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

---

Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A family of mutant amidases has been derived by experimental evolution of the aliphatic amidase of Pseudomonas aeruginosa strain PAC1. Mutation amiE16, in the structural gene for the enzyme, results in the production of the mutant B amidase by strain B6. This strain, unlike the wild-type, can utilize butyramide for growth. Strain B6 gave rise by a single mutational event to strain V9, utilizing valeramide, and strain PhB3, utilizing phenylacetamide. Strain V9 was not itself able to utilize phenylacetamide but gave rise by mutation to the phenylacetamide-utilizing mutant PhV1. Peptide 108 was isolated from chymotryptic digests of mutant amidases from strains B6, PhB3 and PhV1, but could not be detected in chymotryptic digests of the wild-type amidase. The sequence of peptide 108 was established as Met-Arg-His-Gly-Asp-Ile-Phe. Thermolytic digests of mutant amidases from strains B6, PhB3, PhV1 and V9 were compared with digests of the wild-type amidase. A peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val was found in the digest of the wild-type amidase and was replaced in the digests of the mutant amidases by a peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val, Phe. Mutation amiE16 is common to the four mutant enzymes and can be accounted for by the mutation Ser leads to Phe. The sequence of the chymotryptic peptide corresponds with the N-terminal sequence of the amidase protein, and can also be related to the thermolysin peptides. It is concluded that mutation amiE16 is a Ser leads to Phe change at position 7 from the N-terminus and the effect of this on the enzyme conformation is discussed.
...
PMID:Molecular basis of altered enzyme specificities in a family of mutant amidases from Pseudomonas aeruginosa. 11 34

The effect of N10-formyl-H4folate on mitochondrial peptide chain initiation has been studied in isolated mitochondria of Saccharomyces cerevisiae. The addition of N10-formyl-H4-folate strongly stimulates the incorporation of amino acids into mitochondrial protein at both 6 and 15 mm Mg2+. Still higher stimulation (up to 10-fold) has been obtained in the production of de novo synthesized initial peptides, measured as peptidyl puromycin derivatives. The maximum effect is observed at 0.1 mM N10-formyl-H4folate. At 5 mM puromycin, the ratio formylated/unformylated peptides is 3, as shown by electrophoretic analysis. At 10 mM puromycin, the ratio is increased to more than 6. This is due to the presence of deformylase and amidohydrolase activities, which are more effective the longer the initial peptide is synthesized; at increasing puromycin concentrations, progressively shorter peptide chains are formed. Chemically synthesized fMet-puromycin and Met-puromycin are virtually stable when incubated with intact or frozen and thawed mitochondria. More careful kinetic analysis shows an early cessation of the initial peptide formation in the samples without N10-formyl-H4-folate. This indicates that the formylation of methionyl-tRNA formylatable species is an absolute requirement for mitochondrial peptide chain initiation.
...
PMID:Dependence of mitochondrial protein synthesis initiation on formylation of the initiator methionyl-tRNAf. 32 47

A standard procedure for the identification of the N-terminal amino acid in N alpha-acylated proteins has been developed. After exhaustive proteolysis, the amino acids with blocked alpha-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I. Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid. A survey of acylamino acid substrates showed that acylase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl-and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val. Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, N alpha-acyl-derivatives of Arg, Lys or His. Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected. When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle. The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins. When such peptides were treated with 1 N HCl at 110 degrees for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing. The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase.
...
PMID:Studies on N alpha-acylated proteins: the N-terminal sequences of two muscle enolases. 391 71

N alpha-Acyl amino acid releasing enzyme (NAARE), an enzyme cleaving acetylMet-Ala at the Met-Ala bond was purified from rat brain cytosol to apparent homogeneity by salt precipitation, gel filtration, and several steps of ion exchange. Levels of NAARE exceeded acylase measured with acetylmethionine in all brain regions and subcellular fractions examined: 60% was associated with cytosol and the remainder with debris or the crude nuclear and mitochondrial-synaptosomal subfractions. Activity was highest in pituitary and was approximately 0.5-0.6 that of liver or kidney. The purified enzyme preferentially hydrolyzed acetylmethionyl peptides: Km for acetylMet-Ala was 0.93; Vmax, 3.5 nmol-1 (kcat, 1185) with pH optimum of 8.9 as compared with 8.2 for acylases measured in cytosol. The purified enzyme was devoid of acylase and common exo- and endopeptidase contamination. Structure-activity relationships examined with synthetic formylated or acetylated peptides indicated no significant effects for di- or tripeptides if the second substituent was Ala, Ser, Asn, or Thr, but the activity was reduced 0.5-fold for Leu, a branched-chain amino acid. No hydrolysis was observed for polypeptides with five or more residues having N-terminal acetylated Tyr (enkephalin) or Ser (alpha-melanocyte-stimulating hormone, thymosin alpha 1), supporting the notion that the enzyme plays a role only in turnover of smaller peptides formed perhaps as a result of endopeptidase cleavage of proteins or polypeptides containing acetylated Met at the N terminus.
...
PMID:Observations on N alpha-deacetylation of model amino acids and peptides: distribution and purification of a specific N-acyl amino acid releasing enzyme in rat brain. 686 20

