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)

N-Carbamoyl-D-amino acid amidohydrolase was purified 119-fold, with 36% overall recovery from a cell-free extract of Comamonas sp. E222c. The purified enzyme was homogeneous as judged by SDS/PAGE. The relative molecular mass of the native enzyme was 120,000 and that of the subunit was 40,000. The purified enzyme hydrolyzed various N-carbamoyl-D-amino acids to D-amino acids, ammonia and carbon dioxide. N-Carbamoyl-D-amino acids having hydrophobic groups served as good substrates for the enzyme. The Km and Vmax values for N-carbamoyl-D-phenylalanine were 19.7 mM and 13.1 units/mg, respectively, and those for N-carbamoyl-D-p-hydroxyphenylglycine were 13.1 mM and 0.56 units/mg, respectively. The enzyme strictly recognized the configuration of the substrate and only the D-enantiomer of the N-carbamoyl amino acid was hydrolyzed. The enzyme activity was not significantly affected by N-carbamoyl-L-amino acids and ammonia. The enzyme was sensitive to thiol reagents and did not require metal ions for its activity. The enzyme did not hydrolyze N-carbamoyl-beta-alanine or N-carbamoyl-DL-aspartate suggesting that the enzyme is different from the N-carbamoylamide-hydrolyzing enzymes involved in the pyrimidine degradation pathway. The enzyme did not hydrolyze allantoin and allantoic acid, which are intermediates in purine degradation, N-carbamoylsarcosine and citrulline, suggesting that it is a novel N-carbamoylamide amidohydrolase.
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PMID:N-carbamoyl-D-amino acid amidohydrolase from Comamonas sp. E222c purification and characterization. 846 43

Growth promotion and iron transport studies revealed that certain alpha-keto acids generated by amino acid deaminases, by enterobacteria of the Proteus-Providencia-Morganella group (of the tribe Proteeae), show significant siderophore activity. Their iron-binding properties were confirmed by the chrome azurol S assay and UV spectra. These compounds form ligand-to-metal charge transfer bands in the range of 400 to 500 nm. Additional absorption bands of the enolized ligands at 500 to 700 nm are responsible for color formation. Siderophore activity was most pronounced with alpha-keto acids possessing an aromatic or heteroaromatic side chain, like phenylpyruvic acid and indolylpyruvic acid, resulting from deamination of phenylalanine and tryptophan, respectively. In addition, alpha-keto acids possessing longer nonpolar side chains, like alpha-ketoisocaproic acid or alpha-ketoisovaleric acid and even alpha-ketoadipic acid, also showed siderophore activity which was absent or negligible with smaller alpha-keto acids or those possessing polar functional groups, like pyruvic acid, alpha-ketobutyric acid, or alpha-ketoglutaric acid. The fact that deaminase-negative enterobacteria, like Escherichia coli and Salmonella spp., could not utilize alpha-keto acids supports the view that specific iron-carboxylate transport systems have evolved in members of the tribe Proteeae and are designed to recognize ferric complexes of both alpha-hydroxy acids and alpha-keto acids, of which the latter can easily be generated by L-amino acid deaminases in an amino acid-rich medium. Exogenous siderophores, like ferric hydroxamates (ferrichromes) and ferric polycarboxylates (rhizoferrin and citrate), were also utilized by members of the tribe Proteeae.
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PMID:Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases. 847 34

Paecilomyces carneus carboxypeptidase sequentially liberated amino acids from the carboxy-terminus of neurotensin, angiotensin I, bradykinin, and delta sleep-inducing peptide, indicating that the sequential hydrolysis of peptides was limited by the occurrence of intermediates with the structure of -Gly-X (X = L-amino acid), -Pro-X, -X-Gly, and -X-Pro. The enzyme had carboxyamidase and/or amidase activities for the carboxy-terminally amidated peptides. The enzyme essentially acted as a carboxyamidase for the long carboxy-terminally amidated peptides; an amidase became dominant for the substrates in the presence of bulky amino acids such as Arg, Met, Leu, and Phe in the penultimate (P1) and P2 positions, corresponding with the S1 and S2 sites of the enzyme, and the P3 position of carboxy-terminally amidated peptides played a significant role in the action as a carboxyamidase or a amidase.
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PMID:Action of serine carboxypeptidase from paecilomyces carneus on oligopeptides containing carboxy-terminally amidated peptides 940 45

