DOCSLIB.ORG

Determination of Strock Z-GPR-Pna Concentration

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Determination of Strock Z-GPR-Pna Concentration ENHANCING EFFICACY OF PROTEASE DRUGS THROUGH SITE DIRECTED MUTAGENESIS A Thesis submitted to the faculty of A 5 San Francisco State University In partial fulfillment of the requirements for A o n - the Degree •AH Master of Science In Chemistry: Biochemistry by Angela Nicole Amorello San Francisco, California August 2017 CERTIFICATION OF APPROVAL I certify that I have read ENHANCING EFFICACY OF PROTEASE DRUGS THROUGH SITE DIRECTED MUTAGENESIS by Angela Nicole Amorello, and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in Chemistry: Biochemistry at San Francisco State University. ENHANCING EFFICACY OF PROTEASE DRUGS THROUGH SITE-DIRECTED MUTAGENESIS Angela Nicole Amorello San Francisco, California 2017 A modem approach to drug development draws from nature to treat disease. Proteases, historically targeted for the development of inhibitors, show therapeutic potential. Development of protease therapeutics is complicated by the presence of endogenous inhibitors, which result in reduced serum half-lives of these agents. Determining which residues influence inhibitor binding can inform the design of efficacious protease therapeutics that have less favorable interactions with inhibitors through mutagenesis. Trypsin-fold serine proteases account for the majority of commercially available protease therapeutics. Consequently, substitutions that confer inhibitor resistance while maintaining catalytic activity in trypsin may be applied to prospective serine protease drugs. Examination of crystal structures of trypsin and its macromolecular inhibitors have shown a conserved hydrogen bond interaction between Tyr 39, Lys 60 and inhibitor. Using PCR mutagenesis, four trypsin variants were produced with amino acid substitutions at position 39 (Y39E, Y39S, Y39V and Y39L). Single variants Y39S and Y39L display activity that is comparable to wild type [kcat= (8.5 ± 47) x 103 m in'1), (9.1 ± 143) x 10'3 min*1 and (4.9 ± 100) x 103 min'1] respectively. I certify that the Abstract is a correct representation of the content of this thesis. Date TABLE OF CONTENTS List of Tables..................................................................................................................................vii List of Figures............................................................................................................................... viii 1. Introduction....................................................................................................................................1 Background and Significance........................................................................................... 1 Protease Therapeutics.........................................................................................................1 Serine Proteases.................................................................................................................. 2 Protease Inhibitors..............................................................................................................4 Protease Engineering......................................................................................................... 4 Experimental Design.........................................................................................................10 2. Experimental............................................................................................................................... 14 Construction of Variant Trypsin Zymogens.................................................................14 Bacterial Transformation.................................................................................................14 DNA Extraction and Purification....................................................................................15 Yeast Transformation........................................................................................................16 Small Scale Expression.................................................................................................... 17 Large Scale Expression.................................................................................................... 18 Zymogen Activation.........................................................................................................19 Active Site Titration..........................................................................................................19 Separation of Mature Trypsin from Trypsinogen....................................................... 20 Determination of Strock Z-GPR-pNA Concentration.................................................21 Characterization of Wild Type and Variants ..............................................................22 3. Results and Discussion.......................................................................................................... 24 Rationale for Variant Selection...................................................................................... 24 Small and Large Scale Expression.................................................................................25 Fast Protein Liquid Chromatography............................................................................ 26 Activation of Trypsin Variants...................................................................................... 27 Active Site Titration......................................................................................................... 30 Kinetic Characterization of Trypsin Variants..............................................................32 Inhibition Studies..............................................................................................................36 Future Directions........................................................................................................................... 40 References........................................................................................................................................43 LIST OF TABLES Table Page 1. Table 1 ...32 2. Table 2 ...36 LIST OF FIGURES Figures Page 1. Figure 1........... 3 2. Figure 2 ........................................................................................................................... 6 3. Figure 3 ............................................................................................................................7 4. Figure 4 ........................................................................................................................... 