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:3.4.16.2 (PCP)
3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A key step in fungal biosynthesis of lysine, enzymatic reduction of alpha-aminoadipate at C6 to the semialdehyde, requires two gene products in Saccharomyces cerevisiae, Lys2 and Lys5. Here, we show that the 31-kDa Lys5 is a specific posttranslational modification catalyst, using coenzyme A (CoASH) as a cosubstrate to phosphopantetheinylate Ser880 of the 155-kDa Lys2 and activate it for catalysis. Lys2 was subcloned from S. cerevisiae and expressed in and purified from Escherichia coli as a full-length 155-kDa enzyme, as a 105-kDa adenylation/peptidyl carrier protein (A/PCP) fragment (residues 1-924), and as a 14-kDa PCP fragment (residues 809-924). The apo-PCP fragment was covalently modified to phosphopantetheinylated holo-PCP by pure Lys5 and CoASH with a Km of 1 microM and kcat of 3 min-1 for both the PCP and CoASH substrates. The adenylation domain of the A/PCP fragment activated S-carboxymethyl-L-cysteine (kcat/Km = 840 mM-1 min-1) at 16% the efficiency of L-alpha-aminoadipate in [32P]PPi/ATP exchange assays. The holo form of the A/PCP 105-kDa fragment of Lys2 covalently aminoacylated itself with [35S]S-carboxymethyl-L-cysteine. Addition of NADPH discharged the covalent acyl-S-PCP Lys2, consistent with a reductive cleavage of the acyl-S-enzyme intermediate. These results identify the Lys5/Lys2 pair as a two-component system in which Lys5 covalently primes Lys2, allowing alpha-aminoadipate reductase activity by holo-Lys2 with catalytic cycles of autoaminoacylation and reductive cleavage. This is a novel mechanism for a fungal enzyme essential for amino acid metabolism.
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PMID:Lysine biosynthesis in Saccharomyces cerevisiae: mechanism of alpha-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5. 1032 Mar 45

The 207-kDa polyketide synthase (PKS) module (residues 1-1895) and the 143-kDa nonribosomal peptidyl synthetase (NRPS) module (1896-3163) of the 350-kDa HMWP1 subunit of yersiniabactin synthetase have been expressed in and purified from Escherichia coli in soluble forms to characterize the acyl carrier protein (ACP) domain of the PKS module and the homologous peptidyl carrier protein (PCP(3)) domain of the NRPS module. The apo-ACP and PCP domains could be selectively posttranslationally primed by the E. coli ACPS and EntD phosphopantetheinyl transferases (PPTases), respectively, whereas the Bacillus subtilis PPTase Sfp primed both carrier protein domains in vitro or during in vivo coexpression. The holo-NRPS module but not the holo-PKS module was then selectively aminoacylated with cysteine by the adenylation domain embedded in the HMWP2 subunit of yersiniabactin synthetase, acting in trans. When the acyltransferase (AT) domain of HMWP1 was analyzed for its ability to malonylate the holo carrier protein domains, in cis acylation was first detected. Then, in trans malonylation of the excised holo-ACP or holo-PCP(3)-TE fragments by HMWP1 showed both were malonylated with a 3:1 catalytic efficiency ratio, showing a promiscuity to the AT domain.
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PMID:Purification, priming, and catalytic acylation of carrier protein domains in the polyketide synthase and nonribosomal peptidyl synthetase modules of the HMWP1 subunit of yersiniabactin synthetase. 1113 31

The HMWP2 subunit of yersiniabactin (Ybt) synthetase, a 230 kDa nonribosomal peptide synthetase (NRPS) making the N-terminus of the Ybt siderophore of Yersinia pestis, has one cysteine-specific adenylation (A) domain, three carrier protein domains (ArCP, PCP1, PCP2), and two heterocyclization domains (Cy1, Cy2). The A domain loads the two PCP domains with cysteines that get heterocyclized by the Cy domains to yield a tricyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) chain lodged in thioester linkage to the PCP2 domain. The interdomain recognition by the Cy1 and Cy2 domains for the three carrier proteins was tested using inactivating mutations at the conserved serine that is phosphopantetheinylated in each carrier domain (S52A, S1439A, and S1977A). These mutant forms of HMWP2 were tested for in trans complementation by carrier protein fragments: holo-ArCPs (S52A), holo-PCP1 and analogues (S1439A), and holo-PCP2 and analogues (S1977A). The S52A mutant tests the recognition of the Cy1 domain for donor acyl-ArCP substrates, while the S1439A mutant tests the specificity of the same Cy1 domain for downstream substrates presented by distinct PCPs. The S1439A likewise tests the recognition of Cy2 for its upstream PCP-tethered acyl donor. The S1977A mutant analogously tests the Cy2 domain for downstream Cys-PCP recognition. In all cases in trans complementation was successful with the carrier protein fragments, allowing kinetic probes of catalytic efficiency for PCP scaffolds and for uncoupling of the condensation and heterocyclization functions of Cy1 and Cy2. Overall, the Cy domains tested showed a definite selectivity for the upstream protein scaffold but were more relaxed toward the downstream acceptor protein. This work points to the importance of protein-protein interactions in mediating directional chain growth in NRPS and presents the first systematic exploration of how the protein scaffolds affect catalytic efficiency.
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PMID:Yersiniabactin synthetase: probing the recognition of carrier protein domains by the catalytic heterocyclization domains, Cy1 and Cy2, in the chain-initiating HWMP2 subunit. 1131 56

