Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C1832526 (PCC)
 5,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bicarbonate (HCO3-) causes a significant and reversible stimulation of anion-inhibited electron flow in photosystem II of higher plants and cyanobacteria. To test if selected arginine (Arg) residues are involved in the binding of HCO3-, we utilized oligonucleotide-directed mutagenesis to construct Synechocystis sp. PCC 6803 mutants carrying mutations in Arg residues in the D2 protein. Measurements of oxygen evolution showed that the D2 mutants R233Q (arginine-233----glutamine) and R251S (arginine-251----serine) were 10-fold more sensitive to formate than the wild type. The formate concentration giving half-maximal inhibition of the steady-state oxygen evolution rate was 48 mM, 4.5 mM and 4 mM for the wild type, R233Q and R251S, respectively. Measurements of oxygen evolution in single-turnover flashes confirm that the mutants are more sensitive to formate than the wild type. Measurements of chlorophyll a fluorescence decay kinetics after the second saturating actinic flash indicated that, after formate treatment, the halftime of QA- oxidation was decreased by approximately a factor of 2, 4 and 6 in the wild type, R251S and R233Q, respectively. The recombination rate between QA- and S2 was approx. 2-fold slower in R251S and R233Q than in the wild type. In the presence of 100 mM sodium formate, reactivation of the Hill reaction by bicarbonate showed that the wild type had an apparent Km for bicarbonate of 0.5 mM, while the Km values for R233Q and R251S were 1.4 and 1.5 mM, respectively. We suggest that Arg-233 and Arg-251 in the D2 polypeptide contribute to stabilization of HCO3- binding in Photosystem II.
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PMID:Arginine residues in the D2 polypeptide may stabilize bicarbonate binding in photosystem II of Synechocystis sp. PCC. 190 78

Cytoplasmic granules of cytolytic T lymphocytes (CTLs) contain, in addition to the pore-forming protein perforin, a family of highly homologous serine esterases, granzymes A-H. The serine esterase affinity label diisopropyl fluorophosphate reacts strongly with granzymes A and D, to a lesser extent with B, E, F, G, and H, and not at all with C and F. For granzymes A and D, synthetic substrates have been found. Antibodies raised against granzyme B strongly cross-react with A, G, and H, and antibodies to granzyme D recognize C, E, and F. These antigenic relationships correlate with similarities in the N-terminal amino acid sequences. At least 60% homology is observed between the eight proteins, and all are similar to rat mast cell protease 2. Sequence analysis suggests the identity of granzyme A with a protease predicted from a CTL-specific cDNA clone (H factor) and of granzyme B, G, or H with a protein encoded by the CTL-specific cDNA clone CTLA 1/CCP 1.
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PMID:A family of serine esterases in lytic granules of cytolytic T lymphocytes. 355 42

The PII protein in the cyanobacterium Synechococcus sp. strain PCC 7942 signals the cellular state of nitrogen assimilation relative to CO2 fixation by being phosphorylated at a seryl residue. In this study, we first determined the location of the phosphorylated seryl residue within the PII amino acid sequence. The phosphorylation site exhibits an RXS motif, a recognition sequence characteristic for cyclic AMP-dependent protein serine kinases from eukaryotes. We established an in vitro PII phosphorylation assay to further analyze the PII kinase activity in Synechococcus sp. strain PCC 7942. ATP was used specifically as a phosphoryl donor, and the PII kinase activity was shown to be stimulated by alpha-ketoglutarate. Unlike the PII-modifying uridylyltransferase- and uridylyl-removing enzyme characterized in proteobacteria, the activity of the PII kinase from the cyanobacterium did not respond to glutamine.
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PMID:Phosphorylation of the PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942: analysis of in vitro kinase activity. 759 28