The combination method of carboxypeptidase Y digestion and fast atom bombardment (FAB) mass spectrometry is described for the identification of C-terminal amino acid amides in peptides. Carboxypeptidase Y has amidase activity as well as exopeptidase activity in the same digestion buffer condition. Based on this concept, we develop a new technique which can definitively and easily identify the C-terminal amino acid amides. This method obviates the need for several complicated steps occurring in previous methods, but improves sensitivity, and enables exact identification of the amino acid amide by the difference of molecular mass. Analyses of carboxypeptidase Y digested peptides, not liberated free amino acid amides, were carried out by fast atom bombardment mass spectrometry. The use of truncated peptides by fast atom bombardment mass spectrometry in C-terminal amino acid amide determination gives several advantages over analyses of the liberated amino acid amides. The C-terminal amino acid amides of Allantostatin I (Leu-NH2), alpha-Melanocyte Stimulating Hormone (Val-NH2), and Ranatensin (Met-NH2) are unequivocally determined at a level of 0.90-2.3 nmol per peptide. This approach is based on entirely different principles than the previous approaches.
...
PMID:Identification of the C-terminal amino acid amides by carboxypeptidase Y digestion and fast atom bombardment mass spectrometry. 770 6

A cephalosporanic acid acylase from Pseudomonas strain N176 catalyzes hydrolysis of both glutarylcephalosporanic acid and cephalosporin C to 7-amino-cephalosporanic acid. Chemical modification of the enzyme with acidic hydrogen peroxide was performed to investigate residues which play important roles in enzymatic activity. The activity of the enzyme was reduced to 76% of the original by oxidation. From protein chemical analysis combined with site-directed point mutagenesis, modification of Met-164 was found to correspond to the reduction in activity. To study the effect of Met-164 on the enzymatic character, we prepared mutant acylases in which Met-164 was replaced with several other amino acids and obtained the following data: (i) there existed a trend of mutation to noncharged hydrophilic residues, resulting in an increase of activity against glutarylcephalosporanic acid; (ii) the mutation of Met-164 to Gly and Ala resulted in the lowering of both Km values and the optimal pHs against glutarylcephalosporanic acid; (iii) the mutation to Leu enhanced cephalosporin C acylase activity; and (iv) the mutation to Gln improved the k(cat) value for glutarylcephalosporanic acid. In particular, the mutation to Gln resulted in a high rate of conversion of glutarylcephalosporanic acid to 7-amino-cephalosporanic acid under conditions similar to those of a bioreactor system. These results may indicate that Met-164 is located in or near the cephalosporin compound binding pocket on the enzyme.
...
PMID:Oxidative modification of a cephalosporin C acylase from Pseudomonas strain N176 and site-directed mutagenesis of the gene. 870 85

A continuous assay for peptide deformylase has been developed using a formylated dipeptide, formyl-Met-Leu-p-nitroanilide, as substrate. Removal of the formyl group by a peptide deformylase renders the dipeptide product, which contains a free NH2 terminus, a substrate for an aminopeptidase from Aeromonas proteolytica. Sequential hydrolysis of the dipeptide by the aminopeptidase releases a p-nitroaniline, which is monitored spectrophotometrically at 405 nm. This assay was applied to determine the pH optimum and the catalytic activity of a peptide deformylase from Escherichia coli. The E. coli enzyme is most active near neutral pH (pH 7.0) and displays Michaelis-Menten kinetics toward the formylated dipeptide, with K(M) = 20.3 +/- 1.3 microM, k(cat) = 38 +/- 2 s(-1), and k(cat)/K(M) = 1.9 x 10(6) M(-1) s(-1). It also exhibits an acylase activity, capable of deacylating N-acetyl-Met-Leu-p-nitroanilide and N-trifluoroacetyl-Met-Leu-p-nitroanilide, albeit at drastically reduced rates. These results demonstrate that the current assay is a convenient, rapid, and sensitive method for kinetic studies of peptide deformylase. The strategy employed in this work should also be generally applicable to the characterization of other acylases.
...
PMID:Continuous spectrophotometric assay of peptide deformylase. 923 95

