DOCSLIB.ORG

Results and Discussion.-The Metal Binding Site of Apocarboxypeptidase: Ag+, P-Mercuribenzoate, Or Ferricyanide Titrate Only

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Results and Discussion.-The Metal Binding Site of Apocarboxypeptidase: Ag+, P-Mercuribenzoate, Or Ferricyanide Titrate Only CARBOXYPEPTIDASE A: APPROACHES TO THE CHEMICAL NA TURE OF THE ACTIVE CENTER AND THE MECHANISMS OF ACTION* BY BERT L. VALLEE, JAMES F. RIORDANt, AND JOSEPH E. COLEMANt BIOPHYSICS RESEARCH LABORATORY, DIVISION OF MEDICAL BIOLOGY, DEPARTMENT OF MEDICINE, HARVARD MEDICAL SCHOOL AND THE PETER BENT BRIGHAM HOSPITAL, BOSTON Communicated by Eric G. Ball, November 7, 1962 The characteristics of metalloenzymes offer unusual opportunities in the elucida- tion of their active, enzymatic centers. Carboxypeptidasel has proved to be a particularly suitable and experimentally accessible enzyme for such studies, since conclusions concerning the active center2 need not be based on experiments which destroy activity.3 This paper briefly summarizes the implications of recent experi- ments which have been reported and others to be reported elsewhere.4-9 Experimental.-The preparations of the native zinc enzyme and the metal-free apoenzyme, the methods for peptidase and esterase assay, the preparation of metallocarboxypeptidases, the deter- minations of protein concentration, sulfhydryl titrations, gel filtrations and equilibrium dialyses have all been described previously.,, 6, 10-13 Dinitrophenylations of the metalloenzyme and apo- enzyme were carried out according to the method of Sanger'4 or Fraenkel-Conrat.'5 The DNP- amino acid derivatives were isolated by two-dimensional paper chromatography. Phenylisothio- cyanatc was employed for Fraenkel-Conrat's modification", of the Edman degradation.'6 The phenylthiohydantoins were isolated by paper chromatography and identified by their character- istic Rf's as determined bv ultraviolet absorption and starch-iodine. Acylations were performed by adding 15- to 60-fold excess of the various anhydrides mentioned in the text to 10-4 M enzyme in 0.02 M sodium Veronal-2 M NaCl, pH 7.5 buffer at 00 for a reaction time of thirty min. The pH of the reaction was maintained at 7.5 by the addition of 1 M NaGH by means of a pH Stat. Excess anhydride was removed by dialysis against the same buffer at 0° for 48 hr. lodinations were performed with a 35-fold molar excess of I? in 0.5 M KI at 250 for ten min. Photooxidations were carried out in the presence of methylene blue according to the method of Weil and Buchert.'7 All chemicals used in these experiments were reagent grade and used without further purification except for acetic anhydride which was redistilled. Results and Discussion.-The metal binding site of apocarboxypeptidase: Ag+, p-mercuribenzoate, or ferricyanide titrate only one -SH group in the apocarboxy- peptidase and none in the metallocarboxypeptidasel2 (Table 1). A peptide con- taining the single cysteine residue of carboxypeptidase to which zinc is bound has recently been isolated.'8 The sequence and magnitude of the stability constants of different metallo- carboxypeptidases, their comparison to those of simple ligands and complexometric titrations indicated early that the metal atom is also bound to nitrogen.'1 12 The experiments suggesting that the a-amino group of the N-terminal asparagine (AspNH2) (Table 1) may be the second donor group for zinc binding are quite analogous to those employed for the -SH group. On exposure to fluorodinitro- benzene or phenylisothiocyanate, the apoenzyme1' readily forms DNP-aspartic acid (DNP-Asp) or PTH-asparagine (PTN-AspNH2), respectively, while the yields from the metalloenzyme are significantly less. Zinc appears to be displaced from the enzyme in proportion to the reaction of these reagents with the N-terminal asparagine. The sum of the number of gram atoms of zinc bound per mole of enzyme and the moles of product is unity7' 19 (Table 1). Chemical modifications: The search for those groups of carboxypeptidase