8 5. Figure 5 ...........................................................................................................................12 6. Figure 6...........................................................................................................................13 7. Figure 7 .......................................................................................................................... 29 8. Figure 8.......................................................................................................................... 31 9. Figure 9 .......................................................................................................................... 33 10. Figure 10........................................................................................................................12 11. Figure 11.......................................................................................................................38 12. Figure 12.......................................................................................................................39 1 1. Introduction A. Background and Significance Proteases are essential for a wide range of biological processes, including digestion, blood coagulation, and signal transduction pathways (Craik et. al 2011). Protease malfunction can result in various pathologies including bleeding disorders, cancer, and inflammation (Drag et. al 2010). Proteases either activate or inactivate growth factors, cytokines, chemokines, and cellular receptors resulting in downstream signal transduction and gene regulation (Craik et. al 2011). For example, up-regulation of matrix-metalloproteinases and matriptases is often observed in cancer (Drag et. al 2010, p. 2008). The roles of proteases in important biological processes and disease has made them attractive targets for drug design. B. Protease Therapeutics Protease-based drug discovery has traditionally involved development of inhibitors of proteolytic activity, which has resulted in highly successful drugs. Examples of these include Angiotensin Converting Enzyme inhibitors and HIV protease inhibitors, which treat cardiovascular disorders and HIV respectively (Drag et. al 2010). However, a long­ standing goal of the pharmaceutical industry has been to develop a protease therapeutic agent that remains catalytically active (Li et. al 2013). Protease therapeutics can provide unique benefits in contrast to other drugs. As a result of their catalytic activity, protease 2 drugs have the potential to inactivate multiple target proteins with higher efficacy in comparison to small molecule drugs. Exploiting this feature may allow smaller and less frequent doses, as well as lower costs to consumers (Li et. al 2013). A variety of protease therapeutic agents have gained approval from the U.S. Food and Drug Administration for clinical use in the treatment of traumatic bleeding (thrombin), hemophilia (FIX), and psoriasis
Load more

Recommended publications
  • The Serpintine Solution
    & Experim l e ca n i t in a l l C C f a Journal of Clinical & Experimental o r d l i a o n l o r g u Lucas et al., J Clin Exp Cardiolog 2017, 8:1 y o J Cardiology ISSN: 2155-9880 DOI: 10.4172/2155-9880.1000e150 Editorial Open Access The Serpintine Solution Alexandra Lucas, MD, FRCP(C)1,2,*, Sriram Ambadapadi, PhD1, Brian Mahon, PhD3, Kasinath Viswanathan, PhD4, Hao Chen, MD, PhD5, Liying Liu, MD6, Erbin Dai, MD6, Ganesh Munuswami-Ramanujam, PhD7, Jacek M. Kwiecien, DVM, MSc, PhD8, Jordan R Yaron, PhD1, Purushottam Shivaji Narute, BVSc & AH, MVSc, PhD1,9, Robert McKenna, PhD10, Shahar Keinan, PhD11, Westley Reeves, MD, PhD12, Mark Brantly, MD, PhD13, Carl Pepine, MD, FACC14 and Grant McFadden, PhD1 1Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe AZ, USA 2Saint Josephs Hospital, Dignity Health Phoenix, Phoenix, AZ, USA 3NIH/ NIDDK, Bethesda MD, USA 4Zydus Research Centre, Ahmedabad, India 5Department of tumor surgery, The Second Hospital of Lanzhou University, Lanzhou, Gansu, P.R.China 6Beth Israel Deaconess Medical Center, Harvard, Boston, MA, USA 7Interdisciplinary Institute of Indian system of Medicine (IIISM), SRM University, Chennai, Tamil Nadu, India 8MacMaster University, Hamilton, ON, Canada 9Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda MD, USA 10Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA 11Cloud Pharmaceuticals, Durham, North Carolina, USA 12Division of Rheumatology, University of Florida,
    [Show full text]
  • Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice
    pharmaceutics Review Mechanisms of CYP450 Inhibition: Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice Malavika Deodhar 1, Sweilem B Al Rihani 1 , Meghan J. Arwood 1, Lucy Darakjian 1, Pamela Dow 1 , Jacques Turgeon 1,2 and Veronique Michaud 1,2,* 1 Tabula Rasa HealthCare Precision Pharmacotherapy Research and Development Institute, Orlando, FL 32827, USA; [email protected] (M.D.); [email protected] (S.B.A.R.); [email protected] (M.J.A.); [email protected] (L.D.); [email protected] (P.D.); [email protected] (J.T.) 2 Faculty of Pharmacy, Université de Montréal, Montreal, QC H3C 3J7, Canada * Correspondence: [email protected]; Tel.: +1-856-938-8697 Received: 5 August 2020; Accepted: 31 August 2020; Published: 4 September 2020 Abstract: In an ageing society, polypharmacy has become a major public health and economic issue. Overuse of medications, especially in patients with chronic diseases, carries major health risks. One common consequence of polypharmacy is the increased emergence of adverse drug events, mainly from drug–drug interactions. The majority of currently available drugs are metabolized by CYP450 enzymes. Interactions due to shared CYP450-mediated metabolic pathways for two or more drugs are frequent, especially through reversible or irreversible CYP450 inhibition. The magnitude of these interactions depends on several factors, including varying affinity and concentration of substrates, time delay between the administration of the drugs, and mechanisms of CYP450 inhibition. Various types of CYP450 inhibition (competitive, non-competitive, mechanism-based) have been observed clinically, and interactions of these types require a distinct clinical management strategy. This review focuses on mechanism-based inhibition, which occurs when a substrate forms a reactive intermediate, creating a stable enzyme–intermediate complex that irreversibly reduces enzyme activity.
