BIO-ORGANIC and BIOPHYSICAL CHEMISTRY MODULE No. 8: Introduction to Enzymes, Their Nomenclature & Classification

Total Page:16

File Type:pdf, Size:1020Kb

BIO-ORGANIC and BIOPHYSICAL CHEMISTRY MODULE No. 8: Introduction to Enzymes, Their Nomenclature & Classification ____________________________________________________________________________________________________ Subject Chemistry Paper No and Title 16; Bioorganic and Biophysical Chemistry Module No and Title 8; Introduction to Enzymes, their classification and nomenclature Module Tag CHE_P16_M8 Chemistry PAPER No. 16: BIO-ORGANIC AND BIOPHYSICAL CHEMISTRY MODULE No. 8: Introduction to Enzymes, their nomenclature & classification ____________________________________________________________________________________________________ TABLE OF CONTENTS 1. Learning Outcomes 2. Introduction 3. Introduction to Enzymes 3.1 Enzymes are biocatalysts 3.2 History of Enzymology 3.3 Interaction of Enzyme with substrate 3.4 Cofactors and coenzymes 4. Nomenclature and classification of enzymes 4.1 Common names 4.2 IUBMB classification and nomenclature 5. Summary Chemistry PAPER No. 16: BIO-ORGANIC AND BIOPHYSICAL CHEMISTRY MODULE No. 8: Introduction to Enzymes, their nomenclature & classification ____________________________________________________________________________________________________ 1. Learning Outcomes After studying this module, you shall be able to: • Know what are enzymes and how they are different from chemical catalysts • Learn history f discovery of enzymes • How to enzymes recognize their substrates? • Know enzymes require cofactors and coenzymes beyond amino acids which it is made of. • Learn how enzymes are named. 2. Introduction Enzymes are biological catalysts Mostly enzymes are proteins which possess catalytic activity. Enzymes were discovered in 18th century. They recognize their specific substrate and bind to them via a cleft termed as substrate binding site. Once they bind the substrate, they carry out the reaction and release products. For this binding, they require structural as well as electronic complementarity with the substrate. They mediate the reactions at physiological pH and temperature at rates much higher than the uncatalyzed reactions. Some enzymes carry out catalysis by virtue of amino acids that they are made of, while some require additional cofactors and coenzymes for full catalytic activity. Enzymes are named by four digit nomenclature as well as common names. This chapter also discusses the history of enzymology. 3. Introduction to Enzymes 3.1 Enzymes are biocatalysts The term enzyme was coined by Wilhelm Friedrich Kuhne in 1878. Enzymes are biological catalysts that differ from chemical catalysts in following features: (a) They work milder reaction conditions unlike chemical catalysts. Enzymes work physiological temperature and pH for their activity. (b) Enzymes have higher reaction specificity. They bind to specific substrate, carry the catalytic reaction and release specific products. (c) Enzymes enhance the rates of reaction they catalyze by several orders of magnitude when compared with uncatalysed reactions. The rates of enzyme catalyzed reactions are much higher than the reactions catalyzed by chemical catalysts. Chemistry PAPER No. 16: BIO-ORGANIC AND BIOPHYSICAL CHEMISTRY MODULE No. 8: Introduction to Enzymes, their nomenclature & classification ____________________________________________________________________________________________________ Table 1: Enzyme enhance the rates of reactions compared to uncatalyzed reactions. Name of the Enzyme Fold enhancement in rate Carboxypeptidase A 1011 Urease 1014 Triose phosphate isomerase 109 (d) Enzyme catalyzed reactions are well regulated. These reaction rates are not only influenced by substrate or product concentrations but also can be regulated by covalent modifications of enzymes etc. Most enzymes are proteinecous in nature but RNA enzymes also exist. RNA enzymes are ribozymes. 3.2 History of Enzymology Research work on fermentation by Joseph Gay Lussac determined that yeast decomposes sugar into carbon dioxide and ethanol. Jacob Berzelius proposed that the malt extract (diastase) catalyzes starch hydrolysis much efficiently than chemical catalyst H2SO4. However, in mid nineteenth century, Louis Pasteur proposed that fermentation can only take place in living cells. The term enzyme was coined by W.H. Kuhne in 1878. Eduard Buchner in 1897 showed fermentation in cell free extracts illustrating that fermentation can occur outside the living cells. He named the enzyme responsible for fermentation of sucrose ‘zymase’. In 1907, he received Nobel Prize in Chemistry for his pioneer work on discovery of cell free fermentation. In 1926, James Sumner gave identity to the enzymes. His work on jack bean urease (which catalyzes hydrolysis of urea to ammonia and carbon dioxide) proved that enzymes are pure proteins. He crystallized urease. The crystals consisted entirely of proteins. But his work was not accepted till John Northrop and Moses Kunitz showed correlation of activities of enzymes— pepsin, trypsin and chymotrypsin with the amount of protein. In 1946, these three scientists were then awarded Nobel Prize in Chemistry. In 1963, the amino acid sequence of the first enzyme bovie pancreatic ribonuclease A was given and in 1965, the first enzyme whose X-ray structure was worked out was that of hen egg white lysozyme by David Phillips. 