which might function in specificity, catalysis, and binding of the substrate is a logical ex- 109 Downloaded by guest on September 27, 2021 110 BIOCHEMISTRY: VALLEE ET AL. PROC. N. A. S. TABLE 1 COMPLEMENTARITY OF -SH AND a-NH2 GROUPS WITH ZINC CONTENT OF [(CPD) ZN]a Zn++b -SHc Sample (gm-atom/mole) (moles/mole) 2 (Zn++ + -SH) [(CPD) Zn]d 1.00 0 1.00 (CPD) + 0.5 gm-atom Zn++ 0.57 0.42 0.99 (CPD) + 0.25 gm-atom Zn++ 0.27 0.63 0.90 (CPD) 0 0.91 0.91 l)NP-A sp (moles/mole) Z (Zn++ + DNP-Asp) [(CPD)Zn]e 1.0 1.00 [(CPD) Zn]f 0.77 0.25 1.02 (CPD) + 0.5 gin-atom Zn++ 0.47 0.52 0.99 [(CPD) Zn]g 0.23 0.70 0.93 PTH-AspNH2 (moles/mole) 2 (Zn + + + PTH-AspNH2) [(CPD) Zn] + PTCh 2 min 0.72 0.28 1.00 [(CPD) Zn] + PTC 15 min 0.44 0.47 0.91 [(CPD) Zn] + PTC 30 min 0.38 0.69 1.07 a Apocarboxypeptidase is designated (CPD); zinc carboxypeptidase is [(CPD) Zn] in accord with previous usage (Vallee, B. L., Advances in Protein Chem., 10, 317 (1955)). b Determined by chemical analysis.10 c-SH groups titrated with Ag+.l2 d Native and fully reconstituted zinc carboxypeptidase. e Native zinc carboxypeptidase, not treated with 1-fluoro-2,4-dinitrobenzene (FDNB). f Native zinc carboxypeptidase, FDNB, 2 hr, 250, pH 7.0.15 g Native zinc carboxypeptidase, FDNB, pH 9.0, conditions according to Sanger.14 h Native zinc carboxypeptidase, phenylisothiocyanate (PTC) 22°, Whatman No. 1 paper strip, conditions according to Fraenkel-Conrat.'5 The N-terminal sequence is Asp-NH2, Tyr, Ala.7. 19 tension of these studies. Alterations in the general three-dimensional structure of enzymes are not likely to account for the mode of action of procedures which result in marked increases in activity. Though conformational changes at the active site may have occurred, the enhancement of activity provides evidence that the essen- tial chemical and structural features of the active center have been preserved. Replacement of Zn++ by Cd++ and Hg++ at the Active Site of Carboxypeptidase.- Effects on peptidase and esterase activities: Cadmium and mercury carboxypep- tidases are more active as esterases than native zinc carboxypeptidase, but they exhibit no peptidase activity (Table 2). One activity has been abolished while the other is even enhanced as a consequence of the replacement of only one atom, TABLE 2 SIMULTANEOUS ALTERATIONS IN ENZYMATIC ACTIVITY AND SPECIFICITY OF CARBOXYPEPTIDASE INDUCED BY METAL IONS, ANHYDRIDES, IODINATION, AND IRRADIATION Activity Enzyme* Esterase (%) Peptidase (%) [(CPD) Zn] 100 100 [(CPD) Hg] 116 0 [(CPD) Cd] 152 0 Iodinated [(CPD) Zn] 500 0 Acetyl [(CPD) Zn] 610 0 SucCinyl [(CPD) Zn] 150 154 ACetyl-Succinyl [(CPD) Zn] 780 0 Acetyl [(CPD) Zn] + NH20H 138 85 i-Butyryl [(CPD) Zn] 134 83 n-Valeryl [(CPD) Zn] 162 80 n-Butyryl [(CPD) Zn] 191 64 Propionyl [(CPD) Zn] 342 49 Photooxidized [(CPD) Zn] 198 36 * AMetallocarboxypeptidases were prepared as previously described." Acylations were performed as described under Experimental. Iodinations were performed with a 35-fold molar excess of I2 in 0.5 M KI for 10 min at 250. Photooxidations were carried out at 25° for 90 min using a 500-watt photoflood lamp positioned 6" from the sample as previously described."7 Esterase and peptidase activities were measured as previously described." Downloaded by guest on September 27, 2021 VOL. 49, 1963 BIOCHEMISTRY: VALLEE ET AL. 111 zinc, by another, cadmium or mercury; in effect, a curtailment of the dual specificity of the enzyme. Changes in the over-all three-dimensional protein structure would not constitute a likely explanation. Optical rotatory dispersion measurements support this prediction. Other organic chemical modifications of this enzyme enhance this view. Acylation with Monocarboxylic Acid Anhydrides, Iodination, and Photooxidation.