• Title/Summary/Keyword: Site-specificity

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Substrate Ground State Binding Energy Concentration Is Realized as Transition State Stabilization in Physiological Enzyme Catalysis

  • Britt, Billy Mark
    • BMB Reports
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    • v.37 no.5
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    • pp.533-537
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    • 2004
  • Previously published kinetic data on the interactions of seventeen different enzymes with their physiological substrates are re-examined in order to understand the connection between ground state binding energy and transition state stabilization of the enzyme-catalyzed reactions. When the substrate ground state binding energies are normalized by the substrate molar volumes, binding of the substrate to the enzyme active site may be thought of as an energy concentration interaction; that is, binding of the substrate ground state brings in a certain concentration of energy. When kinetic data of the enzyme/substrate interactions are analyzed from this point of view, the following relationships are discovered: 1) smaller substrates possess more binding energy concentrations than do larger substrates with the effect dropping off exponentially, 2) larger enzymes (relative to substrate size) bind both the ground and transition states more tightly than smaller enzymes, and 3) high substrate ground state binding energy concentration is associated with greater reaction transition state stabilization. It is proposed that these observations are inconsistent with the conventional (Haldane) view of enzyme catalysis and are better reconciled with the shifting specificity model for enzyme catalysis.

Modulation of the Metal(loid) Specificity of Whole-Cell Bioreporters by Genetic Engineering of ZntR Metal-Binding Loops

  • Kim, Hyojin;Jang, Geupil;Kim, Bong-Gyu;Yoon, Youngdae
    • Journal of Microbiology and Biotechnology
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    • v.30 no.5
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    • pp.681-688
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    • 2020
  • Bacterial cell-based biosensors, or whole-cell bioreporters (WCBs), are an alternative tool for the quantification of hazardous materials. Most WCBs share similar working mechanisms. In brief, the recognition of a target by sensing domains induces a biological event, such as changes in protein conformation or gene expression, providing a basis for quantification. WCBs targeting heavy metal(loid)s employ metalloregulators as sensing domains and control the expression of genes in the presence of target metal(loid) ions, but the diversity of targets, specificity, and sensitivity of these WCBs are limited. In this study, we genetically engineered the metal-binding loop (MBL) of ZntR, which controls the znt-operon in Escherichia coli. In the MBL of ZntR, three Cys sites interact with metal ions. Based on the crystal structure of ZntR, MBL sequences were modified by site-directed mutagenesis. As a result, the metal-sensing properties of WCBs differed depending on amino acid sequences and the new selectivity to Cr or Pb was observed. Although there is room for improvement, our results support the use of currently available WCBs as a platform to generate new WCBs to target other environmental pollutants including metal(loid)s.

Reactivity of Prototype Foamy Virus Integrase to the Mutants of the Highly Conserved Terminal Sequence of U5 LTR (원조포미바이러스 U5 LTR 말단의 보존적인 잔기의 돌연변이에 대한 인테그라제의 반응성)

  • Hyun, U-Sok;Lee, Dong-Hyun;Ko, Hyun-Tak;Shin, Cha-Gyun
    • YAKHAK HOEJI
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    • v.52 no.2
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    • pp.125-130
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    • 2008
  • The long terminal repeat (LTR) of retroviral DNA genome plays an important role in the integration process by providing substrate recognition site for viral integrase (IN). The dinucleotide CA near the 3'-end of the LTR termini is completely conserved among retoviruses. In order to study specificity of interaction between prototype foamy virus (PFV) IN and its U5 LTR DNA, the effect of mutagenesis of the CA sequence was investigated by studying reactivity of PFV IN to the mutant LTR substrates. Replacement of only the C or the A allowed 60 to 100% of the reactivity of the wild type LTR substrate. In addition, replacement of the C and the A showed 50 to 80% of the reactivity of the wild type LTR substrate, indicating that PFV IN has less specificity on the conserved CA sequence when it is compared to the other retroviral INs. Therefore it is suggested that PFV IN is less dependent on the conserved sequence of LTR termini for its enzymatic reaction.

Base Specificity for DNA Interstrand Cross-Linking Induced by Anticancer Agent Bizelesin

  • Lee, Chong-Soon;Myung, Pyung-Keun;Gibson, Neil W.
    • Archives of Pharmacal Research
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    • v.19 no.3
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    • pp.191-196
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    • 1996
  • Bizelesin is a promising novel anticancer agent which is known to alkylate N3 of adenine to induce DNA interstrand cross-links (ISC) with in $5^I-TAATTA\; and\; 5^I-TAAAAAA$. We have investigated the base specificity for DNA ISC induced by bizelesin using oligomers containing the cross-linkable sequence $5^I-TAATTA\; and\; 5^I-TAAAAAA$. in which "N" was either A, C, G, or T. An analysis of denaturing polyacrylamide gel showed that bizelesin is able to induce DNA ISC in the duplex oligomer containing sequences $5^I-TAATTA\; and\; 5^I-TAAAAAA$. The formation of interstrand crosslinking did not occur in the sequences $5^I-TAATTA\; and\; 5^I-TAAAAAA$. DNA strand cleavage assay to determine the cross-linking site within $5^I-TAATTA$sequence showed that bizelesin alkylates guanine. These results demonstrate that bizelesin is able to induce DNA ISC at guanine but not at cytosine or thymine. In addition, guanine adducts have been found to be susceptible to DNA strand cleavage by exposure to hot piperidine. The extent of DNA strand cleavage, however, was not 100% efficient in either neutral pH buffer or hot piperidine.