    [Show full text]
  • Insights Into the Role of Tick Salivary Protease Inhibitors During Ectoparasite–Host Crosstalk
    International Journal of Molecular Sciences Review Insights into the Role of Tick Salivary Protease Inhibitors during Ectoparasite–Host Crosstalk Mohamed Amine Jmel 1,† , Hajer Aounallah 2,3,† , Chaima Bensaoud 1, Imen Mekki 1,4, JindˇrichChmelaˇr 4, Fernanda Faria 3 , Youmna M’ghirbi 2 and Michalis Kotsyfakis 1,* 1 Laboratory of Genomics and Proteomics of Disease Vectors, Biology Centre CAS, Institute of Parasitology, Branišovská 1160/31, 37005 Ceskˇ é Budˇejovice,Czech Republic; [email protected] (M.A.J.); [email protected] (C.B.); [email protected] (I.M.) 2 Institut Pasteur de Tunis, Université de Tunis El Manar, LR19IPTX, Service d’Entomologie Médicale, Tunis 1002, Tunisia; [email protected] (H.A.); [email protected] (Y.M.) 3 Innovation and Development Laboratory, Innovation and Development Center, Instituto Butantan, São Paulo 05503-900, Brazil; [email protected] 4 Faculty of Science, University of South Bohemia in Ceskˇ é Budˇejovice, 37005 Ceskˇ é Budˇejovice, Czech Republic; [email protected] * Correspondence: [email protected] † These authors contributed equally. Abstract: Protease inhibitors (PIs) are ubiquitous regulatory proteins present in all kingdoms. They play crucial tasks in controlling biological processes directed by proteases which, if not tightly regulated, can damage the host organism. PIs can be classified according to their targeted proteases or their mechanism of action. The functions of many PIs have now been characterized and are showing clinical relevance for the treatment of human diseases such as arthritis, hepatitis, cancer, AIDS, and cardiovascular diseases, amongst others. Other PIs have potential use in agriculture as insecticides, anti-fungal, and antibacterial agents.
    [Show full text]
  • Competitive Inhibition That Barrier, As We Shall See
    Chapter 4 Catalysis In living systems, speed is everything. Providing the reaction speeds necessary to support life are the catalysts, mostly in the form of enzymes. Catalysis Introduction Introduction Activation Energy General Mechanisms of Action If there is a magical component to life, an argument can surely be made for it Substrate Binding being catalysis. Thanks to catalysis, reactions that could take hundreds of years Enzyme Flexibility to complete in the “real world,” occur in seconds in the presence of a catalyst. Active Site Chymotrypsin Chemical catalysts, like platinum, speed reactions, but enzymes (which are simply Enzyme Parameters super-catalysts with a twist) put chemical catalysts to shame. To understand VMAX & KCAT enzymatic catalysis, we must first understand energy. In Chapter 2, we noted the KM tendency for processes to move in the direction of lower energy. Chemical Perfect Enzymes Lineweaver-Burk Plots reactions follow this universal trend, but they often have a barrier in place that Enzyme Inhibition must be overcome. The secret to catalytic action is reducing the magnitude of Competitive Inhibition that barrier, as we shall see. No Effect On VMAX Increased KM Non-Competitive Inhibition Uncompetitive Inhibition Suicide Inhibition Control of Enzymes Allosterism Covalent Control of Enzymes Other Controls of Enzymes Ribozymes Enzyme Rate Enhancements 81 Activation Energy Another feature to note about catalyzed reactions is Click HERE, HERE, and HERE for The figure below schematically depicts the energy changes that the reduced energy barrier Kevin’s Catalytic Mechanism occur during the progression of a simple reaction. In the figure, lectures on YouTube (also called the activation the energy differences during the reaction are compared for a energy or free energy of catalyzed (plot on the right) and an uncatalyzed reaction (plot on activation) to reach the transition state of the catalyzed reaction.