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.
...
PMID:Action of serine carboxypeptidase from paecilomyces carneus on oligopeptides containing carboxy-terminally amidated peptides 940 45

Penicillin G acylase is an important enzyme in the commercial production of semisynthetic penicillins used to combat bacterial infections. Mutant strains of Providencia rettgeri were generated from wild-type cultures subjected to nutritional selective pressure. One such mutant, Bro1, was able to use 6-bromohexanamide as its sole nitrogen source. Penicillin acylase from the Bro1 strain exhibited an altered substrate specificity consistent with the ability of the mutant to process 6-bromohexanamide. The X-ray structure determination of this enzyme was undertaken to understand its altered specificity and to help in the design of site-directed mutants with desired specificities. In this paper, the structure of the Bro1 penicillin G acylase has been solved at 2.5 A resolution by molecular replacement. The R-factor after refinement is 0.154 and R-free is 0.165. Of the 758 residues in the Bro1 penicillin acylase heterodimer (alpha-subunit, 205; beta-subunit, 553), all but the eight C-terminal residues of the alpha-subunit have been modeled based on a partial Bro1 sequence and the complete wild-type P. rettgeri sequence. A tightly bound calcium ion coordinated by one residue from the alpha-subunit and five residues from the beta-subunit has been identified. This enzyme belongs to the superfamily of Ntn hydrolases and uses Ogamma of Ser beta1 as the characteristic N-terminal nucleophile. A mutation of the wild-type Met alpha140 to Leu in the Bro1 acylase hydrophobic specificity pocket is evident from the electron density and is consistent with the observed specificity change for Bro1 acylase. The electron density for the N-terminal Gln of the alpha-subunit is best modeled by the cyclized pyroglutamate form. Examination of aligned penicillin acylase and cephalosporin acylase primary sequences, in conjunction with the P. rettgeri and Escherichia coli penicillin acylase crystal structures, suggests several mutations that could potentially allow penicillin acylase to accept charged beta-lactam R-groups and to function as a cephalosporin acylase and thus be used in the manufacture of semi-synthetic cephalosporins.
...
PMID:Crystal structure of penicillin G acylase from the Bro1 mutant strain of Providencia rettgeri. 1054 42

In many GL-7ACA acylases, the first Ser residue at the N-terminal of beta-subunit is the catalytic center. In order to investigate relationship between the N-terminal structure and catalytic activities, peptide replacement and site-directed mutagenesis were performed at the N-terminal of beta-subunit of GL-7ACA acylase C130. When the N-terminal 8 amino acid residues of C130 were replaced by the corresponding sequence of penicillin acylases PAC and PGA, respectively, the first mutant B8PAC lost the activity of the acylase, and the second mutant B8PGA had lower activity with the K(m) value increasing from 0.44x10(-3)mol.L(-1) to 0.55x10(-3) mol.L(-1), and the k(cat) decreasing from 4.92 s(-1) to 1.64 s(-1). Although the substitution of Trp (beta4) by Tyr did not change the K(m) value, the k (cat) decreased to 2.29 s(-1). When the Trp was substitued by Leu, both the K ( m ) and k ( cat ) values decreased. Compared with the wild type, mutations of Ser (beta3) to Met, Ala and Cys caused decrease of K(m) values by 52.27%, 43.18% and 38.64%, respectively. Mutation of Asn (beta2) to Gln caused the K ( m ) value being increased by 5-fold, and k ( cat ) decreased by 10-fold. These results suggested that the N-terminal amino acid residues of beta-subunit in GL-7ACA acylase C130 are important for enzyme function.
...
PMID:Mutagenesis of N-terminal Amino Acid Residues in beta-subunit of Glutaryl-7-amino-cephalosporanic Acid Acylase C130. 1203 60


1 2 Next >>

---