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Influence of Coating Ligands onf Enzyme-linked Immunosorbent Assay of Toluene

  • Kim, So Yeong;Lee, Nam Taek;Choe, Myeong Ja
    • Bulletin of the Korean Chemical Society
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    • v.22 no.9
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    • pp.953-957
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    • 2001
  • The specificity of conjugation site for coating ligands was investigated using toluene-bovine serum albumin (BSA) conjugates in which BSA was conjugated at the position of o-, m-, and ${\rho}-toluic$ acid. Toluene-BSA conjugated at ${\rho}-position$ showed a binding activity of about 89-95%, whereas, those conjugated at o- and m-position of toluene exhibited a binding activity of 5 and 11%, respectively. On the basis of the above result, coating ligands with various proteins (OVA, BSA, KLH) were compared by conjugating with $\rho-toluic$ acid, and toluene-KLH was considered as the best coating ligand for this ELISA. Indirect competitive ELISA method was developed using anti-toluene antibody and $\rho-position$ conjugated toluene-KLH. The dose-response curve constructed after kinetic and optimization studies showed a 1${\times}$10-4 - 1${\times}$102 mM detectable response range with 0.1 ${\mu}M$ detectability. In specificity test of the antibody, the binding capabilities of aromatic compounds substituted with nitro-, alkyl-, chloro-, and hydroxyl group were larger rather than those of aromatic compounds (benzene, toluene and xylene) themselves. Also, tests with soil and water samples that had been spiked with toluene resulted in 102.7-113.7% recovery.

Molecular determinants of the host specificity by Xanthomonas spp.

  • Heu, Sunggi;Choi, Min-Seon;Park, Hyoung-Joon;Lee, Seung-Don;Ra, Dong-Soo
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2004.10a
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    • pp.65-67
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    • 2004
  • During initial interactions of bacteria with their host plants, most plants recognize the bacterial infections and repel the pathogen by plant defense mechanism. The most active plant defense mechanism is the hypersensitive response (HR) which is the localized induced cell death in the plant at the site of infection by a pathogen. A primary locus induced in gram-negative phytopathogenic bacteria during this initial interaction is the Hrp locus. The Hrp locus is composed of a cluster of genes that encodes the bacteral Type 111 machinery that is involved in the secretion and translocation of effector proteins to the plant cell. DNA sequence analysis of hrp gene in phytopathogenic bacteria has revealed a Hrp pathogenicity is]and (PAI) with a tripartite mosaic structure. For many gram-negative pathogenic bacteria, colonization of the host's tissue depends on the type III protein secretion system (TTSS) which secrets and translocates effector proteins into the host cell. Effectors can be divided into several groups including broad host range effectors, host specific effectors, disease specific effectors, and effectors inhibit host defenses. The role of effectors carrying LRR domain in plant resistance is very elusive since most known plant resistance gene carry LRR domain. Host specific effectors such as several avr gene products are involved in the determination of the host specificity. Almost all the phytopathogenic Xanthomonas spp. carry avrBs1, avrBs2, and avrBs3 homologs. Some strains of X. oryzae pv. oryzae carry more than 10 copies of avrBs3 homologs. However, the functions of all those avr genes in host specificity are not characterized well.;

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The Evaluation of Historical and Cultural Authenticity of the Three Major Cultural Areas Projects - Targeting Korean culture theme park in Yeongju-Zone and World confucian scholar culture park in Bonghwa-Zone - (3대문화권 사업 계획의 역사문화적 진정성 평가 - 한국문화테마파크 영주지구와 세계유교선비문화공원 봉화지구를 대상으로 -)

  • Jung, Kyoung-Ah;So, Hyun-Su
    • Journal of Korean Society of Rural Planning
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    • v.22 no.1
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    • pp.25-35
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    • 2016
  • This study targets Korean culture theme park in Yeongju-Zone and World confucian scholar culture park in Bonghwa-Zone among the Three Major Cultural Areas Projects supervised by Ministry of Culture, Sports and Tourism. The study analyzes the Historical and Cultural Authenticity of each project on the basis of the reports and the report materials for meeting with related organizations. The results of the study are drawn as follows. First, through the theoretical consideration, this study drew the types of authenticity: originality, identity, specificity and visibility, which are evaluation items on the Historical and Cultural Authenticity. Second, Bonghwa-Zone succeeded in acquiring originality with tangible cultural properties but Yeongju-Zone chose a project site without it. Third, with originality, Bonghwa-zone was evaluated as having resources and concept with high traditional culture connectivity and fulfilled identity. It led the feature showing the high affinity between originality and identity. Fourth, compared to the projects of Andong-Zone in the Three Major Cultural Areas Projects, these two projects failed to acquire the distinctions since the primary and the secondary influence area and major facilities & programs coincided with those of the projects of Andong-Zone. Fifth, compared to Bonghwa-Zone, Yeongju-Zone realized visibility faithfully by the conceptual flexibility of "Korean Culture" and a large-scale development. Sixth, in terms of the Historical and Cultural Authenticity of project plan, it is evaluated that Yeongju-Zone and Bonghwa-Zone only fulfilled visibility and specificity respectively.

