Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Porphobilinogen deaminase has been purified and crystallized from an overproducing recombinant strain of Escherichia coli harbouring a hemC-containing plasmid which has permitted the purification of milligram quantities of the enzyme. Determination of the Mr of the enzyme by SDS/polyacrylamide-gel electrophoresis (35,000) and gel filtration (32,000) agrees with the gene-derived Mr of 33,857. The enzyme has a Km of 19 +/- 7 microM, an isoelectric point of 4.5 and an N-terminal sequence NH2-MLDNVLRIAT. The substrate, porphobilinogen, binds to the active-site dipyrromethane cofactor to form three intermediate complexes: ES, ES2 and ES3. The gene-derived primary structure of the E. coli deaminase is compared with that derived from the cDNA of the human enzyme.
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PMID:Purification, crystallization and properties of porphobilinogen deaminase from a recombinant strain of Escherichia coli K12. 305 34

The active site of porphobilinogen (PBG)1 deaminase (EC 4.3.1.8) from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-224, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA- strain of E. coli the enzyme was enriched from [5-13C]ALA and examined by 1H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked head to tail and terminating in a CH2-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-[2,11-13C2]PBG reveals that the aninomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the alpha-free pyrrole. NMR spectroscopy of the ES2 complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the alpha-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.
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PMID:Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase. 306 24

Porphobilinogen deaminase from Escherichia coli becomes progressively more susceptible to inactivation by the thiophilic reagent N-ethylmaleimide (NEM) as the catalytic cycle proceeds through the enzyme-intermediate complexes ES, ES2, ES3, and ES4. Site-directed mutagenesis of potentially reactive cysteines has been used to identify cysteine-134 as the key residue that becomes modified by the reagent and leads to inactivation. Since cysteine-134 is buried at the interface between domains 2 and 3 of the E. coli deaminase molecule, the observations suggest that a stepwise conformational change occurs between these domains during each stage of tetrapyrrole assembly. Interestingly, mutation of the invariant active-site cysteine-242 to serine leads to an enzyme with up to a third of the catalytic activity found in the wild-type enzyme. Electrospray mass spectrometry indicates that serine can substitute for cysteine as the dipyrromethane cofactor attachment site.
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PMID:Evidence for conformational changes in Escherichia coli porphobilinogen deaminase during stepwise pyrrole chain elongation monitored by increased reactivity of cysteine-134 to alkylation by N-ethylmaleimide. 766 87

The biosynthesis of the uroporphyrinogen III macrocycle from porphobilinogen requires the sequential participation of two enzymes--porphobilinogen deaminase (1-hydroxymethylbilane synthase, EC 4.3.1.8) and uroporphyrinogen III synthase (cosynthase, EC 4.2.1.75). The product of the deaminase-catalysed reaction is a highly unstable 1-hydroxymethylbilane called preuroporphyrinogen which acts as the substrate for the uroporphyrinogen III synthase, resulting in the exclusive formation of uroporphyrinogen III. In the absence of the synthase, preuroporphyrinogen cyclizes spontaneously to give uroporphyrinogen I. Porphobilinogen deaminase contains a dipyrromethane cofactor that acts as a primer onto which the tetrapyrrole chain is built. The assembly process occurs in stages through enzyme-intermediate complexes, ES, ES2, ES3 and ES4. The negatively charged carboxylates of the cofactor, substrate and intermediate complexes interact with positively charged amino acid side chains in the catalytic cleft. Mutagenesis of conserved arginines has dramatic effects on the assembly of the dipyrromethane cofactor and on the tetrapolymerization process. During the polymerization, the enzyme changes conformation to accommodate the elongating pyrrole chain. The structure of the deaminase from Escherichia coli has been determined by X-ray crystallography at 1.9A resolution and gives important insight into the enzymic mechanism. Aspartate 84 plays a key role in catalysis and its substitution by glutamate reduces kcat by two orders of magnitude.
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PMID:The biosynthesis of uroporphyrinogen III: mechanism of action of porphobilinogen deaminase. 784 63

The assembly process of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase holoenzyme is initiated by the reaction of the porphobilinogen deaminase apoenzyme with preuroporphyrinogen. The resulting enzyme-bound tetrapyrrole (bilane) is equivalent to the holoenzyme intermediate complex ES2 and yields the dipyrromethane cofactor by reactions of the normal catalytic cycle. These observations indicate that preuroporphyrinogen, rather than porphobilinogen, is the preferred precursor for the dipyrromethane cofactor and explain the existence of the D84A and D84N deaminase mutants as catalytically inactive ES2 complexes.
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PMID:Discovery that the assembly of the dipyrromethane cofactor of porphobilinogen deaminase holoenzyme proceeds initially by the reaction of preuroporphyrinogen with the apoenzyme. 868 74