- Effects on peptidase and esterase activities: Under appropriate conditions, acylation with monocarboxylic acid anhydrides, iodination, and photooxidation of carboxy- peptidase all markedly increase esterase and decrease peptidase activity (Table 2). Acetylation increases esterase activity to about 610 per cent of the native enzyme and peptidase activity is abolished completely; f3-phenylpropionate, a competitive inhibitor of the enzyme, selectively prevents both of these changes8 (Fig. 1). Acetylation overcomes the inhibition of ester hydrolysis at higher substrate con- centrations.9 When acetylcarboxypeptidase is treated with hydroxylamine, both peptidase and esterase activities are restored almost to their original rates (Table 2). Acylations with i-butyric, n-butyric, n-valeric, and propionic anhydrides also increase esterase and decrease peptidase activity. Though qualitatively the same, these changes are quantitatively smaller, as might be expected in view of the known reactivities of these anhydrides. In each case the increase in esterase activity is commensurate with the decrease in peptidase activity. At present, the evidence suggests that acylation of e-amino groups does not account for these reversible alterations of activity.9 Histidyl or tyrosyl residues or even the single atypical sulfur-containing residue of carboxypeptidase'8 could react with all of these reagents. But alkylation of this atypical sulfur group does not affect activity nor does ,B- phenylpropionate prevent this alkylation. "8 Esterase activity is markedly en- hanced and peptidase activity is diminished or abolished either by acylation or replacement of zinc by cadmium or mercury." Chemical interactions of all of these reagents with a specific group in the active center would be the simplest postu- late for the alterations in rate and catalytic specificities of the modified enzyme. Succinylation: The catalytic properties of succinylcarboxypeptidase differ significantly from those of the other acylenzymes studied so far. Succinylation increases both esterase and peptidase activities by 50 per cent, but f3-phenylpropio- nate does not prevent these changes. Acetylated succinylcarboxypeptidase is completely inactive toward peptides. However, its esterase activity is 780 per cent of the native enzyme and incorporates the increases due to both modifications. Succinic anhydride apparently does not react with the group affected by the other reagents listed in Table 2, nor does it prevent their reaction.
Load more

Recommended publications
  • Control of Enzyme Activity Using Chemical Rescue of Protein Structure
    Control of Enzyme Activity using Chemical Rescue of Protein Structure by Yang Du A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science in Engineering Baltimore, Maryland May 2016 ii Abstract A cell can modulate the level of protein activity by the regulation of the amount of protein. On the other hand, strategies for controlling of cellular protein activity may lie in regulating the protein’s specific activity directly. Based on this idea, we demonstrated a method for designing an effector site directly into the catalytic domain of an enzyme and using a chemical compound to regulate the protein’s activity. This approach is different from the traditional chemical rescue of enzymes because it relies on disruption and restoration of protein structure instead of disrupting the active site and having the chemical compound restore the chemical functionality to the active site as a means to achieve modulated function. In detail, we demonstrate the activity of TEM-1 can be disrupted by removing buried tryptophan or leucine side chain that serve as a buttress supporting the protein structure. Three cases of site-direct mutations (W227G & W286G, L247G & W286G, and L282G & W286G) to TEM-1 β-lactamase enzyme (BLA) were made. In all cases, we observe disruption of enzyme function. To test the theory of chemical rescue of protein structure, we then assay β-lactamase activity in the presence of specific chemical compounds designed to bind in place of the iii side chains of the mutated amino acids. We observed an 8- fold restoration of W227G/W286G enzyme function in the presence of 2- (1-naphthylmethyl)-isoquinoline (NMIQ) and 3 -methyl-2- (1-naphthylmethyl)-1,3-benzothiazol-3- ium (MNMBT).