    [Show full text]
  • Optimizing Defence, Counter-Defence and Counter-Counter Defence in Parasitic and Trophic Interactions – a Modelling Study
    1 Optimizing defence, counter-defence and counter-counter defence in parasitic and trophic interactions – A modelling study Stefan Schuster1*, Jan Ewald1,2, Thomas Dandekar2, Sybille Dühring1 1Dept. of Bioinformatics, Friedrich Schiller University of Jena Ernst-Abbe-Platz 2, 07743 Jena, Germany 2Dept. of Bioinformatics, Biocenter Julius Maximilian University of Würzburg Am Hubland, 97074 Würzburg *Corresponding author, e-mail: [email protected] Abstract In host-pathogen interactions, often the host (attacked organism) defends itself by some toxic compound and the parasite, in turn, responds by producing an enzyme that inactivates that compound. In some cases, the host can respond by producing an inhibitor of that enzyme, which can be considered as a counter-counter defence. An example is provided by cephalosporins, β-lactamases and clavulanic acid (an inhibitor of β-lactamases). Here, we tackle the question under which conditions it pays, during evolution, to establish a counter- counter defence rather than to intensify or widen the defence mechanisms. We establish a mathematical model describing this phenomenon, based on enzyme kinetics for competitive inhibition. We use an objective function based on Haber’s rule, which says that the toxic effect is proportional to the time integral of toxin concentration. The optimal allocation of defence and counter-counter defence can be calculated in an analytical way despite the nonlinearity in the underlying differential equation. The calculation provides a threshold value for the dissociation constant of the inhibitor. Only if the inhibition constant is below that threshold, that is, in the case of strong binding of the inhibitor, it pays to have a counter-counter defence.
    [Show full text]
  • Bsc Chemistry
    Subject Chemistry Paper No and Title 16, Bio-organic and Biophysical Chemistry Module No and Title 30, Enzymes as targets or drug design Module Tag CHE_P16_M30 Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design TABLE OF CONTENTS 1. Learning outcomes 2. Why Enzymes as Drug Targets? 3. Enzyme structure and Catalysis 4. Concept of Enzyme Inhibition 5. Types of Enzyme Inhibition 5.1. Nonspecific Inhibitors 5.2. Specific Inhibitors 6. Summary Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design 1. Learning outcomes After studying this module you shall be able to: Know about enzymes as drug targets Learn about enzyme structure and catalysis Know the concept of enzyme inhibition Understand the different types of enzyme inhibition 2. Introduction 2.1 Why Enzymes as Drug Targets? Medicine in twenty first century has become a science in which drug molecules are directed against the macromolecules. Enzymes hold a prominent position among the biological macromolecules that can be used as drug targets. Some features of enzyme structures and reaction pathways which make them attractive and primary target for drug action are as follows They play an essential role in biological life processes and pathophysiology The structures of active sites of enzymes and the ligand binding pockets are highly amenable for high affinity interactions with small drug like molecules Presence of potential allosteric sites Conformational variations in the binding sites Can be directly used as a therapeutic agent 3. Enzyme structure and catalysis Enzymes are biological catalysts as well as can act as receptors by acting with the substrates.
    [Show full text]
  • Imexon and Gemcitabine: Mechanisms of Synergy Against Human Pancreatic Cancer
    Imexon and Gemcitabine: Mechanisms of Synergy against Human Pancreatic Cancer Item Type text; Electronic Dissertation Authors Roman, Nicholas Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 02/10/2021 18:39:06 Link to Item http://hdl.handle.net/10150/194494 IMEXON AND GEMCITABINE: MECHANISMS OF SYNERGY AGAINST HUMAN PANCREATIC CANCER by NICHOLAS ORDUÑO ROMÁN _________________________ A Dissertation Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE) In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2 0 0 5 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the disser tation prepared by Nicholas Ordu ño Rom án entitled Imexon and Gemcitabine: Mechanisms of Synergy against Human Pancreatic Cancer and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy _____________________________________ ___________________________ Date:_______________ Robert T. Dorr, Ph.D. 4/12/05 _________ _______________________________________________________ Date:_______________ Terrence J. Monks. Ph.D. 4/12/05 _____ ___________________________________________________________ Date:_______________ Nathan J. Cherrington, Ph.D. 4/12/05 __________________________________________ ______________________ Date:_______________ Garth Powis, D. Phil. 4/12/05 ________ __ ______________________________________________________ Date:_______________ Terry H. Landowski, Ph.D. 4/12/05 Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertatio n to the Graduate College.