3.3 Interaction of Enzyme with Substrate Chemistry PAPER No. 16: BIO-ORGANIC AND BIOPHYSICAL CHEMISTRY MODULE No. 8: Introduction to Enzymes, their nomenclature & classification ____________________________________________________________________________________________________ 3.3.1 Geometric and Electronic complementarity Van der Waals forces, hydrophobic interactions and H-bonding are the noncovalent forces driving interactions between substrate and product. Substrate binding site in enzyme is a cleft in the enzyme in which the substrate fits. This is called the geometric or physical complementarity (Figure 1). Figure 1. Depiction of geometric and electronic complementarity between substrate and enzyme. The amino acids that form the substrate binding site of the enzyme form attractive interaction with the substrate. This is termed as electronic complementarity (Figure 1). 3.3.2 Models of substrate binding to enzyme Two models have been proposed for substrate binding to enzyme: (a) Lock and key model In 1894, Emil Fischer proposed that both enzyme and substrate possess geometric shapes complementary to each other such that substrate perfectly fits the substrate binding site of the enzyme just like a key fits into the lock. Most importantly, enzyme possesses the substrate binding site even in the absence of the substrate. Chemistry PAPER No. 16: BIO-ORGANIC AND BIOPHYSICAL CHEMISTRY MODULE No. 8: Introduction to Enzymes, their nomenclature & classification ____________________________________________________________________________________________________ A B Figure 2. (A) The lock and key model of substrate binding to enzyme. (B) induced fit model. (b) Induced fit model Induced fit model was proposed by Daniel Koshland in 1958. He suggested that the substrate binding site is not rigid but reshapes or moulds itself after initial interaction with the substrate (Figure 2B) to bind the substrate perfectly. 3.4 Cofactors and Coenzymes Most enzymes are proteins composed entirely of amino acids. Few enzymes require additional chemical moieties other than amino acids. These chemical moieties are termed as cofactors. For example: Hexokinase requires Mg2+ for its catalytic activity. These cofactors can be inorganic or organic moieties. If they are organic in nature, they are termed as ‘coenzymes’. These coenzymes are derived from vitamins. Few examples of cofactors and coenzymes have been listed in Table 1. Cofactor/ Coenzymes Enzymes Mg2+ Hexokinase Zn2+ Carbonic anhydrase Ni2+ Urease Biotin (Coenzyme form-Biocytin) Pyruvate carboxylase Vitamin B / Riboflavin (Flavin adenine 2 Succinate dehydrogenase dinucleotide) Vitamin B12 (Coenzyme B12) Methionine synthase Vitamin B /Thiamin (Thiamine 1 Pyruvate dehydrogenase pyrophosphate) Chemistry PAPER No. 16: BIO-ORGANIC AND BIOPHYSICAL CHEMISTRY MODULE No. 8: Introduction to Enzymes, their nomenclature & classification ____________________________________________________________________________________________________ Some enzymes can require both metal ions and coenzymes for their activity. If the metal ion or coenzyme is tightly bound to the enzyme, it is termed as prosthetic group. The enzyme without the prosthetic group is called the apoenzyme. Along with the prosthetic group, it is called holoenzyme. Holoenzyme is thus the complete catalytically active form of the enzyme. ���������� → ��������� + �������� 4. Nomenclature and Classification of Enzymes 4.1 Common names The enzymes are named by adding suffix ‘-ase’ to the name of the substrate or their activity. For example: Hexokinase mediates phosphorylation of hexoses; Urease catalyzes the hydrolysis of urea. RNA polymerase catalyzes the polymerization of ribonucleotides to form RNA. 4.2 IUBMB classification and nomenclature With the increasing number of enzymes, common names became less popular to avoid same names of two enzymes and one enzyme carrying different names. Hence, to avoid this confusion, International Union of Biochemistry and Molecular Biology (IUBMB) adopted a systematic classification and nomenclature of enymes. According to this classification, enzymes are functionally classified into six classes (Table 3). Class 1 belongs to all the oxidoreductases, Class 2 to transferases and so on. These classes have been subdivided into subclasses and sub- subclasses. Each enzyme has been allotted two names and four digit classification.