    [Show full text]
  • What Are the Preferred Ways for Proteins to Interact?
    Principles of Protein−Protein Interactions: What are the Preferred Ways For Proteins To Interact? Ozlem Keskin,*,† Attila Gursoy,† Buyong Ma,‡ and Ruth Nussinov*,‡,§ Koc University, Center for Computational Biology and Bioinformatics and College of Engineering, Rumelifeneri Yolu, 34450 Sariyer Istanbul, Turkey; Basic Research Program, SAIC−Frederick, Inc., Center for Cancer Research Nanobiology Program, NCIsFrederick, Frederick, Maryland 21702; and Sackler Institute of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel Received July 13, 2007 Contents 7.3. Chemistry of the Interactions: How Are 15 Subtle Differences Distinguished? 1. Introduction 1 8. Allostery 15 − 1.1. Protein Protein Interactions: Toward 1 9. Large Assemblies 16 Functional Prediction and Drug Design 10. Crystal Interfaces 16 1.2. Proteins are Flexible Molecules Even Though 3 We Frequently Treat Them as Rigid 11. Concluding Remarks: Preferred Organization in 17 Protein Interactions 1.3. Proteins Interact through Their Surfaces 5 12. Acknowledgment 18 2. Cooperativity in Protein Folding and in 5 Protein−Protein Associations 13. References 18 3. Protein−Protein Interfaces Have Preferred 6 Organization 1. Introduction 3.1. Description of Protein−Protein Interfaces 6 3.2. Some Amino Acids at the Interface Are Hot 7 1.1. Protein−Protein Interactions: Toward Spots Since They Contribute Significantly to Functional Prediction and Drug Design the Stability of the Protein−Protein Association Proteins are the working horse of the cellular machinery. 3.3. Protein Binding Sites Can Be Described as 8 They are responsible for diverse functions ranging from Consisting of a Combination of molecular motors to signaling.
    [Show full text]
  • Enzyme Substrate Binding Two Models Have Been Proposed to Explain How an Enzyme Binds Its Substrate: the Lock-And –Key Model and the Induced-Fit Model
    ENZYMOLOGY 1 Introduction إعداد محمود بركات Enzymology 1 - Definition: Biologic (organic catalysts) polymers that catalyze the chemical reactions. - Nature: Enzymes are proteins except catalytic RNA molecules (Ribozymes) (Ribozymes: short segment of RNA molecule which act as enzyme in processing RNA molecules (From immature to mature RNA)) - Characteristics: i. Enzymes are neither consumed nor permanently altered as a consequence of their participation in a reaction. Throughout their life span ii. Highly efficient. iii. Extremely selective catalysts. Specific iv. Thermolabile. (Enzymes are affected by the change of temperature when the temperature increases the enzymes will denatured) v. Site specific. (Enzymes found in cytoplasm differ from those found in organelles or membrane) vi. High turnover number compared to inorganic catalysts and other organic catalysts (Turnover number: the number of substrate molecules that can be converted by one molecule of catalyst into product molecule in a unit time) - Inorganic Catalysts such as Ni, Pb, Pt. • Thermostable • Lower turnover number Nomenclature of enzymes (In most cases, enzyme names end in –ase) 1. The common name for a hydrolase is derived from the substrate. ➢ Urea: remove -a, replace with -ase = urease ➢ Lactose: remove - ose, replace with - ase = lactase 2. Other enzymes are named for the substrate and the reaction catalyzed. ➢ Lactate dehydrogenase ➢ Pyruvate decarboxylase 3. Some names are historical - no direct relationship to substrate or reaction type. (Digestive Enzymes) Catalase, Pepsin, Chymotrypsin, Trypsin Classification of Enzymes - Enzyme Commission (EC) – according to International Union of Biochemistry & Molecular Biology (IUBMB) - Each enzyme was given 4-digit numbes [1.2.3.4] 1st one of the 6 major classes of enzyme activity 2nd the subclass (type of substrate or bond cleaved) 3rd the sub-subclass (group acted upon, cofactor required, etc...) 4th a serial number… (order in which enzyme was added to list) The 6 Magor Classes are: 1) Oxidoreductases (EC.1) catalyze redox reactions.