    [Show full text]
  • Identification of Regulatory Mechanisms of the Hepatic
    Available online at www.sciencedirect.com Computers and Chemical Engineering 32 (2008) 356–369 Identification of regulatory mechanisms of the hepatic response to thermal injury E. Yang a, T.J. Maguire a, M.L. Yarmush a, F. Berthiaume b, I.P. Androulakis a,∗ a Biomedical Engineering Department, Rutgers University, Piscataway, NJ, USA b Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, Boston, MA, USA Received 30 October 2006; received in revised form 11 February 2007; accepted 13 February 2007 Available online 25 February 2007 Abstract The purpose of this paper is to evaluate the hypothesis that a systems biology approach can be developed such that a set of transcription factors, relevant to burn-induced inflammatory response can be identified and modulated to control the host response. We explore a novel method for identifying coherent and informative expression motifs and we subsequently determine conserved transcription factor binding sites for the sub-sets of co-expressed genes. The responses are rationalized in the context of burn-induced inflammation and the putative transcription factors are rationalized in the context of intervention targets for controlling gene expression. © 2007 Published by Elsevier Ltd. Keywords: Gene expression; Burn; Inflammation; Transcritpion factors 1. Introduction to thermal injury predispose to the later hypermetabolic state would make it possible to define points of intervention by which Deep thermal injury over greater than 20 percent of the total such outcomes can be avoided. body surface area is one of the most severe forms of trauma. The response to burn injury and trauma results from a com- Following the early acute phase response dealing with the initial plex interplay between inflammation caused by the initial injury injury and shock (Carter, Tompkins, Yarmush, Walker, & Burke, and hypermetabolism.
    [Show full text]
  • Reversible and Mechanism-Based Irreversible Inhibitor Studies on Human Steroid Sulfatase and Protein Tyrosine Phosphatase 1B
    Reversible and Mechanism-Based Irreversible Inhibitor Studies on Human Steroid Sulfatase and Protein Tyrosine Phosphatase 1B by F. Vanessa Ahmed A thesis presented to the University of Waterloo in fulfilment of the thesis requirement for the degree of Doctor of Philosophy in Chemistry Waterloo, Ontario, Canada, 2009 © F. Vanessa Ahmed 2009 Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4- mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity- based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4- difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor.
    [Show full text]
  • Cefoxitin, N-Formimidoyl Thienamycin, Clavulanic
    1594 THE JOURNAL OF ANTIBIOTICS NOV. 1982 CEFOXITIN, N-FORMIMIDOYL THIENAMYCIN, CLAVULANIC ACID, AND PENICILLANIC ACID SULFONE AS SUICIDE INHIBITORS FOR DIFFERENT TYPES OF ~-LACTAMASES PRODUCED BY GRAM-NEGATIVE BACTERIA TETSUO SAWAI and KIKUO TSUKAMOTO Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan (Received for publication July 30, 1982) Cefoxitin, N-formimidoyl thienamycin (MK0787), clavulanic acid, and penicillanic acid sulfone (CP45,899) were studied to determine their potency as suicide inhibitors of three very different kinds of Vii-lactamases produced by Gram-negative bacteria: type Ib penicillinase (TEM-2 type), a typical cephalosporinase (the enzyme of Proteus morganii), and a cephalo- sporinase with broad substrate specificity (the enzyme of Proteus vulgaris). All these ~-lactams were confirmed to be quite stable to the three 19-lactamases. The absolute values of the turn- over numbers (kcat) for these enzyme-catalyzed hydrolyses were determined, the values ranged from 0.25 minute-1 to 660 minute-1. All the j3-lactams studied, except cefoxitin, acted as suicide inhibitors of the typical cephalosporinase. Although cefoxitin did not exhibit such an effect, it was a powerful competitive inhibitor of this enzyme. Although the four j3-lactams acted as suicide inhibitors of the P. vulgaris cephalosporinase, the inactivated enzyme partially regained its activity after removing the effect of the free inhibitor. Type lb penicillinase was also inactivated by the four p-lactams, and the activity of the inactivated enzyme, except in the case of cefoxitin, was partially restored. The rate constants for enzyme inactivation or reacti- vation were calculated and are presented. The information obtained from this study suggests that the catalytic center of the P.