Recommended publications
  • (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002
    USOO6395889B1 (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002 (54) NUCLEIC ACID MOLECULES ENCODING WO WO-98/56804 A1 * 12/1998 ........... CO7H/21/02 HUMAN PROTEASE HOMOLOGS WO WO-99/0785.0 A1 * 2/1999 ... C12N/15/12 WO WO-99/37660 A1 * 7/1999 ........... CO7H/21/04 (75) Inventor: fish E. Robison, Wilmington, MA OTHER PUBLICATIONS Vazquez, F., et al., 1999, “METH-1, a human ortholog of (73) Assignee: Millennium Pharmaceuticals, Inc., ADAMTS-1, and METH-2 are members of a new family of Cambridge, MA (US) proteins with angio-inhibitory activity', The Journal of c: - 0 Biological Chemistry, vol. 274, No. 33, pp. 23349–23357.* (*) Notice: Subject to any disclaimer, the term of this Descriptors of Protease Classes in Prosite and Pfam Data patent is extended or adjusted under 35 bases. U.S.C. 154(b) by 0 days. * cited by examiner (21) Appl. No.: 09/392, 184 Primary Examiner Ponnathapu Achutamurthy (22) Filed: Sep. 9, 1999 ASSistant Examiner William W. Moore (51) Int. Cl." C12N 15/57; C12N 15/12; (74) Attorney, Agent, or Firm-Alston & Bird LLP C12N 9/64; C12N 15/79 (57) ABSTRACT (52) U.S. Cl. .................... 536/23.2; 536/23.5; 435/69.1; 435/252.3; 435/320.1 The invention relates to polynucleotides encoding newly (58) Field of Search ............................... 536,232,235. identified protease homologs. The invention also relates to 435/6, 226, 69.1, 252.3 the proteases. The invention further relates to methods using s s s/ - - -us the protease polypeptides and polynucleotides as a target for (56) References Cited diagnosis and treatment in protease-mediated disorders.
    [Show full text]
  • Structure of Human Aspartyl Aminopeptidase Complexed With
    Chaikuad et al. BMC Structural Biology 2012, 12:14 http://www.biomedcentral.com/1472-6807/12/14 RESEARCH ARTICLE Open Access Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family Apirat Chaikuad1, Ewa S Pilka1, Antonio De Riso2, Frank von Delft1, Kathryn L Kavanagh1, Catherine Vénien-Bryan2, Udo Oppermann1,3 and Wyatt W Yue1* Abstract Backround: Aspartyl aminopeptidase (DNPEP), with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated. Results: The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β- hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids. Conclusions: The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference.
    [Show full text]
  • Anaphylatoxin-Mediated Shock Susceptibility to C5a Carboxypeptidase N (CPN1) Causes the Murine Small Subunit of Targeted Disrupt
    Targeted Disruption of the Gene Encoding the Murine Small Subunit of Carboxypeptidase N (CPN1) Causes Susceptibility to C5a This information is current as Anaphylatoxin-Mediated Shock of September 28, 2021. Stacey L. Mueller-Ortiz, Dachun Wang, John E. Morales, Li Li, Jui-Yoa Chang and Rick A. Wetsel J Immunol 2009; 182:6533-6539; ; doi: 10.4049/jimmunol.0804207 Downloaded from http://www.jimmunol.org/content/182/10/6533 References This article cites 43 articles, 9 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/182/10/6533.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Targeted Disruption of the Gene Encoding the Murine Small Subunit of Carboxypeptidase N (CPN1) Causes Susceptibility to C5a Anaphylatoxin-Mediated Shock1 Stacey L. Mueller-Ortiz,* Dachun Wang,* John E.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 8,603,824 B2 Ramseier Et Al