    [Show full text]
  • Deciphering Molecular Mechanisms of Adverse Reactions of Drugs
    UNIVERSITY OF CALIFORNIA, SAN DIEGO Deciphering Molecular Mechanisms of Adverse Reactions of Drugs A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioinformatics and Systems Biology by Yu-Chen Chen Committee in charge: Professor Ruben Abagyan, Chair Professor Grace M. Kuo, Co-Chair Professor Nuno F. Bandeira Professor Sanjoy Dasgupta Professor Lucila Ohno-Machado 2014 Copyright Yu-Chen Chen, 2014 All rights reserved. Signature Page The Dissertation of Yu-Chen Chen is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Co-Chair _____________________________________________________________________ Chair University of California, San Diego 2014 iii TABLE OF CONTENTS Table of Contents Signature Page ............................................................................................................... iii Table of Contents .......................................................................................................... iv List of Figures ................................................................................................................ ix List of Tables ...............................................................................................................
    [Show full text]
  • Structural Biochemistry/Enzyme/Active Site 1 Structural Biochemistry/Enzyme/Active Site
    Structural Biochemistry/Enzyme/Active Site 1 Structural Biochemistry/Enzyme/Active Site Overview An active site is the part of an enzyme that directly binds to a substrate and carries a reaction. It contains catalytic groups which are amino acids that promote formation and degradation of bonds. By forming and breaking these bonds, enzyme and substrate interaction promotes the formation of the transition state structure. Enzymes help a reaction by stabilizing the transition state intermediate. This is accomplished by lowering the energy barrier or activation energy- the energy that is required to promote the formation of transition state intermediate. The three dimensional cleft is formed by the groups that come from different part of the amino acid sequences. The active site is only a small part of the total enzyme volume. It enhances the enzyme to bind to substrate and catalysis by many differnet weak interactions because of its nonpolar microenvironment. The weak interactions includes the Van der Waals, hydrogen bonding, and electrostatic interactions. The arrangement of atoms in the active site is crucial for binding spectificity. The overall result is the acceleration of the reaction process and increasing the rate of reaction. Furthermore, not only do enzymes contain catalytic abilities, but the active site also carries the recognition of substrate. The enzyme active site is the binding site for catalytic and inhibition reactions of enzyme and substrate; structure of active site and its chemical characteristic are of specific for the binding of a particular substrate. The binding of the substrate to the enzyme causes changes in the chemical bonds of the substrate and causes the reactions that lead to the formation of products.