    [Show full text]
  • Chapter 8. Enzymes: Basic Concepts and Kinetics
    CHAPTER 8. ENZYMES: BASIC CONCEPTS AND KINETICS coelenterazine The activity of an enzyme is responsible for the glow of the luminescent jellyfish at left. The enzyme aequorin catalyzes the oxidation of a compound by oxygen in the presence of calcium to release CO2 and light CHAPTER 8. ENZYMES: BASIC CONCEPTS AND KINETICS Introduction . Enzymes are large biological molecules that catalyze chemical transformations . The molecule also mediates the transformation of one form of energy into another . 25% of the genes in the human genome encode enzymes . Catalytic power and specificity – important characteristics . Active site – the place where enzymatic reactions take place CHAPTER 8. ENZYMES: BASIC CONCEPTS AND KINETICS Introduction . Nearly all known enzymes are proteins • RNA can function as a catalyst . Proximity effect – enzymes bring substrates closer together in an optimal orientation . They catalyze reactions by stabilizing transition states 8.1 CATALYTIC POWER AND SPECIFICITY Rate enhancement by enzymes . Enzymes accelerate reactions by factors of as much as a million or more 8.1 CATALYTIC POWER AND SPECIFICITY Substrate specificity . A substrate is a molecule upon which an enzyme acts . Some proteases cleaves specific amide bonds in a peptide . The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme . This precision is a result of the intricate 3D structure of the enzyme protein Fig 8.1 Enzyme specificity. (A) Trypsin cleaves on the carboxyl side of arginine and lysine residues, whereas (B) thrombin cleaves Arg-Gly bonds in particular sequences only 8.1 CATALYTIC POWER AND SPECIFICITY Cofactors . The catalytic activity of enzymes depends on the presence of small molecules termed cofactors .
    [Show full text]
  • Pdf (734.95 K)
    Biochemistry Letters, 11(20) 2016, pages 259-274 Scientific Research & Studies Center-Faculty of Science- Zagazig University- Egypt Biochemistry Letters Journal home page: Interactions of Monoamine Oxidase from Human And Rat Liver Mitochondria with (S)-2-[4-(3-Flouro-Benzyloxy)-Benzylamino]-Propionamide Zakia Ben Ramadan*1 , Keith Tipton2 , Safaa khater3 1-Department of Biochemistry, Faculty of Medicine University of Tripoli-Libya. 2-Department of Biochemistry, Trinity College, Dublin 2, Ireland . 3- Department of Biochemistry, Zagazig University –Egypt. ARTICLE INFO ABSTRACT Article history: Background:The interactions of the anticonvulsant (S)-2-[4- Received : (3-Flouro-Benzyloxy) Benzylamino]- Propionamide (FCE Accepted : 26743) with MAO-A and MAO -B from human and rat liver Available online : mitochondria has been studied. Objective: This compound is an alanine derivative that differs from the parent anticonvulsant Keywords: Monoamine compound, 2-n-pentylamino acetamide in that alaninamide oxidase-A and-B (MAO-A& residue replaces the glycinamide moiety, and in the presence of MAO -B), kinetics of inhibition, a phenyl ring substituted in the para-position by a 3-flouro- (S)-2-[4-(3-flourobenzyloxy)- benzyloxy group. Results: The results indicated that, The Ki benzylamino] propionamide, values for rat and human MAO-A were > 1000 times higher (FCE 26743), Inhibition than their corresponding values for rat and human MAO- Studies. Consistent with the IC50values that showed this compound to be > 1000 times more potent as an inhibitor of MAO-B than of MAO-A of the same species. There was an increase in the strength of inhibition when MAO-B was incubated with this compound. This was confirmed by the extended time-courses studies which showed a rather rapid increase in the degree of inhibition of MAO-B, despite incubation being continued for a total of 4h.
    [Show full text]