloys, and pigments. Deficiency in animals leads to anemia; its excess in humans can lead to erythrocytosis.	Cobalt-59,Cobalt 59
D004789	Enzyme Activation	Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.	Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006863	Hydrogen-Ion Concentration	The normality of a solution with respect to HYDROGEN ions; H+. It is related to acidity measurements in most cases by pH	pH,Concentration, Hydrogen-Ion,Concentrations, Hydrogen-Ion,Hydrogen Ion Concentration,Hydrogen-Ion Concentrations
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000623	Porphobilinogen Synthase	An enzyme that catalyzes the formation of porphobilinogen from two molecules of 5-aminolevulinic acid. EC 4.2.1.24.	Aminolevulinate Hydro-Lyase,Aminolevulinic Acid Dehydratase,ALA-Dehydrase,delta-Aminolevulinate Dehydratase,delta-Aminolevulinic Acid Dehydratase,ALA Dehydrase,Acid Dehydratase, Aminolevulinic,Acid Dehydratase, delta-Aminolevulinic,Aminolevulinate Hydro Lyase,Dehydratase, Aminolevulinic Acid,Dehydratase, delta-Aminolevulinate,Dehydratase, delta-Aminolevulinic Acid,Hydro-Lyase, Aminolevulinate,Synthase, Porphobilinogen,delta Aminolevulinate Dehydratase,delta Aminolevulinic Acid Dehydratase
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D015032	Zinc	A metallic element of atomic number 30 and atomic weight 65.38. It is a necessary trace element in the diet, forming an essential part of many enzymes, and playing an important role in protein synthesis and in cell division. Zinc deficiency is associated with ANEMIA, short stature, HYPOGONADISM, impaired WOUND HEALING, and geophagia. It is known by the symbol Zn.