The side chain of residue threonine 65 within the active site of ribulosebisphosphate carboxylase participates in a network of hydrogen bonds and ionic interactions involving the phosphate moiety attached to C-1 of the substrate. This residue was replaced with serine, alanine, and valine in the enzyme from Synechococcus PCC 6301. The mutant enzymes were stable, expressed abundantly by Escherichia coli, and retained the ability to form gel-filterable complexes with the reaction-intermediate analog, 2'-carboxyarabinitol-1,5-bisphosphate. The substitutions reduced the kcat/Km(CO2) (where kcat is the substrate-saturated turnover rate) of the enzyme from 17- to 340-fold with the more radical substitutions causing more severe reductions. The CO2/O2 specificity also deteriorated progressively, the valine replacement causing a 2.3-fold reduction. In concert with these changes, a compound tentatively identified as 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate, the product of beta elimination of the 2,3-enediol(ate) intermediate of the catalytic reaction, appeared among the reaction products in progressively increasing amounts. In the case of the valine substitution, it comprised 13% of the ribulose bisphosphate consumed. The mutant enzymes also partitioned more of their reaction flux to pentulose bisphosphate isomers of ribulose bisphosphate. By contrast, the diversion of carboxylated product to pyruvate, as a result of beta elimination of the three-carbon aci-carbanion intermediate of the carboxylation reaction, was ameliorated by the replacements, the valine mutant showing a 5-fold improvement in this parameter. These observations focus attention on a geometric conflict which exists between the requirements for stabilization of the 5-carbon enediol(ate) and 3-carbon aci-carbanion intermediates. This conflict must be resolved by a change in the angle of the C-1/bridge oxygen bond during each catalytic cycle. The network of hydrogen bonds involving the side chain of threonine 65 must play a crucial role in facilitating reaction of the enediol(ate) with the gaseous substrate and in shepherding this subsequent movement.
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PMID:Mutations of an active site threonyl residue promote beta elimination and other side reactions of the enediol intermediate of the ribulosebisphosphate carboxylase reaction. 813 34

The type A domain of the von Willebrand Factor is found also in the complement proteins factor B (FB), C2, CR3 and CR4, the integrins, collagen types VI, VII, XII and XIV, and other proteins. FB is a component of the alternative pathway of the complement system of immune defence, and is cleaved into the fragments Bb and Ba during complement activation. Bb contains a von Willebrand Factor type A (vWF) domain of unknown secondary structure and a serine proteinase (SP) domain, whereas Ba contains three short consensus repeat/complement control protein (SCR/CCP) domains. Fourier transform infrared (FT-IR) spectroscopy on a recombinant vWF domain and on FB and its Bb and Ba fragments shows a broad amide I band. In H2O buffer, second derivative spectra of the amide I band show subcomponents at 1654 to 1657 cm-1, which is typical of alpha-helix, and at 1676 to 1685 cm-1 and 1636 to 1637 cm-1, which are typical of beta-strand. alpha-Helix was detected in the vWF domain, the Bb fragment and FB, and the proportion of alpha-helix present decreased in that order. This shows that the vWF domain contains appreciable amounts of alpha-helix, while the SP and SCR/CCP domains are almost entirely beta-sheet in their secondary structures. Quantitative integration of the vWF FT-IR spectrum showed that this contained 31% alpha-helix and 36% beta-sheet. In 2H2O buffer, the alpha-helix content in the vWF domain is sensitive to the solvent, while the beta-sheet content is less so. An alignment of 75 vWF type A sequences from 25 proteins was used for averaged secondary structure predictions of the total length of 206 residues by the Robson and Chou-Fasman methods. In support of the FT-IR analysis, a total of at least five well-predicted alpha-helices (35% of residues) and at least five well-predicted beta-strands (21% of residues) were identified by both predictive methods, all of which were interspersed by regions of coil or turn conformations. Eight of the ten predicted alpha-helices and beta-strands form an alternating arrangement with each other. Since the predicted alpha-helices are mostly amphipathic, and since the alpha-helix FT-IR band is sensitive to solvent, the alpha-helices are inferred to be on the protein surface.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The secondary structure of the von Willebrand factor type A domain in factor B of human complement by Fourier transform infrared spectroscopy. Its occurrence in collagen types VI, VII, XII and XIV, the integrins and other proteins by averaged structure predictions. 814 50