Acceptor Specificity of Amylosucrase from Deinococcus radiopugnans and Its Application for Synthesis of Rutin Derivatives

  • Kim, Myo-Deok;Jung, Dong-Hyun;Seo, Dong-Ho;Jung, Jong-Hyun;Seo, Ean-Jeong;Baek, Nam-In;Yoo, Sang-Ho;Park, Cheon-Seok
    • Journal of Microbiology and Biotechnology
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    • v.26 no.11
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    • pp.1845-1854
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    • 2016
  • The transglycosylation activity of amylosucrase (ASase) has received significant attention owing to its use of an inexpensive donor, sucrose, and broad acceptor specificity, including glycone and aglycone compounds. The transglycosylation reaction of recombinant ASase from Deinococcus radiopugnans (DRpAS) was investigated using various phenolic compounds, and quercetin-3-O-rutinoside (rutin) was found to be the most suitable acceptor molecule used by DRpAS. Two amino acid residues in DRpAS variants (DRpAS Q299K and DRpAS Q299R), assumed to be involved in acceptor binding, were constructed by site-directed mutagenesis. Intriguingly, DRpAS Q299K and DRpAS Q299R produced 10-fold and 4-fold higher levels of rutin transglycosylation product than did the wild-type (WT) DRpAS, respectively. According to in silico molecular docking analysis, the lysine residue at position 299 in the mutants enables rutin to more easily position inside the active pocket of the mutant enzyme than in that of the WT, due to conformational changes in loop 4.

A Study on the Effect of Experience-specificity and Uncertainty on Choice in Experiential Products -From Transaction Cost Perspective- (경험재 거래의 경험특유성, 불확실성이 선택에 주는 영향에 관한 연구 -거래비용적 관점에서-)

  • Jeong, Yun-Hee;Park, JI-Yeon
    • Journal of Convergence for Information Technology
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    • v.9 no.4
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    • pp.152-159
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    • 2019
  • The purpose of this study is to investigate the effect of transaction characteristics on transaction cost and choice intention by applying transaction cost theory to experiential product. Experience-specificity, transaction uncertainty, and personal uncertainty are proposed to reflect the characteristics of experiential products, and the effects of these variables on transaction costs and transaction costs are assumed to have an influence on the choice intention. The results of this study are summarized as follows. First, experience-specificity(site, physical equipment, knowledge skill, temporal), transactional uncertainty(product-, process-), personal uncertainty (preference-, and situation-) have a significant positive effect on transaction cost. Second, transaction costs (search, comparison, examination, negotiation, payment, delivery) have a significant negative effect on the choice intention of the experiential product. The results of this study show that the increase of transaction costs can reduce the choice of experiential products and the strategic consideration of experience specificity, transaction uncertainty and individual uncertainty are required to reduce transaction costs. In addition, experiential products lacked access from a transactional and cost-based point of view, and this study contributes theoretically by compensating for the lack.

Occurrence of Vesicular-Arbuscular Mycorrhizal (VAM) Fungi and Their Effect on Plant Growth in Endangered Vegetations

  • Selvaraj, Thangaswamy;Padmanabhan, Chellappan;Jeong, Yu-Jin;Kim, Hoon
    • Journal of Microbiology and Biotechnology
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    • v.14 no.4
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    • pp.885-890
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    • 2004
  • A survey for vesicular-arbuscular mycorrhizae (VAM) occurrence was undertaken in three endangered vegetation sites in the area of Kudankulam atomic power station. Fifteen VAM fungal species were isolated from the root-zone soils of fourteen different plant species. There was a significant correlation observed between the number of spores and of percentage root colonization as exemplified by Phyllanthus niruri and Paspalum vaginatum (450, 95%; 60, 25%). Although VAM species are not known to be strictly site specific, the fact that Acaulospora elegans was observed only in site 1, Glomus pulvinatum in site 2 only, and Gl. intraradices in site 3 only, showed site-specificity in this study. To confirm the infection efficiency, two host plant species in the sites, P. niruri and Eclipta alba, were selected and inoculated in field with three selected VAM fungal spores. Gl. fasciculatum was found to be the most efficient VAM species in percentage root colonization, number of VAM spores, and dry matter content. When the nutrients in roots of P. niruri and E. alba were analyzed, there was higher uptake of K (4.2 and 3.4 times, respectively) and Ca (5.3 and 4.9 times, respectively), the analogues for $^{137}Cs$ and $^{90}Sr$, respectively. From the results, it might be concluded that VAM association helps the plants survive in a disturbed ecosystem and enhances uptake and cycling of radionuclides from the ecosystem.