    [Show full text]
  • On the Specificity of Protein-Protein Interactions in the Context of Disorder
    On the specificity of protein-protein interactions in the context of disorder Teilum, Kaare; Olsen, Johan G.; Kragelund, Birthe B. Published in: Biochemical Journal DOI: 10.1042/BCJ20200828 Publication date: 2021 Document version Publisher's PDF, also known as Version of record Document license: CC BY-NC-ND Citation for published version (APA): Teilum, K., Olsen, J. G., & Kragelund, B. B. (2021). On the specificity of protein-protein interactions in the context of disorder. Biochemical Journal, 478(11), 2035-2050. https://doi.org/10.1042/BCJ20200828 Download date: 29. sep.. 2021 Biochemical Journal (2021) 478 2035–2050 https://doi.org/10.1042/BCJ20200828 Review Article On the specificity of protein–protein interactions in the context of disorder Kaare Teilum1,2, Johan G. Olsen1,2,3 and Birthe B. Kragelund1,2,3 1The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark; 2Structural Biology and NMR Downloaded from http://portlandpress.com/biochemj/article-pdf/478/11/2035/914573/bcj-2020-0828c.pdf by Copenhagen University user on 24 June 2021 Laboratory, Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark; 3REPIN, Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark Correspondence: Birthe B. Kragelund ([email protected]) With the increased focus on intrinsically disordered proteins (IDPs) and their large interac- tomes, the question about their specificity — or more so on their multispecificity — arise. Here we recapitulate how specificity and multispecificity are quantified and address through examples if IDPs in this respect differ from globular proteins. The conclusion is that quantitatively, globular proteins and IDPs are similar when it comes to specificity.
    [Show full text]
  • Studen Talk P Ste Session
    Nu Biophysical Society presents MBU In-House Symposium 2020 ❖ Studen talk ❖ Pste session Plenar Talk Dr. John Briggs Dr. Gautam Basu MRC LMB, UK Bose Institute, India ‘Studying proteins within viral ‘Protein functional evolution driven particles using cryo-electron by genome plasticity’ tomography’ Join us on: 18th December 4 pm - 6.30 pm IST 19th December 9 am - 5.45 pm IST SYMPOSIUM SCHEDULE DATE SESSION CHAIR FROM TO AGENDA STUDENTS DAY-1 1600 1605 Chairman Inauguration 1605 1720 John Briggs 1720 1735 Anand Srivastava Gaurav ORAL 1735 1750 Aravind Penmatsa Archishman 18/12/2020 Ishika 1750 1757 Mahavir Singh Kavyashree Pramanick 1757 1804 A. Surolia Soujanya 1804 1811 K Suguna Sreeparna POSTER 1811 1818 N Srinivasan Sohini 1818 1825 S K Sikdar Ashish DAY-2 9000 1015 Gautam Basu 1015 1030 Ashok Sekhar Vaishali 1030 1045 B Gopal Amit ORAL 1045 1100 Dipankar Chatterji Sudhanshu 1100 1107 Siddhartha P Sarma Mihir Arunabh Athreya 1107 1114 Saraswathi Vishweshwara Arinnia 1114 1121 Rishikesh Narayanan Rituparna POSTER 1121 1128 Rahul Roy Rohit 1128 1135 Raghavan Varadarajan Priyanka 1135 1142 N Srinivasan Adithyan 1145 1200 BREAK 1200 1215 Jayanta Chatterjee Venkateshwar Rao Malyasree 1215 1230 Mahavir Singh Niranjan ORAL Giri 1230 1245 M Vijayan Sivaji 1245 1400 LUNCH 1400 1415 N Srinivasan Sandhya 1415 1430 Raghavan Varadarajan Gopinath ORAL 19/12/2020 1430 1445 Rishikesh Narayanan Sameera 1445 1500 Siddhartha P Sarma Mihir Priyanka 1500 1507 N Srinivasan Ashraya Bajaj 1507 1514 Somnath Dutta Alakta 1514 1521 N Srinivasan Yazhini POSTER 1521 1528 M Vijayan Prateek 1528 1535 Jayanta Chatterjee Swati 1535 1542 Dipankar Chatterji Anirban 1545 1600 BREAK 1600 1615 S K Sikdar Monica 1615 1630 Somnath Dutta Suman ORAL 1630 1645 A.