    USOO8603824B2 (12) United States Patent (10) Patent No.: US 8,603,824 B2 Ramseier et al. (45) Date of Patent: Dec. 10, 2013 (54) PROCESS FOR IMPROVED PROTEIN 5,399,684 A 3, 1995 Davie et al. EXPRESSION BY STRAIN ENGINEERING 5,418, 155 A 5/1995 Cormier et al. 5,441,934 A 8/1995 Krapcho et al. (75) Inventors: Thomas M. Ramseier, Poway, CA 5,508,192 A * 4/1996 Georgiou et al. .......... 435/252.3 (US); Hongfan Jin, San Diego, CA 5,527,883 A 6/1996 Thompson et al. (US); Charles H. Squires, Poway, CA 5,558,862 A 9, 1996 Corbinet al. 5,559,015 A 9/1996 Capage et al. (US) 5,571,694 A 11/1996 Makoff et al. (73) Assignee: Pfenex, Inc., San Diego, CA (US) 5,595,898 A 1/1997 Robinson et al. 5,610,044 A 3, 1997 Lam et al. (*) Notice: Subject to any disclaimer, the term of this 5,621,074 A 4/1997 Bjorn et al. patent is extended or adjusted under 35 5,622,846 A 4/1997 Kiener et al. 5,641,671 A 6/1997 Bos et al. U.S.C. 154(b) by 471 days. 5,641,870 A 6/1997 Rinderknecht et al. 5,643,774 A 7/1997 Ligon et al. (21) Appl. No.: 11/189,375 5,662,898 A 9/1997 Ligon et al. (22) Filed: Jul. 26, 2005 5,677,127 A 10/1997 Hogan et al. 5,683,888 A 1 1/1997 Campbell (65) Prior Publication Data 5,686,282 A 11/1997 Lam et al.
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2006/0110747 A1 Ramseier Et Al
    US 200601 10747A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0110747 A1 Ramseier et al. (43) Pub. Date: May 25, 2006 (54) PROCESS FOR IMPROVED PROTEIN (60) Provisional application No. 60/591489, filed on Jul. EXPRESSION BY STRAIN ENGINEERING 26, 2004. (75) Inventors: Thomas M. Ramseier, Poway, CA Publication Classification (US); Hongfan Jin, San Diego, CA (51) Int. Cl. (US); Charles H. Squires, Poway, CA CI2O I/68 (2006.01) (US) GOIN 33/53 (2006.01) CI2N 15/74 (2006.01) Correspondence Address: (52) U.S. Cl. ................................ 435/6: 435/7.1; 435/471 KING & SPALDING LLP 118O PEACHTREE STREET (57) ABSTRACT ATLANTA, GA 30309 (US) This invention is a process for improving the production levels of recombinant proteins or peptides or improving the (73) Assignee: Dow Global Technologies Inc., Midland, level of active recombinant proteins or peptides expressed in MI (US) host cells. The invention is a process of comparing two genetic profiles of a cell that expresses a recombinant (21) Appl. No.: 11/189,375 protein and modifying the cell to change the expression of a gene product that is upregulated in response to the recom (22) Filed: Jul. 26, 2005 binant protein expression. The process can improve protein production or can improve protein quality, for example, by Related U.S. Application Data increasing solubility of a recombinant protein. Patent Application Publication May 25, 2006 Sheet 1 of 15 US 2006/0110747 A1 Figure 1 09 010909070£020\,0 10°0 Patent Application Publication May 25, 2006 Sheet 2 of 15 US 2006/0110747 A1 Figure 2 Ester sers Custer || || || || || HH-I-H 1 H4 s a cisiers TT closers | | | | | | Ya S T RXFO 1961.
