Investigation of the Catalytic and Regulatory Domains Of
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On the Active Site Thiol of Y-Glutamylcysteine Synthetase
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 2464-2468, April 1988 Biochemistry On the active site thiol of y-glutamylcysteine synthetase: Relationships to catalysis, inhibition, and regulation (glutathione/cystamine/Escherichia coli/kidney/enzyme inactivation) CHIN-SHIou HUANG, WILLIAM R. MOORE, AND ALTON MEISTER Cornell University Medical College, Department of Biochemistry, 1300 York Avenue, New York, NY 10021 Contributed by Alton Meister, December 4, 1987 ABSTRACT y-Glutamylcysteine synthetase (glutamate- dithiothreitol, suggesting that cystamine forms a mixed cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia disulfide between cysteamine and an enzyme thiol (15). coli strain enriched in the gene for this enzyme by recombinant Inactivation of the enzyme by the L- and D-isomers of DNA techniques. The purified enzyme has a specific activity of 3-amino-1-chloro-2-pentanone, as well as that by cystamine, 1860 units/mg and a molecular weight of 56,000. Comparison is prevented by L-glutamate (14). Treatment of the enzyme of the E. coli enzyme with the well-characterized rat kidney with cystamine prevents its interaction with the sulfoxi- enzyme showed that these enzymes have similar catalytic prop- mines. Titration of the enzyme with 5,5'-dithiobis(2- erties (apparent Km values, substrate specificities, turnover nitrobenzoate) reveals that the enzyme has a single exposed numbers). Both enzymes are feedback-inhibited by glutathione thiol that reacts with this reagent without affecting activity but not by y-glutamyl-a-aminobutyrylglycine; the data indicate (16). 5,5'-Dithiobis(2-nitrobenzoate) does not interact with that glutathione binds not only at the glutamate binding site but the thiol that reacts with cystamine. -
Molecular Markers of Serine Protease Evolution
The EMBO Journal Vol. 20 No. 12 pp. 3036±3045, 2001 Molecular markers of serine protease evolution Maxwell M.Krem and Enrico Di Cera1 ment and specialization of the catalytic architecture should correspond to signi®cant evolutionary transitions in the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, history of protease clans. Evolutionary markers encoun- MO 63110-1093, USA tered in the sequences contributing to the catalytic apparatus would thus give an account of the history of 1Corresponding author e-mail: [email protected] an enzyme family or clan and provide for comparative analysis with other families and clans. Therefore, the use The evolutionary history of serine proteases can be of sequence markers associated with active site structure accounted for by highly conserved amino acids that generates a model for protease evolution with broad form crucial structural and chemical elements of applicability and potential for extension to other classes of the catalytic apparatus. These residues display non- enzymes. random dichotomies in either amino acid choice or The ®rst report of a sequence marker associated with serine codon usage and serve as discrete markers for active site chemistry was the observation that both AGY tracking changes in the active site environment and and TCN codons were used to encode active site serines in supporting structures. These markers categorize a variety of enzyme families (Brenner, 1988). Since serine proteases of the chymotrypsin-like, subtilisin- AGY®TCN interconversion is an uncommon event, it like and a/b-hydrolase fold clans according to phylo- was reasoned that enzymes within the same family genetic lineages, and indicate the relative ages and utilizing different active site codons belonged to different order of appearance of those lineages. -
Crystallography Captures Catalytic Steps in Human Methionine Adenosyltransferase Enzymes
Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes Ben Murraya,b, Svetlana V. Antonyuka, Alberto Marinab, Shelly C. Luc, Jose M. Matod, S. Samar Hasnaina,1, and Adriana L. Rojasb,1 aMolecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZX, England; bStructural Biology Unit, Center for Cooperative Research in Biosciences, 48160 Derio, Spain; cDivision of Gastroenterology, Cedars-Sinai Medical Center, Los Angeles, CA 90048; and dCIC bioGUNE, CIBERehd, Parque Tecnologico Bizkaia, 801A-1.48160 Derio, Spain Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved January 8, 2016 (received for review June 4, 2015) The principal methyl donor of the cell, S-adenosylmethionine catalytic mechanism (13, 14) in which the reaction is initiated (SAMe), is produced by the highly conserved family of methionine through a nucleophilic attack by the sulfur atom of methionine adenosyltranferases (MATs) via an ATP-driven process. These en- on the C5′ atom of ATP, which produces the intermediate tri- zymes play an important role in the preservation of life, and their polyphosphate (PPPi). Hydrolysis of the PPPi into pyrophos- dysregulation has been tightly linked to liver and colon cancers. We phate (PPi) and orthophosphate (Pi) then occurs (Fig. S1). present crystal structures of human MATα2 containing various bound A common feature of MAT enzymes is a gating loop that α – ligands, providing a “structural movie” of the catalytic steps. High- to flanks the active site (in human MAT 2 residues 113 131), atomic-resolution structures reveal the structural elements of the which has been postulated to act in a dynamic way to allow ac- enzyme involved in utilization of the substrates methionine and cess to the active site. -
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. -
Spring 2013 Lecture 13-14
CHM333 LECTURE 13 – 14: 2/13 – 15/13 SPRING 2013 Professor Christine Hrycyna INTRODUCTION TO ENZYMES • Enzymes are usually proteins (some RNA) • In general, names end with suffix “ase” • Enzymes are catalysts – increase the rate of a reaction – not consumed by the reaction – act repeatedly to increase the rate of reactions – Enzymes are often very “specific” – promote only 1 particular reaction – Reactants also called “substrates” of enzyme catalyst rate enhancement non-enzymatic (Pd) 102-104 fold enzymatic up to 1020 fold • How much is 1020 fold? catalyst time for reaction yes 1 second no 3 x 1012 years • 3 x 1012 years is 500 times the age of the earth! Carbonic Anhydrase Tissues ! + CO2 +H2O HCO3− +H "Lungs and Kidney 107 rate enhancement Facilitates the transfer of carbon dioxide from tissues to blood and from blood to alveolar air Many enzyme names end in –ase 89 CHM333 LECTURE 13 – 14: 2/13 – 15/13 SPRING 2013 Professor Christine Hrycyna Why Enzymes? • Accelerate and control the rates of vitally important biochemical reactions • Greater reaction specificity • Milder reaction conditions • Capacity for regulation • Enzymes are the agents of metabolic function. • Metabolites have many potential pathways • Enzymes make the desired one most favorable • Enzymes are necessary for life to exist – otherwise reactions would occur too slowly for a metabolizing organis • Enzymes DO NOT change the equilibrium constant of a reaction (accelerates the rates of the forward and reverse reactions equally) • Enzymes DO NOT alter the standard free energy change, (ΔG°) of a reaction 1. ΔG° = amount of energy consumed or liberated in the reaction 2. -
Chemical Tools for Residue-Specific and Secondary Amine Selective Petasis (SASP) Bioconjugation of Peptides and Proteins
Seton Hall University eRepository @ Seton Hall Seton Hall University Dissertations and Theses (ETDs) Seton Hall University Dissertations and Theses Spring 5-4-2020 Chemical tools for Residue-Specific and Secondary Amine Selective Petasis (SASP) bioconjugation of Peptides and Proteins Yonnette Sim [email protected] Follow this and additional works at: https://scholarship.shu.edu/dissertations Part of the Biochemistry Commons Recommended Citation Sim, Yonnette, "Chemical tools for Residue-Specific and Secondary Amine Selective Petasis (SASP) bioconjugation of Peptides and Proteins" (2020). Seton Hall University Dissertations and Theses (ETDs). 2776. https://scholarship.shu.edu/dissertations/2776 Chemical tools for Residue-Specific and Secondary Amine Selective Petasis (SASP) bioconjugation of Peptides and Proteins A dissertation submitted to Seton Hall University in partial fulfillment for the Doctor of Philosophy Degree By: Yonnette E. Sim May 2020 Department of Chemistry and Biochemistry Seton Hall University South Orange, NJ. 07079 USA © 2020 Yonnette E. Sim DocuSign Envelope ID: 9EF6018D-72DB-4316-B86E-3A103260E910 We certify that we have read this dissertation and in our opinion it is adequate in scientific scope and quality as dissertation for the degree of Doctor of Philosophy Dr. Gregory R. Wiedman Mentor Dr. Monika Raj Co-Mentor (No Longer SHU Faculty) Dr. Joseph Badillo Member of Dissertation Committee Dr. Stephen Kelty Department Chair Seton Hall University I dedicate this thesis to my husband, Ebo, my children Ebony and Ethan for their tremendous love and support & My late parents Rupert and Theresa for their love and wisdom. “The only limit to the height of your achievements is the reach of your dreams and your willingness to work for them” – Michelle Obama i ACKNOWLEDGEMENTS First I would like to express my appreciation and thanks to my research mentor, Dr. -