    [Show full text]
  • F.Y. B.Sc. (Sem. II) (CBCS) MICROBIOLOGY [201]: BASICS OF
    Shree H. N. Shukla College of Science, (Affiliated to Saurashtra university) Nr. Lalpari Lake, B/H Marketing Yard, Rajkot-360 003 F.Y. B.Sc. (Sem. II) (CBCS) MICROBIOLOGY [201]: BASICS OF BIOCHEMISTRY AND MICROBIAL CONTROL Unit 3 ENZYMOLOGY Prepared By KRUPA BARAVADIYA Shree H. N. Shukla college of Science F.Y.BSc. MICRO 201 UNIT 3 INTRODUCTION TO ENZYME ➢ Enzymes are biological catalysts. They increase the rate of chemical reactions taking ➢ place within living cells without themselves suffering any overall change. The ➢ reactants of enzyme-catalysed reactions are termed substrates. Each enzyme is ➢ quite specific in character, acting on a particular substrate or substrates to produce a particular product or products. ➢ All enzymes are proteins. However, without the presence of a non-protein ➢ component called a cofactor, many enzyme proteins lack catalytic activity. When this is the case, the inactive protein component of an enzyme is termed the apoenzyme, and the active enzyme, including cofactor, the holoenzyme. ➢ The cofactor may be an organic molecule, when it is known as a coenzyme, or it may be a metal ion. Some enzymes bind cofactors more tightly than others. When a cofactor is bound so tightly that it is difficult to remove without damaging the enzyme, it is sometimes called a prosthetic group. To summarize diagrammatically: ENZYME < INACTIVE PROTEIN+ COFACTOR < METAL ENZYME ACTIVE PROTEIN COENZYME As we shall see later, both the protein and cofactor components may be directly involved in the catalytic processes taking place. A BRIEF HISTORY OF ENZYMES ➢ Until the nineteenth century, it was considered that processes such as the souring of milk and the fermentation of sugar to alcohol could only take place through the action of a living organism.
    [Show full text]
  • The Structure-Based Virtual Screening for Natural Compounds That Bind with the Activating Receptors of Natural Killer Cells
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.19.160861; this version posted June 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The Structure-Based Virtual Screening for Natural Compounds that Bind with the Activating Receptors of Natural Killer Cells Adekunle Babajide Rowaiye1,2, Solomon Oni*¹, Ikemefuna Chijioke Uzochukwu3, Alex Akpa1, Charles Okechukwu Esimone2 1Department of Medical Biotechnology, National Biotechnology Development Agency, Abuja, Nigeria 2Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Anambra State, Nigeria 3Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Anambra State, Nigeria. Abstract Aim: This study is aimed at prospecting for natural compounds that have strong binding affinity for the Activating Receptors of Natural Killer (NK) cells. Background: NK cells are responsible for the immunosurveillance of tumor and virally- infected cells. The cytotoxic potentials of this unique population of immune cells are triggered by the activating receptors. Through ligand-binding, these receptors induce the tyrosine phosphorylation of adapter proteins through their Immunoreceptor Tyrosine–based Activation Motif ITAM sequences and this triggers direct cytotoxicity and the production of cytokines through different signal pathways. Objective: To computationally predict the selectivity, specificity, and efficacy of natural compounds to be used as immunostimulatory agents for cancer treatment. Method: In this study, 1,697 natural compounds were obtained from 82 edible tropical plants through data mining.
    [Show full text]
  • Rum Aroma Descriptive Analysis Sabina Maza Gomez Louisiana State University and Agricultural and Mechanical College, [email protected]
    Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2002 Rum aroma descriptive analysis Sabina Maza Gomez Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Life Sciences Commons Recommended Citation Maza Gomez, Sabina, "Rum aroma descriptive analysis" (2002). LSU Master's Theses. 2770. https://digitalcommons.lsu.edu/gradschool_theses/2770 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. RUM AROMA DESCRIPTIVE ANALYSIS A Thesis Submitted to the Graduate School Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements of the degree of Master of Science in The Department of Food Science by Sabina Maza Gómez B.S., La Salle University, Mexico City, 1998 December, 2002 ACKNOWLEDGMENTS I would like to thank the many people that contributed one way or another to the realization of this work. To Dr. Witoon Prinyawiwatkul, I cannot thank him enough for his support, guidance, and example throughout the course of my studies, and for patiently advising me. To Dr. Willem H. Kampen and Dr. Donal F. Day, for all their valuable time and advice. To my friends Gabriela Rosales, Denise Pallais, Sireesha Bhattiprolu, Sirisha Sonti, Boris Castro, Guillermo Duque, Sandeep Bhale, Noemi Pavón, Patricio Paz, María del Pilar Paz, Manuel Rodriguez, and to Dr.