    [Show full text]
  • 1 No. Affymetrix ID Gene Symbol Genedescription Gotermsbp Q Value 1. 209351 at KRT14 Keratin 14 Structural Constituent of Cyto
    1 Affymetrix Gene Q No. GeneDescription GOTermsBP ID Symbol value structural constituent of cytoskeleton, intermediate 1. 209351_at KRT14 keratin 14 filament, epidermis development <0.01 biological process unknown, S100 calcium binding calcium ion binding, cellular 2. 204268_at S100A2 protein A2 component unknown <0.01 regulation of progression through cell cycle, extracellular space, cytoplasm, cell proliferation, protein kinase C inhibitor activity, protein domain specific 3. 33323_r_at SFN stratifin/14-3-3σ binding <0.01 regulation of progression through cell cycle, extracellular space, cytoplasm, cell proliferation, protein kinase C inhibitor activity, protein domain specific 4. 33322_i_at SFN stratifin/14-3-3σ binding <0.01 structural constituent of cytoskeleton, intermediate 5. 201820_at KRT5 keratin 5 filament, epidermis development <0.01 structural constituent of cytoskeleton, intermediate 6. 209125_at KRT6A keratin 6A filament, ectoderm development <0.01 regulation of progression through cell cycle, extracellular space, cytoplasm, cell proliferation, protein kinase C inhibitor activity, protein domain specific 7. 209260_at SFN stratifin/14-3-3σ binding <0.01 structural constituent of cytoskeleton, intermediate 8. 213680_at KRT6B keratin 6B filament, ectoderm development <0.01 receptor activity, cytosol, integral to plasma membrane, cell surface receptor linked signal transduction, sensory perception, tumor-associated calcium visual perception, cell 9. 202286_s_at TACSTD2 signal transducer 2 proliferation, membrane <0.01 structural constituent of cytoskeleton, cytoskeleton, intermediate filament, cell-cell adherens junction, epidermis 10. 200606_at DSP desmoplakin development <0.01 lectin, galactoside- sugar binding, extracellular binding, soluble, 7 space, nucleus, apoptosis, 11. 206400_at LGALS7 (galectin 7) heterophilic cell adhesion <0.01 2 S100 calcium binding calcium ion binding, epidermis 12. 205916_at S100A7 protein A7 (psoriasin 1) development <0.01 S100 calcium binding protein A8 (calgranulin calcium ion binding, extracellular 13.
    [Show full text]
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
    [Show full text]
  • ABSTRACT MUDIGANTI, USHARANI. Insect Response to Alphavirus Infection. (Under the Direction of Prof
    ABSTRACT MUDIGANTI, USHARANI. Insect response to alphavirus infection. (Under the Direction of Prof. Dennis T. Brown.) Invertebrate cells survive Alphavirus infections to establish viral persistence, in contrast to cell death seen soon after infection in mammalian cells. Invertebrate response to prototype alphavirus, Sindbis, has been studied to a certain extent, using mosquitoes and cell lines derived from mosquitoes. Some of the observations made in studies using mosquito systems include formation of intracellular vesicles soon after infection with Sindbis, identification of antiviral activity in the media used to grow the mosquito cell lines and in Sindbis-infected mosquito cell lysates, controlled levels of virus production as persistence is established and superinfection exclusion by Sindbis-infected cells. The study presented here is designed to utilize array of genomic and genetic information available in Drosophila model to identify the candidate genes / gene products playing a role in establishment of alphavirus persistence. Observations described in Chapter I establish Drosophila S2 cells as a suitable invertebrate system to study alphavirus-insect interactions. Gene expression analysis identified increased expression of 18 transcripts coding for membrane trafficking and cytoskeletal components and 10 transcripts coding for Notch pathway components, at 5 days post-infection. Identification of upregulation of Notch pathway suggests similarities between mechanism of establishment of persistence of Alphaviruses and Herpesviruses. Transcript coding for TEP II, a wide-spectrum protease inhibitor is increased in expression at 5 days post- infection and upon superinfection at 5 days post-infection. We probed for inhibition of viral protease activity during early persistence and upon superinfection of Sindbis- infected cells with Sindbis.
    [Show full text]
  • Transcriptome-Wide Survey of Gene Expression Changes and Alternative Splicing in Trichophyton Rubrum in Response to Undecanoic Acid
    Transcriptome-wide survey of gene expression changes and alternative splicing in Trichophyton rubrum in response to undecanoic acid Niege S. Mendes1, Tamires A. Bitencourt1, Pablo R. Sanches1, Rafael Silva-Rocha2, Nilce M. Martinez-Rossi1* & Antonio Rossi1 1Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, 14049-900 Ribeirão Preto, SP, Brazil 2Department of Molecular and Cellular Biology, Ribeirão Preto Medical School, University of São Paulo, 14049-900 Ribeirão Preto, SP, Brazil * Correspondence and requests for materials should be addressed to N.M.M.-R. (email: [email protected]) Tel: +55 16 33153150; Fax: +55 16 33150222; Niege S. Mendes, Tamires A. Bitencourt, and Pablo R. Sanches contributed equally to this work. Supplementary Figures Supplementary Figure S1. Volcano plot of the different time points analysed. The log2 fold changes for modulated genes are plotted against the –log10 P-values under each condition. Supplementary Figure S2. Intron retention at each time point analysed. The distribution frequency is plotted against –log10 P-values. Supplementary Figure S3. RT-PCR showing the intron 2 retention in impdh gene in T. rubrum. The PCR amplicons were generated after electrophoresis in a 2% agarose gel containing ethidium bromide. The samples presented in this gel, starting from left side border: Molecular weight ladder (1kb plus, Thermo Scientific), 0h (control), 3h, and 12h time points of UDA exposure, assessed in first biological replicate, as well as for second, and third biological replicates, respectively. The last well is related to genomic DNA amplicon (PCR control). The expected size of each amplicon was 481 bp for the elimination of intron 2 and 684 bp for the retention of intron 2 (Full-length version agarose gel related to Fig.