The involvement of tyrosine residues in the allosteric function of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli was first proposed on the basis of a theoretical analysis of the sequence and demonstrated by spectrophotometric experiments. Two tyrosine residues, Tyr121 and Tyr254, were indicated as involved in the mechanism of cooperativity and in the allosteric regulation of the enzyme [Altamirano et al. (1994) Eur. J. Biochem. 220, 409-413]. Tyr121 replacement by threonine or tryptophan altered the symmetric character of the T --> R transition [Altamirano et al. (1995) Biochemistry 34, 6074-6082]. From crystallographic data of the R allosteric conformer, Tyr254 has been shown to be part of the allosteric pocket [Oliva et al. (1995) Structure 3, 1323-1332]. Although it is not directly involved in binding the allosteric activator, N-acetylglucosamine 6-phosphate, Tyr 254 is hydrogen bonded through its phenolic hydroxyl to the backbone carbonyl from residue 161 in the neighboring polypeptide chain. Kinetic and binding experiments with the mutant form Tyr254-Phe of the enzyme reveal that this replacement caused an uncoupling of the homotropic and heterotropic effects. Homotropic cooperativity diminished and the allosteric activation pattern changed from one of the K-type in the wild-type deaminase to a mixed K-V pattern. On the other hand, Tyr254-Trp deaminase is kinetically closer to a K-type enzyme and it has a higher catalytic efficiency than the wild-type protein. These results show that the interactions of Tyr254 are fundamental in coupling binding in the active site to events occurring in the allosteric pocket of E. coli glucosamine 6-P deaminase.
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PMID:Tyr254 hydroxyl group acts as a two-way switch mechanism in the coupling of heterotropic and homotropic effects in Escherichia coli glucosamine-6-phosphate deaminase. 960 Oct 45

The conversion of trypsin into a protease with chymotrypsin-like activity and specificity required substitution of fifteen residues in the S1 site and two surface loops with their chymotrypsin counterparts [Hedstrom,L., Szilagyi,L. and Rutter,W.J. (1992) Science, 255, 1249-1253]. These residues may define a set of general structural determinants of specificity in the trypsin family. In order to test this hypothesis, we have attempted to convert trypsin into a protease with specificity for substrates containing small aliphatic residues by replacing the S1 site and these surface loops with the analogous residues of elastase. Five elastase-like mutant enzymes were constructed with various combinations of these substitutions. Four mutant enzymes catalyze the hydrolysis of MeOSuc-Ala-Ala-Pro-Ala-SBzl more efficiently than the hydrolysis of Suc-Ala-Ala-Pro-Phe-SBzl. This observation indicates that the mutant enzymes have elastase-like esterase specificity. The best mutant, Tr-->E1-2, is a more specific esterase than elastase: the ratio of the values of kcat/Km for MeOSuc-Ala-Ala-Pro-Ala-SBzl and Suc-Ala-Ala-Pro-Phe-SBzl is greater than 160 for Tr-->E1-2 and 50 for elastase. However, the esterase activity of Tr-->E1-2 is 300-fold less than elastase; in addition, Tr-->E1-2 has no measurable amidase activity. Thus these substitutions do not construct a protease with elastase-like activity. These experiments indicate that a unique structural solution is required for each different specificity. Previous work suggested that instability of the S1 site is a major barrier to redesigning the specificity of trypsin. This view is corroborated by preliminary structural studies of Tr-->E1-2. One dimensional 1H NMR spectrum of Tr-->E1-2 suggests that the S1 site and the two surface loops of this mutant trypsin may be disordered.
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PMID:Converting trypsin to elastase: substitution of the S1 site and adjacent loops reconstitutes esterase specificity but not amidase activity. 974 19

With the present study, evidence is provided that prekallikrein (PK) in human plasma might be present in two different states, one of them removed along with IgG on Protein G columns. At a plasma dilution of 1 + 2.5, small amounts of an IgG fraction were left in plasma along with all of the PK. At a dilution of 1 + 11, nearly all IgG was removed. The removal in parallel of part of the PK was shown in immunoblot experiments and confirmed in amidase assays. One monoclonal antibody against PK (13G11) and two preparations of polyclonal antibodies were used for the immunoblot experiments. Different peptide substrates (S-2302, S-2222, Bz-Pro-Phe-Arg-pNA), along with protease inhibitors (soybean trypsin inhibitor, corn trypsin inhibitor, lima bean trypsin inhibitor) were used for the amidase assays. The amidase assays indicated that factors XII and XI were reduced by Protein G columns. In all experiments with extensive removal of IgG, protein recognized by the factor XII light chain mAb C6B7 was removed at the same time. This antibody preparation did not detect purified contact factors, but it did recognize a preparation of purified beta-FXIIa, and also significant amounts of protein present in plasma deficient in factor XII and not detectable in plasma deficient in PK. This protein accordingly seems to be connected with the PK fraction removed with IgG.
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PMID:Part of prekallikrein removed from human plasma together with IgG--immunoblot experiments and functional tests. 1020 98

Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)-induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspensionswith ADA. In contrast, the selective A2a receptor agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS15943. The removal of Ado by ADA or the blockade of its action by CSC or CGS15943, markedly increased the membrane recruitment of cytosolic protein kinase Calpha (PKCalpha), RhoA, and ADP-ribosylation factor (ARF) in response to fMLP. As shown for PLD activity, the stimulatory effect of Ado receptor antagonists on PLD cofactors translocation was abolished by a pretreatment of the cells with ADA. Moreover, the membrane translocation of both PKCalpha, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this effect of the A2a receptor agonist was antagonized by CSC or CGS15943. These data demonstrate that Ado released by neutrophils in the extracellular milieu inhibits PLD activation by blocking membrane association of ARF, RhoA, and PKCalpha through Ado A2a receptor occupancy. (Blood. 2000;95:519-527)
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PMID:Adenosine receptor occupancy suppresses chemoattractant-induced phospholipase D activity by diminishing membrane recruitment of small GTPases. 1062 57

Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.
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PMID:Purification and characterization of a unique alkaline elastase from Micrococcus luteus. 1064 68

Spermatozoa of paddlefish and sturgeon fishes (Acipenseriformes), unlike teleost fish, have an acrosome. The objectives of this study were to characterize acrosin-like activity of cryopreserved sperm of paddlefish (Polyodon spathula) and to test and compare stability of paddlefish acrosin-like activity with that of lake sturgeon and bull spermatozoa. Mean acrosin-like activity of cryopreserved paddlefish sperm was 0.372 +/- 0.067 microU/10(6) spermatozoa. This activity was 79% higher in the whole semen than in spermatozoa. Highest activity was recorded at pH 8.0 and 8.5. Triton X-100, zinc ions and 4'-acetamidophenyl 4-guanidinobenzoate (AGB) inhibited the activity. Amidase activity was also inhibited by N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). TLCK at concentrations of 0.1 and 1.0 mM gave a significant decrease in activity of 19 and 61%, respectively. However, TPCK significantly inhibited amidase activity (by 19%) only at concentration 1.0 mM. After acidification and 60 min incubation at 4 degrees C of sperm suspensions only 4% of the activity was retained. A similar phenomenon was observed in the case of lake sturgeon but not bull sperm. These results suggest that trypsin-like activity of Acipenserid fish resembles rather fish trypsin that mammalian one. In frozen-thawed paddlefish sperm a minute chymotrypsin-like activity was also indicated, when GPNA was used as substrate. This activity amounted to 0.0415 +/- 0.0138 microU/10(6) spermatozoa and was 18% of total amidase activity. This suggests that chymotrypsin-like activity may also be present in paddlefish spermatozoa.
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PMID:Characteristics of sperm acrosin-like activity of paddlefish (Polyodon spathula Walbaum). 1081 6

Polyclonal Immunoglobulin (Ig) G from patients with rheumatoid arthritis (RA) and healthy subjects hydrolyzed carbobenzoxy-Val-Gly-Arg p-nitroanilide and D-Pro-Phe-Arg p-nitroanilide. RA IgG exhibited higher activity against the former substrate, but not the latter. On the other hand, RA IgG showed reduced activity against D-Pro-Phe-Arg methylcoumarinamide, when compared with those of the healthy controls. These results suggest that RA IgGs differ from normal IgGs in the substrate specificity of amidase activity. Preliminary studies have shown that two out of three RA IgG samples cleaved a pentapeptide--Gln-Arg-Arg-Ala-Ala--which is assumed to be associated with the risk of developing RA (Gregersen, P. K. et al. (1987), Arthritis Rheum. 30, 1205-1213). By contrast, virtually no cleavage of the same peptide was observed with IgG from healthy controls. A peptide analog, Gln-Arg-Arg-Trp-Ala, was not cleaved at all by any IgGs examined either from RA patients or healthy controls.
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PMID:Amidase and peptidase activities of polyclonal immunoglobulin G present in the sera of patients with rheumatoid arthritis. 1082 53


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