    [Show full text]
  • Histone Deacetylases (Hdacs): Evolution, Specificity, Role In
    G C A T T A C G G C A T genes Review Histone Deacetylases (HDACs): Evolution, Specificity, Role in Transcriptional Complexes, and Pharmacological Actionability Giorgio Milazzo, Daniele Mercatelli , Giulia Di Muzio, Luca Triboli, Piergiuseppe De Rosa, Giovanni Perini and Federico M. Giorgi * Department of Pharmacy and Biotechnology, University of Bologna, Via Selmi 3, 41026 Bologna, Italy; [email protected] (G.M.); [email protected] (D.M.); [email protected] (G.D.M.); [email protected] (L.T.); [email protected] (P.D.R.); [email protected] (G.P.) * Correspondence: [email protected] Received: 30 April 2020; Accepted: 11 May 2020; Published: 15 May 2020 Abstract: Histone deacetylases (HDACs) are evolutionary conserved enzymes which operate by removing acetyl groups from histones and other protein regulatory factors, with functional consequences on chromatin remodeling and gene expression profiles. We provide here a review on the recent knowledge accrued on the zinc-dependent HDAC protein family across different species, tissues, and human pathologies, specifically focusing on the role of HDAC inhibitors as anti-cancer agents. We will investigate the chemical specificity of different HDACs and discuss their role in the human interactome as members of chromatin-binding and regulatory complexes. Keywords: histone deacetylases; HDAC; chromatin; epigenetics; epigenomics; HDAC inhibitors; HDACi; gene networks; cancer; phylogenesis 1. Introduction Histone deacetylases (HDACs) constitute a family of proteins highly conserved across all eukaryotes [1]. Their main action consists in removing acetyl groups from DNA-binding histone proteins, which is generally associated to a decrease in chromatin accessibility for transcription factors (TFs) and specific, repressive effects on gene expression [2].
    [Show full text]
  • Methods to Prepare DNA for Efficient Massive Sequencing
    Methods to Prepare DNA for Efficient Massive Sequencing Sverker Lundin Royal Institute of Technology School of Biotechnology Stockholm 2012 © Sverker Lundin Stockholm 2012 Royal Institute of Technology (KTH) School of Biotechnology Division of Gene Technology Science for Life Laboratory SE-171 21 Solna Sweden Printed by E-PRINT Oxtorgsgatan 9-11 SE-111 57 Stockholm Sweden ISBN 978-91-7501-548-4 TRITA-BIO Report 2012:22 ISSN 1654-2312 Sverker Lundin (2012): Methods to Prepare DNA for Efficient Massive Sequencing, Division of Gene Technology, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden ABSTRACT Massive sequencing has transformed the field of genome biology due to the continuous introduction and evolution of new methods. In recent years, the technologies available to read through genomes have undergone an unprecedented rate of development in terms of cost-reduction. Generating sequence data has essentially ceased to be a bottleneck for analyzing genomes instead to be replaced by limitations in sample preparation and data analysis. In this work, new strategies are presented to increase both the throughput of li- brary generation prior to sequencing, and the informational content of libraries to aid post-sequencing data processing. The protocols developed aim to enable new possibilities for genome research concerning project scale and sequence complexity. The first two papers that underpin this thesis deal with scaling library production by means of automation. Automated library preparation is first described for the 454 se- quencing system based on a generic solid-phase polyethylene-glycol precipitation protocol for automated DNA handling. This was one of the first descriptions of automated sample handling for producing next generation sequencing libraries, and substantially improved sample throughput.
    [Show full text]