We reported earlier [Smart, L. B., Warren, P. V., Golbeck, J. H., & McIntosh, L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1132-1136] that the site-directed conversion of cysteine-565 to serine (C565S) in PsaB of Synechocystis sp. PCC 6803 leads to an accumulation of photosystem I polypeptides and the low-temperature photoreduction of the terminal electron acceptors FA and FB. In this paper, we report the occurrence of a [3Fe-4S]1 + ,0 cluster in dodecyl maltoside-solubilized photosystem I complexes prepared from the C565S mutant. The [3Fe-4S] cluster is reducible with dithionite at pH 6.5, implying a midpoint potential considerably more oxidizing than either FA or FB. Similar to the behavior of FX, the [3Fe-4S] cluster undergoes partial, reversible photoreduction when the complex is illuminated at 15 K, and complete photoreduction when the sample is illuminated during freezing. Contrary to the result expected in the presence of a relatively high-potential FX, there is significant low-temperature and room temperature photoreduction of FA and FB in the C565S complex. Although the FA and FB resonances are more intense when the complex is frozen during illumination, they still account for < 60% of FA and FB found by chemical reduction. When the FA and FB clusters are prereduced with dithionite at pH 10.0, a new set of resonances appear upon illumination at g = 2.015, 1.941, and 1.811, and disappear on subsequent darkness. The species giving rise to this signal is most likely a mixed-ligand [4Fe-4S]2+,1+ cluster located in the FX site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed conversion of cysteine-565 to serine in PsaB of photosystem I results in the assembly of [3Fe-4S] and [4Fe-4S] clusters in Fx. A mixed-ligand [4Fe-4S] cluster is capable of electron transfer to FA and FB. 838 46

Dipeptidyl peptidase II (DPP II) was purified to homogeneity from porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approx. 185,000 and 200,000 on Superdex 200 column chromatography and non-denatured PAGE, respectively, and to be 58,000 and 61,000 on SDS-PAGE in the absence and presence of beta-mercaptoethanol (beta-ME), respectively. These findings suggested that the enzyme is composed of three identical subunits. The enzyme rapidly hydrolyzed the substrates Lys-Ala-MCA and Gly-Pro-MCA at acidic pH. The Km and V(max) values of DPP II at optimal pH (pH 6.0) were 1330 microM and 2.9 mumol/mg per min for Gly-Pro-MCA, and 360 microM and 1.43 mumol/mg per min for Lys-Ala-MCA, respectively. It was strongly inhibited by diisopropylphosphofluoride (DFP), and moderately by 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). These findings suggest that DPP II is a serine peptidase. Furthermore, the enzyme activity was also strongly inhibited by copper ions. The amino-acid sequence of the first 41 residues of the enzyme was determined as Ala1-Ser-Pro-Pro-Glu-Pro-Gly-Phe-Arg- Glu10-Val-Tyr-Phe-Glu-Gln-Leu-Leu-Asp-His-Phe20-Asn-Phe-Glu- Arg-Phe- Gly-Lys-Lys-Thr-Phe30-Arg-Gln-Arg-Phe-Leu-Val-Ser-Asp-Lys-Phe40 -Trp. This sequence showed homology (11.6-30.2%) to the N-terminal amino-acid sequences of cytotoxic cell proteinases (CCP 1-4), granzymes. Other properties of DPP II including pH optimum, pH stability, and heat stability were characterized.
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PMID:Dipeptidyl peptidase II from porcine seminal plasma: purification, characterization, and its homology to granzymes, cytotoxic cell proteinases (CCP 1-4). 864 18

Two [4Fe-4S] clusters, FA and FB, function as terminal electron carriers in Photosystem I (PS I), a thylakoid membrane-bound protein-pigment complex. To probe the function of these two clusters in photosynthetic electron transport, site-directed mutants were created in the transformable cyanobacterium Synechocystis sp. PCC 6803. Cysteine ligands in positions 14 or 51 to FB and FA, respectively, were replaced with aspartate, serine, or alanine, and the effect on the genetic, physiological, and biochemical characteristics of PS I complexes from the mutant strains were studied. All mutant strains were unable to grow photoautotrophically, and compared with wild type, mixotrophic growth was inhibited under normal light intensity. The mutant cells supported lower rates of whole-chain photosynthetic electron transport. Thylakoids isolated from the aspartate and serine mutants have lower levels of PS I subunits PsaC, PsaD, and PsaE and lower rates of PS I-mediated substrate photoreduction compared with the wild type. The alanine and double aspartate mutants have no detectable levels PsaC, PsaD, and PsaE. Electron transfer rates, measured by cytochrome c6-mediated NADP+ photoreduction, were lower in purified PS I complexes from the aspartate and serine mutants. By measuring the P700(+) kinetics after a single turnover flash, a large percentage of the backreaction in the aspartate and serine mutants was found to be derived from A1 and FX, indicating an inefficiency at the FX --> FA/FB electron transfer step. The alanine and double aspartate mutants failed to show any backreaction from [FA/FB]-. These results indicate that the various mutations of the cysteine 14 and 51 ligands to FB and FA affect biogenesis and electron transfer differently depending on the type of substitution, and that the effects of mutations on biogenesis and function can be biochemically separated and analyzed.
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PMID:Strains of synechocystis sp. PCC 6803 with altered PsaC. I. Mutations incorporated in the cysteine ligands of the two [4Fe-4S] clusters FA and FB of photosystem I. 906 76

