Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 (PBG) deaminase catalyzes the polymerization of four PBG monopyrrole units into the linear tetrapyrrole hydroxymethylbilane necessary for the formation of chlorophyll and heme in plant cells. Degenerate oligonucleotide primers were designed based on amino acid sequence data (generated by mass spectrometry) for purified PBG deaminase from pea (Pisum sativum L.) chloroplasts. These primers were used in TaqI polymerase-catalyzed polymerase chain reaction (PCR) amplification to produce partial cDNA and nuclear genomic fragments encoding the enzyme. Subsequently, a 1.6-kb cDNA was isolated by screening a cDNA library constructed in lambda gt11 from leaf poly(A)+ RNA with the PCR products. The cDNA encodes an approximately 40-kD polypeptide containing a 46-amino acid NH2-terminal transit peptide and a mature protein of 323 amino acids. The deduced amino acid sequence of the mature pea enzyme is similar to PBG deaminases from other species and contains the conserved arginine and cysteine residues previously implicated in catalysis. Northern blot analysis indicates that the pea gene encoding PBG deaminase is expressed to varying levels in chlorophyll-containing tissues and is subject to light induction.
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PMID:Structure and expression of chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.) isolated by redundant polymerase chain reaction. 751 80

A database search indicated homology between some members of the nitrilase/cyanide hydratase family, Pseudomonas aeruginosa and Rhodococcus erythropolis amidases and several other proteins, some of unknown function. BLOCK and PROFILE searches confirmed these relationships and showed that four regions of the P. aeruginosa amidase had significant homology with corresponding regions of nitrilases. A phylogenetic tree placed the P. aeruginosa and R. erythropolis amidases in a group with nitrilases but separated other amidases into three groups. The active site cysteine in nitrilases is conserved in the P. aeruginosa amidase indicating that Cys166 is the active site nucleophile.
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PMID:Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys166 is predicted to be the active site nucleophile of the catalytic mechanism. 760 22

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 N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We isolated a Saccharomyces cerevisiae gene, termed NTA1, that encodes an amidase (Nt-amidase) specific for N-terminal asparagine and glutamine. Alterations at the putative active-site cysteine of the 52-kDa Nt-amidase inactivate the enzyme. Null nta1 mutants are viable but unable to degrade N-end rule substrates that bear N-terminal asparagine or glutamine. The effects of overexpressing Nt-amidase and other components of the N-end rule pathway suggest interactions between these components and the existence of a multienzyme targeting complex.
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PMID:Yeast N-terminal amidase. A new enzyme and component of the N-end rule pathway. 774 55

Penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) is widely distributed among microorganisms, including bacteria, yeast and filamentous fungi. It is used on an industrial scale for the production of 6-aminopenicillanic acid, the starting material for the synthesis of semi-synthetic penicillins. Its in vivo role remains unclear, however, and the observation that expression of the Escherichia coli enzyme in vivo is regulated by both temperature and phenylacetic acid has prompted speculation that the enzyme could be involved in the assimilation of aromatic compounds as carbon sources in the organism's free-living mode. The mature E. coli enzyme is a periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids, respectively) synthesized as a single cytoplasmic precursor containing a 26-amino-acid signal sequence to direct export to the cytoplasm and a 54-amino-acid spacer between the A and B chains which may influence the final folding of the chains. The N-terminal serine of the B chain reacts with phenylmethylsulphonyl fluoride, which is consistent with a catalytic role for the serine hydroxyl group. Modifying this serine to a cysteine inactivates the enzyme, whereas threonine, arginine or glycine substitution prevents in vivo processing of the enzyme, indicating that this must be an important recognition site for cleavage. Here we report the crystal structure of penicillin acylase at 1.9 A resolution. Our analysis shows that the environment of the catalytically active N-terminal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases. The nearest base to the hydroxyl of this serine is its own alpha-amino group, which may act by a new mechanism to endow the enzyme with its catalytic properties.
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PMID:Penicillin acylase has a single-amino-acid catalytic centre. 781 45

Glucosamine-6-phosphate deaminase is an oligomeric protein composed of six identical 29.7 kDa subunits. Each subunit has four cysteine residues located at positions 118, 219, 228 and 239. We have previously shown that Cys-118 and Cys-239 form a pair of vicinal thiols, the reactivity of which changes with the allosteric transition. The site-directed mutations Cys-->Ser corresponding to the other two cysteine residues have been constructed, as well as some selected multiple mutations involving the four cysteines. Thiol and disulphide measurements on the wild-type and mutant enzymes indicate that thiols from Cys-219 are oxidized and form interchain disulphide bonds. The disulphide-linked dimer was demonstrated by SDS/PAGE. This result is consistent with preliminary crystallographic data and thermal denaturation studies, and strongly suggests that glucosamine-6-phosphate deaminase is a trimer of disulphide-linked dimers. The mutant forms of the deaminase lacking the interchain disulphide bond or the thiol at Cys-228 are both stable hexamers showing the same sensitivity to urea denaturation as the wild-type protein. Furthermore, these Cys-->Ser mutants display the same kinetics and allosteric properties as those already described for the wild-type enzyme.
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PMID:Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides. 824 Feb 71