Bactrim/Septra is a drug used for treating and preventing PCP (Pneumocystis carinii pneumonia) and toxoplasmosis. However, people with HIV are more likely to develop hypersensitivity reactions to Bactrim/Septra. NAC (N-acetyl-cysteine) is being studied to determine if its detoxifying properties could reduce the risk of hypersensitivity to Bactrim/Septra. However, a Canadian study found no statistically significant difference in the rates of hypersensitivity among the nearly 200 subjects.
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PMID:Study finds NAC fails to prevent Bactrim/Septra hypersensitivity. 1136 23

A novel mucoadhesive drug carrier system has been generated which protects a model polypeptide antigen from degradation by the most abundant intestinal proteases. The enzyme inhibitors antipain, chymostatin and elastatinal, respectively, were covalently attached to the mucoadhesive polymer sodium carboxymethylcellulose (NaCMC) and the inhibitory efficacy of the resulting polymer-inhibitor conjugates was evaluated in vitro. When these inhibitor conjugates were combined with the thiolated polymer polycarbophil-cysteine (PCP-Cys), 95.8 +/- 3.8% (mean +/- SD, n = 3) of the incorporated model antigen ovalbumin (OVA) was protected from enzymatic degradation within 90 min incubation in the presence of an artificial intestinal fluid containing the pancreatic serine proteases trypsin, chymotrypsin and elastase. Replacing the CMC-inhibitor conjugates in the dosage form by unmodified CMC significantly reduced the protective effect to 78.8 +/- 4.7% (mean +/- SD, n = 3), whereas incorporation of the model antigen in a CMC dosage form omitting PCP-Cys protected 72.5 +/- 3.2% (mean +/- SD, n = 3) of OVA from degradation within a 90 min incubation period. Further, the incorporation of PCP-Cys resulted in higher cohesiveness within the dosage form and controlled drug release of the antigen for a time period of more than 9 h. Results suggest that a delivery system combining thiolated polymer and polymer-inhibitor conjugates improves the metabolic stability of the model polypeptide antigen and may therefore be a useful tool for oral protein vaccination.
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PMID:Design and in vitro evaluation of a mucoadhesive oral delivery system for a model polypeptide antigen. 1159 93

The purpose of this study was to evaluate the potential of polycarbophil-cysteine conjugates (PCP-Cys) as an oral excipient to protect leucine enkephalin (leu-enkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 microM (pH 6) significantly (p < 0.05) inhibited aminopeptidase N activity, and PCP-Cys (0.25% w/v, pH 6) had a significantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP-Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 +/- 4 and 60 +/- 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP-Cys (0.25% w/v, pH 6) 11 +/- 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides.
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PMID:Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N. 1174 48

In order to understand the thermodynamic and kinetic basis of the intrinsic stability of proteins from hyperthermophiles, the folding-unfolding reactions of cysteine-free pyrrolidone carboxyl peptidase (Cys142/188Ser) (PCP-0SH) from Pyrococcus furiosus were examined using circular dichroism (CD) and differential scanning calorimetry (DSC) at pH 2.3, where PCP-0SH exists in monomeric form. DSC showed a strong dependence of the shape and position of the unfolding profiles on the scan rate, suggesting the stability of PCP-0SH under kinetic control. On DSC timescales, even at a scan rate of 1 deg. C/hour, heat denaturation of PCP-0SH was non-equilibrium. However, over a long period of incubation of the heat-denatured PCP-0SH at pre-transition temperatures, it refolded completely, indicating reversibility with very slow relaxation kinetics. The rates of refolding of the heat-denatured PCP-0SH determined from the time-resolved DSC and CD spectroscopic progress curves were found to be similar within experimental error, confirming the mechanism of refolding to be a two-state process. The equilibrium established with a relaxation time of 5080 seconds (at t(m)=46.5 degrees C), which is unusually higher than the relaxation times observed for mesophilic and hyperthermophilic proteins. The long relaxation time may lead to the apparent irreversibility of an unfolding process occurring on the DSC experiment timescale. The refolding rate (9.8 x 10(-5) s(-1)) peaked near the t(m) (=46.5 degrees C), whereas the stability profile reached maxima (11.8 kJ mol(-1)) at 17 degrees C. The results clearly indicate the unusual mode of protein destabilization via a drastic decrease in the rate of folding at low pH and still maintaining a high activation energy barrier (284 kJ mol(-1)) for unfolding, which provides an effective kinetic advantage to unusually stable proteins from hyperthermophiles.
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PMID:The unusually slow relaxation kinetics of the folding-unfolding of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus. 1188 37