A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues that ligate the FA and FB iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14DPsaC, C51DPsaC, C14D/C51DPsaC), serine (C14SPsaC, C51SPsaC), and alanine (C14APsaC, C51APsaC), and the properties of FA and FB were characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14DPsaC-PS I and C14SPsaC-PS I complexes showed high levels of photoreduction of FA with g values of 2.045, 1. 944, and 1.852 after illumination at 15 K, but there was no evidence of reduced FB in the g = 2 region. The C51DPsaC-PS I and C51SPsaC-PS I complexes showed low levels of photoreduction of FB with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced FA in the g = 2 region. The presence of FB was inferred in C14DPsaC-PS I and C14SPsaC-PS I, and the presence of FA was inferred in C51DPsaC-PS I and C51SPsaC-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700(+) cation generated by illumination at 77 K. Flash-induced optical absorbance changes at 298 K in the presence of a fast electron donor indicate that two electron acceptors function after FX in the four mutant PS I complexes at room temperature. These data suggest that a mixed-ligand [4Fe-4S] cluster is present in the mutant sites of C14X-PS I and C51X-PS I (where X = D or S), but that the proposed spin state of S = 3/2 renders the resonances undetectable in the g = 2 region. The C14APsaC-PS I, C51APsaC-PS I and C14D/C51DPsaC-PS I complexes show only the photoreduction of FX, consistent with the absence of PsaC. These results show that only those PsaC proteins that contain two [4Fe-4S] clusters are capable of assembling onto PS I cores in vivo.
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PMID:Strains of Synechocystis sp. PCC 6803 with altered PsaC. II. EPR and optical spectroscopic properties of FA and FB in aspartate, serine, and alanine replacements of cysteines 14 and 51. 906 77

The authors previously reported the isolation and partial characterization of a periplasmically located dihydrolipoamide dehydrogenase (LPD) from the cyanobacterium Synechocystis sp. strain PCC 6803. In the present work the gene (lpdA; database accession number Z48564) encoding the apoprotein of this LPD in Synechocystis PCC 6803 has been identified, sequenced and analysed. The lpdA gene codes for a protein starting with methionine, which is post-translationally removed. The mature protein contains an N-terminal serine and consists of 473 amino acids with a deduced molecular mass of 51421 Da (including one FAD). The LPD is an acidic protein with a calculated isoelectric point of 5.17. Comparison of the amino acid sequence of the Synechocystis LPD with protein sequences in the databases revealed that the enzyme shares identities of 31-35% with all 18 LPDs so far sequenced and published. As a first step in determining the role of this cyanobacterial LPD, attempts were made to generate an LPD-free Synechocystis mutant by insertionally inactivating the lpdA gene with a kanamycin-resistance cassette. However, the selected transformants appeared to be heteroallelic, containing both the intact lpdA gene and the lpdA gene inactivated by the drug-resistance cassette. The heteroallelic mutant studied, which had about 50% of the wild-type LPD activity, caused acidification of the growth medium. Growth over a prolonged time was only possible after an increased buffering of the medium. Since it is reported in the literature that inactivation of the pyruvate dehydrogenase complex (PDC) leads to acidosis, a function of the LPD in a cytoplasmic-membrane-associated PDC is conceivable.
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PMID:Characterization of a gene encoding dihydrolipoamide dehydrogenase of the cyanobacterium Synechocystis sp. strain PCC 6803. 938 33


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