Cysteine-Mediated Decyanation of Vitamin B12 by the Predicted Membrane Transporter Btum
ARTICLE DOI: 10.1038/s41467-018-05441-9 OPEN Cysteine-mediated decyanation of vitamin B12 by the predicted membrane transporter BtuM S. Rempel 1, E. Colucci 1, J.W. de Gier 2, A. Guskov 1 & D.J. Slotboom 1,3 Uptake of vitamin B12 is essential for many prokaryotes, but in most cases the membrane proteins involved are yet to be identified. We present the biochemical characterization and high-resolution crystal structure of BtuM, a predicted bacterial vitamin B12 uptake system. 1234567890():,; BtuM binds vitamin B12 in its base-off conformation, with a cysteine residue as axial ligand of the corrin cobalt ion. Spectroscopic analysis indicates that the unusual thiolate coordination allows for decyanation of vitamin B12. Chemical modification of the substrate is a property other characterized vitamin B12-transport proteins do not exhibit. 1 Groningen Biomolecular and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4, 9474 AG Groningen, The Netherlands. 2 Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden. 3 Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Correspondence and requests for materials should be addressed to D.J.S. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:3038 | DOI: 10.1038/s41467-018-05441-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05441-9 fi obalamin (Cbl) is one of the most complex cofactors well as re nement statistics are summarized in Table 1. BtuMTd C(Supplementary Figure 1a) known, and used by enzymes consists of six transmembrane helices with both termini located catalyzing for instance methyl-group transfer and ribo- on the predicted cytosolic side (Fig. -
The Crotonase Superfamily: Divergently Related Enzymes That Catalyze Different Reactions Involving Acyl Coenzyme a Thioesters
Acc. Chem. Res. 2001, 34, 145-157 similar three-dimensional architectures. In each protein, The Crotonase Superfamily: a common structural strategy is employed to lower the Divergently Related Enzymes free energies of chemically similar intermediates. Catalysis of the divergent chemistries is accomplished by both That Catalyze Different retaining those functional groups that catalyze the com- mon partial reaction and incorporating new groups that Reactions Involving Acyl direct the intermediate to new products. Indeed, as a Coenzyme A Thioesters specific example, the enolase superfamily has served as a paradigm for the study of catalytically diverse superfami- HAZEL M. HOLDEN,*,³ lies.3 The active sites of proteins in the enolase superfamily MATTHEW M. BENNING,³ are located at the interfaces between two structural TOOMAS HALLER,² AND JOHN A. GERLT*,² motifs: the catalytic groups are positioned in conserved Departments of Biochemistry, University of Illinois, regions at the ends of the â-strands forming (R/â) 8-barrels, Urbana, Illinois 61801, and University of Wisconsin, while the specificity determinants are found in flexible Madison, Wisconsin 53706 loops in the capping domains formed by the N- and Received August 9, 2000 C-terminal portions of the polypeptide chains. While the members of the enolase superfamily share similar three- ABSTRACT dimensional architectures, they catalyze different overall Synergistic investigations of the reactions catalyzed by several reactions that share a common partial reaction: abstrac- members of an enzyme superfamily provide a more complete tion of an R-proton from a carboxylate anion substrate understanding of the relationships between structure and function than is possible from focused studies of a single enzyme alone. -
Synthesis and Characterization of Doxorubicin Carrying Cetuximab-Pamam Dendrimer Bioconjugates
Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2012 SYNTHESIS AND CHARACTERIZATION OF DOXORUBICIN CARRYING CETUXIMAB-PAMAM DENDRIMER BIOCONJUGATES GUNJAN SAXENA Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Biomedical Engineering and Bioengineering Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/2788 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. © Gunjan Saxena 2012 All Rights Reserved SYNTHESIS AND CHARACTERIZATION OF DOXORUBICIN CARRYING CETUXIMAB- PAMAM DENDRIMER BIOCONJUGATES A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biomedical Engineering at Virginia Commonwealth University By Gunjan Saxena Bachelor of Engineering, Rajiv Gandhi Technological Institute, India, 2008 Director: Dr. Hu Yang, Ph.D., Associate Professor, Biomedical Engineering Virginia Commonwealth University Richmond, Virginia May 2012 ii Acknowledgement To my parents - Every bit of me is a little bit of you. This thesis is dedicated to my father, Nawal Saxena, who taught me that the best kind of knowledge to have is that which is learned for its own sake. It is also dedicated to my mother, Preety Saxena, who taught me to dream big and that even the largest task can be accomplished if it is done one step at a time. I am most grateful to them and my sister, Nikita Saxena, for having faith in me. -
An Introduction to Metabolism
CAMPBELL BIOLOGY IN FOCUS URRY • CAIN • WASSERMAN • MINORSKY • REECE 6 An Introduction to Metabolism Lecture Presentations by Kathleen Fitzpatrick and Nicole Tunbridge, Simon Fraser University © 2016 Pearson Education, Inc. SECOND EDITION The Energy of Life . The living cell is a miniature chemical factory where thousands of reactions occur . The cell extracts energy and applies energy to perform work . Some organisms even convert energy to light, as in bioluminescence © 2016 Pearson Education, Inc. Figure 6.1 © 2016 Pearson Education, Inc. Concept 6.1: An organism’s metabolism transforms matter and energy . Metabolism is the totality of an organism’s chemical reactions . Metabolism is an emergent property of life that arises from interactions between molecules within the cell © 2016 Pearson Education, Inc. Metabolic Pathways . A metabolic pathway begins with a specific molecule and ends with a product . Each step is catalyzed by a specific enzyme © 2016 Pearson Education, Inc. Figure 6.UN01 Enzyme 1 Enzyme 2 Enzyme 3 A B C D Reaction 1 Reaction 2 Reaction 3 Starting Product molecule © 2016 Pearson Education, Inc. Catabolic pathways release energy by breaking down complex molecules into simpler compounds . One example of catabolism is cellular respiration, the breakdown of glucose and other organic fuels to carbon dioxide and water © 2016 Pearson Education, Inc. Anabolic pathways consume energy to build complex molecules from simpler ones . The synthesis of proteins from amino acids is an example of anabolism . Bioenergetics is the study of how energy flows through living organisms © 2016 Pearson Education, Inc. Forms of Energy . Energy is the capacity to cause change . Energy exists in various forms, some of which can perform work © 2016 Pearson Education, Inc. -
Proteolytic Cleavage—Mechanisms, Function
Review Cite This: Chem. Rev. 2018, 118, 1137−1168 pubs.acs.org/CR Proteolytic CleavageMechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification Theo Klein,†,⊥ Ulrich Eckhard,†,§ Antoine Dufour,†,¶ Nestor Solis,† and Christopher M. Overall*,†,‡ † ‡ Life Sciences Institute, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ABSTRACT: Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissuefrom 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C- termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms -
Activity-Based Profiling of Proteases
BI83CH11-Bogyo ARI 3 May 2014 11:12 Activity-Based Profiling of Proteases Laura E. Sanman1 and Matthew Bogyo1,2,3 Departments of 1Chemical and Systems Biology, 2Microbiology and Immunology, and 3Pathology, Stanford University School of Medicine, Stanford, California 94305-5324; email: [email protected] Annu. Rev. Biochem. 2014. 83:249–73 Keywords The Annual Review of Biochemistry is online at biochem.annualreviews.org activity-based probes, proteomics, mass spectrometry, affinity handle, fluorescent imaging This article’s doi: 10.1146/annurev-biochem-060713-035352 Abstract Copyright c 2014 by Annual Reviews. All rights reserved Proteolytic enzymes are key signaling molecules in both normal physi- Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org ological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the ap- plication of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers. 249 BI83CH11-Bogyo ARI 3 May 2014 11:12 gens that contain inhibitory prodomains that Contents must be removed for the protease to become active.