    [Show full text]
  • Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases
    Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases Alan J. Barrett Neil D. Rawlings J. Fred Woessner Editor biographies xxi Contributors xxiii Preface xxxi Introduction ' Abbreviations xxxvii ASPARTIC PEPTIDASES Introduction 1 Aspartic peptidases and their clans 3 2 Catalytic pathway of aspartic peptidases 12 Clan AA Family Al 3 Pepsin A 19 4 Pepsin B 28 5 Chymosin 29 6 Cathepsin E 33 7 Gastricsin 38 8 Cathepsin D 43 9 Napsin A 52 10 Renin 54 11 Mouse submandibular renin 62 12 Memapsin 1 64 13 Memapsin 2 66 14 Plasmepsins 70 15 Plasmepsin II 73 16 Tick heme-binding aspartic proteinase 76 17 Phytepsin 77 18 Nepenthesin 85 19 Saccharopepsin 87 20 Neurosporapepsin 90 21 Acrocylindropepsin 9 1 22 Aspergillopepsin I 92 23 Penicillopepsin 99 24 Endothiapepsin 104 25 Rhizopuspepsin 108 26 Mucorpepsin 11 1 27 Polyporopepsin 113 28 Candidapepsin 115 29 Candiparapsin 120 30 Canditropsin 123 31 Syncephapepsin 125 32 Barrierpepsin 126 33 Yapsin 1 128 34 Yapsin 2 132 35 Yapsin A 133 36 Pregnancy-associated glycoproteins 135 37 Pepsin F 137 38 Rhodotorulapepsin 139 39 Cladosporopepsin 140 40 Pycnoporopepsin 141 Family A2 and others 41 Human immunodeficiency virus 1 retropepsin 144 42 Human immunodeficiency virus 2 retropepsin 154 43 Simian immunodeficiency virus retropepsin 158 44 Equine infectious anemia virus retropepsin 160 45 Rous sarcoma virus retropepsin and avian myeloblastosis virus retropepsin 163 46 Human T-cell leukemia virus type I (HTLV-I) retropepsin 166 47 Bovine leukemia virus retropepsin 169 48
    [Show full text]
  • Dna Polymerase Beta, Carboxypeptidase, and Acetyl Coenzyme-A Decarbonylase/Synthase
    CHARACTERIZATION AND STRUCTURAL DETERMINATION OF METALLOENZYMES: DNA POLYMERASE BETA, CARBOXYPEPTIDASE, AND ACETYL COENZYME-A DECARBONYLASE/SYNTHASE DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Joseph W. Arndt, B.S. ***** The Ohio State University 2003 Dissertation Committee: Approved by Professor Michael K. Chan, Advisor Professor Joseph Krzycki ___________________________ Professor Ming-Daw Tsai Advisor Department of Chemistry ABSTRACT My research focused on the structure determination of proteins from three metalloenzyme systems by X-ray crystallography. The first target was rat DNA polymerase β, which catalyzes the template-directed nucleotidyl transfer reaction required for DNA replication. We have determined the crystal structures of two intermediate complexes in the reaction pathway of this enzyme, (i) a pre-chemistry ternary complex containing protein, DNA, and a chromium dNTP analog and (ii) a post- chemistry complex after nucleotide incorporation. These intermediate structures have allowed us to dissect the role of the two essential magnesium ions in initiating the enzyme’s conformational change. Based on these structures, a revised mechanism for replication and fidelity is proposed. The second part of this research involved structural studies on a carboxypeptidase (PfuCP) from the hyperthermophilic archaeon, Pyrococcus furiosus. Like other carboxypeptidases, it catalyzes the removal of amino acids from the C-terminus of protein and peptide chains. In this project we have solved three different structures of this enzyme, an apo form and two metal-bound forms. The overall fold of this enzyme is distinct from all other known structures of carboxypeptidase. It differs significantly in sequence, however, with one important feature being a consensus HEXXH metal-binding ii motif at its active site.
    [Show full text]