Excretion of mercapturic acids in the urine is indicative of the formation of electrophiles in the metabolism of xenobiotics. The determination of these mercapturic acids thus may be a useful method to estimate the exposure. We identified the nephrotoxic and mutagenic mercapturic acids N-acetyl-S-(1,2-dichlorovinyl)-L- cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine in the urine of workers exposed to 1,1,2-trichloroethene. A method to quantify these mercapturic acids by gas chromatography-mass spectrometry-selected ion monitoring was developed and appreciable amounts (2.8-3.8 mumole/L were found in human urine samples. Because deacetylation determines notably the amount of the excreted mercapturic acids, the formation of the resulting cysteine S-conjugates was comparably measured in subcellular fractions of rodent and human kidneys; significant species differences in acylase activity were found. The formation of mutagenic and nephrotoxic metabolites during 1,1,2-trichloroethene metabolism mandates a revision of the risk assessment of trichloroethene exposure.
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PMID:Nephrotoxic and genotoxic N-acetyl-S-dichlorovinyl-L-cysteine is a urinary metabolite after occupational 1,1,2-trichloroethene exposure in humans: implications for the risk of trichloroethene exposure. 831 44

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.
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PMID:The peptidase activity of human serum butyrylcholinesterase: studies using monoclonal antibodies and characterization of the peptidase. 842 27

Stereoselective glutathione conjugation and amidase-catalyzed hydrolysis of [(R)- and (S)-]2-bromoisovalerylurea (BIU), yielding bromoisovaleric acid (BI) and urea, have been observed in the rat in vivo and in isolated rat hepatocytes. The metabolism of enantiomeric (R)- and (S)-BIU was presently examined in the single-pass perfused rat liver with varying input concentrations (8-250 microM). Steady-state hepatic extraction ratios for (R)-BIU (0.6) were constant and higher than those for (S)-BIU, whose extraction ratio decreased from 0.36 (8 microM) to 0.23 (236 microM). (R)- and (S)-BIU were excreted unchanged only minimally into bile. [14C-Urea](R)-BIU underwent amidase-catalyzed hydrolysis to yield [14C]urea (15-24% of rate in) and conjugation to form the (S)-glutathionyl conjugate (31-35% of rate in); two metabolites, most likely the cysteinyl and dipeptide conjugates of BIU (10% of rate in), were found. [3H-Isovaleryl](S)-BIU formed much less amidase-hydrolyzed product, [3H]BI (1-2% of rate in) less (R)-glutathionyl conjugate (9-18% of rate in), but appreciable amounts (14-17% of rate in) of three other metabolites, of which two were most likely the cysteinyl and glycinylcysteinyl conjugates of BIU. When the glutathione conjugation products (glutathione, cysteine and cysteinylglycine conjugates) were summed, the total glutathione conjugation rate for (R)-BIU (44% of rate in) exceeded that for (S)-BIU (34 to 24% of rate in). Fitting of data to the Michaelis-Menten equation revealed similar Km for glutathione conjugation, but a 2-fold higher Vmax for (R)-BIU.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stereoselectivity in glutathione conjugation and amidase-catalyzed hydrolysis of the 2-bromoisovalerylurea enantiomers in the single-pass perfused rat liver. 851 17

A peptide nitrile hydratase activity has been engineered into the cysteine protease papain by a single carefully selected mutation at the active site of the enzyme. The papain variant Gln19Glu hydrolyzes the substrate MeOCO-PheAla-CN to the corresponding amide with a kcat/KM value of 1.15 x 10(3) M-1 s-1. The reaction leads to an accumulation of the corresponding amide, which is then further hydrolyzed to the acid by the natural amidase activity of the enzyme. The pH-dependency of the nitrile hydratase activity of Gln19Glu supports the involvement of the acid form of the Glu19 residue in the reaction. The wild type enzyme displays very weak nitrile hydratase activity, and the introduction of a glutamic acid residue in the oxyanion hole of papain causes the kcat at pH 5 to increase by a factor of at least 4 x 10(5). Peptide nitriles react with cysteine proteases to form thioimidates, and the role of the glutamic acid residue introduced at position 19 in the Gln19Glu enzyme is to participate in the acid-catalyzed hydrolysis of the thiomidate to the amide by the provision of a proton to form the more reactive protonated thioimidate. This dramatically decreases the energy barrier for the hydrolysis of the thioimidate, as shown by the impressive increase in kcat. The success of the rational approach undertaken is a consequence of the level of understanding of the basic catalytic properties of cysteine proteases of the papain family.
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PMID:Engineering nitrile hydratase activity into a cysteine protease by a single mutation. 884 64


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