The objective of this study was to evaluate the influence of the molecular mass and accordingly the polymer chain length on mucoadhesion and cohesion of thiolated polymers. Linear poly(acrylic acid)-cysteine (PAA-Cys) conjugates of 2-, 45-, 250- and 450 kDa (PAA(2)-Cys, PAA(45)-Cys, PAA(250)-Cys and PAA(450)-Cys) and polycarbophil-cysteine (PCP-Cys, 750-3000 kDa), all displaying on average 404.1+/-65.5 microMol thiol groups per gram polymer were compressed into tablets to perform disintegration tests, mucoadhesion studies and viscosity measurements. Moreover, the influence of free unbound cysteine on mucoadhesion was evaluated. Disintegration tests showed a stability of the tablets as following: PAA(2)-Cys<PAA(45)-Cys<PAA(250)-Cys<PAA(450)-Cys=PCP-Cys. According to tensile studies and tests on the rotating cylinder the following rank order in mucoadhesive properties could be established: PAA(2)-Cys<PAA(45)-Cys<PCP-Cys<PAA(250)-Cys<PAA(450)-Cys. Evidence for the formation of disulphide bonds between thiolated polymers and mucin could be provided by the addition of free cysteine resulting in strongly decreased mucoadhesion and by viscosity studies showing comparatively higher viscosity of conjugate/mucin mixtures than of unthiolated polymer/mucin mixtures. The results of the present study contribute to the development of new polymers displaying further improved mucoadhesive properties.
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PMID:Mucoadhesive and cohesive properties of poly(acrylic acid)-cysteine conjugates with regard to their molecular mass. 1255 77

Cyclization (Cy) domains in NRPS catalyze the heterocyclization of cysteine and serine/threonine to thiazoline and oxazoline rings. A model system consisting of the first two modules of bacitracin synthetase A fused to the thioesterase (Te) domain of tyrocidine synthetase was constructed (BacA1-2-Te) and shown to be active in production of the heterocyclic IleCys(thiazoline). Based on this model system, the feasibility of Cy domain module fusions was investigated by replacing the BacA2 Cy-A-PCP-module with modules of MbtB and MtaD from the biosynthesis systems of mycobactin and myxothiazol, revealing the formation of novel heterocyclic dipeptides. To dissect the reaction sequence of the Cy domain in peptide bond formation and heterocyclization, several residues of the BacA1-2-Te Cy domain were analyzed by mutagenesis. Two mutants exhibited formation of the noncyclic dipeptide, providing clear evidence for the independence of condensation and cyclization.
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PMID:Rational design of a bimodular model system for the investigation of heterocyclization in nonribosomal peptide biosynthesis. 1512 87

This study was aimed at investigating the potential of a new polycarbophil-cysteine (PCP-Cys)/glutathione (GSH) gel formulation to enhance the permeation of the model drug human growth hormone (hGH) across nasal mucosa in vitro and in vivo. The aqueous nasal gel contained PCP-Cys, GSH, and hGH in a final concentration of 0.3%, 0.5%, and 0.6% (m/v), respectively. In vitro permeation studies were performed in Ussing chambers on freshly excised bovine nasal mucosa using fluorescence-labeled dextran (molecular mass: 4.3 kDa; FD-4) and hGH (FITC-hGH). The release profile of FITC-hGH from the gel formulation and an unmodified PCP control formulation was determined. Furthermore, in vivo studies in rats were performed comparing the PCP-Cys/GSH/hGH gel with PCP/hGH control gel and physiological saline. The permeation of FD-4 and FITC-hGH across the nasal mucosa was improved two-fold and three-fold, respectively, in the presence of PCP-Cys/GSH. The PCP-Cys/GSH/hGH gel and the PCP/hGH control gel showed the same biphasic and matrix-controlled drug release. The nasal administration of the PCP-Cys/GSH/hGH gel formulation to rats resulted in a significantly increased and prolonged hGH plasma concentration-time profile versus unmodified PCP gel and physiological saline. According to these results, PCP-Cys gels might represent a promising new strategy for systemic nasal polypeptide delivery.
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PMID:Thiomers in noninvasive polypeptide delivery: in vitro and in vivo characterization of a polycarbophil-cysteine/glutathione gel formulation for human growth hormone. 1517 58


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