DOCSLIB.ORG

Substrate and Nucleotide

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Substrate and Nucleotide ClpX interactions with ClpP, SspB, protein substrate and nucleotide by OF ECHNOLOGY Greg Louis Hersch FEB 0 1RIE2006 B.S. Biochemistry LIBRAR IES University of California at Davis, 2001 Submitted to the Department of Biology in partial fulfillment of the requirementsfor the degreeof Doctor of Philosophy in Biochemistry McM0.l at the Massachusetts Institute of Technology February 2006 © 2005 Greg L. Hersch. All rights reserved The authors herebygrants to MITpermission to reproduceand to distributepublicly Paper and electronic copies of the thesis document in whole or in part. Signature of Author: .1 - · -- - Department of Biology Certified by: I/ Robert T. Sauer Salvador E. Luria Professor of Biology Thesis Supervisor Accepted by: / - t R11 (_2~ ,tenhen P RPellI Professor of Biology Co-Chair, Biology Graduate Committee CIpX interactions with CIpP, SspB, protein substrate and nucleotide By Greg Louis Hersch Submitted to the Department of Biology on February 6th, 2006 in partial fulfillment of the requirements for the degree of doctor of philosophy in biochemistry ABSTRACT ClpXP and related ATP-dependent proteases are implements of cytosolic protein destruction. They couple chemical energy, derived from ATP hydrolysis, to the selection, unfolding, and degradation of protein substrates with the appropriate degradation signals. The ClpX component of ClpXP is a hexameric enzyme that recognizes protein substrates and unfolds them in an ATP-dependent reaction. Following unfolding, ClpX translocates the unfolded substrate into the ClpP peptidase for degradation. The best characterized degradation signal is the ssrA-degradation tag, which contains a binding site for ClpX and an adjacent binding site for the SspB adaptor protein. I show that the close proximity of these binding elements causes SspB binding to mask signals needed for ssrA-tag recognition by ClpX. The SspB dimer overcomes this signal masking by tethering itself and bound substrate to ClpX, via docking sites located in the dimeric N-terminal domain of ClpX. Because this N-domain dimer binds only a single SspB subunit, the ClpX hexamer can accommodate just one SspB dimer per hexamer. Other adaptor proteins that use these same tethering sites must compete with SspB for access to ClpXP. Substrates bearing ssrA tags with increased spacing between the SspB and ClpX binding elements are degraded more efficiently at low concentrations by ClpXP. This mechanism in which the adaptor first obstructs and then stimulates substrate recognition may have evolved to permit an additional level of regulation of substrate choice. SspB binding to ssrA-tagged substrate is a highly dynamic process, allowing rapid transfer of substrates from SspB to ClpX. Although the ClpX hexamer is composed of six identical polypeptides, individual subunits assume at least three distinct conformations. Using a hexamer that was engineered to prevent nucleotide hydrolysis, I show that some nucleotide-binding sites in ClpX release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Occupancy of both the slow sites by ATP and the fast sites by either ATP or ADP is required to bind the degradation tags of protein substrates. The ability of ClpX to retain binding of substrate with ATP or ADP in the fast sites suggests that nucleotide hydrolysis in the fast sites, but not in the slow sites, will allow repeated unfolding attempts without substrate release over multiple ATPase cycles. My results rule out ATPase models including ClpX 6eATP 6 or ADP 6 and also suggest that the enzyme hydrolyzes only a fraction of bound ATP in a single turnover event. Short peptide motifs of ClpX, known as IGF loops, interact with ClpP and change conformation as a response to nucleotide binding by ClpX. As ClpX varies its nucleotide content during the ATP hydrolysis cycle, it also varies its affinity for ClpP. Processing of substrates is coupled to the ATP-hydrolysis cycle of ClpX and appears to modulate ClpX's affinity for ClpP by changing how long each ClpX subunit spends in each nucleotide state. Thesis Supervisor: Robert T. Sauer Title: Salvador Luria Professor of Biology 2 TABLE OF CONTENTS Page Abstract 2 Chapter One An introduction to ClpX, ClpP, SspB, and the AAA+ superfamily of ATPases 7 Part I: Introduction 8 Part II: Interactions between ClpX, ClpP, adaptors, and substrate 14 Part III: The AAA+ superfamily 19 Part IV: Nucleotide utilization by AAA+ and related proteins 24 Part V: Interpretations and Conclusions 39 Chapter Two SspB Delivery of Substrates for ClpXP Proteolysis Probed by the Design of Improved Degradation Tags 56 Introduction and Methods 58 Design of extended-spacing ssrA tags 63 Improved SspB delivery to ClpXP 65 Dynamic interactions between SspB and ssrA tags 69 Discussion 72 Chapter Three Characterization of contacts with the adaptor protein, SspB mediated by the N-Domain dimer of ClpX 84 Introduction 86 Interactions of SspB with the ClpX N Domain 87 Discussion 89 Where does the XB peptide bind on the N-Domain? 92 Is SspB a ClpX specific adaptor protein? 94 Chapter Four Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine 99 An ATP-hydrolysis defective ClpX variant 102 Strength and stoichiometry of ATP binding 104 Cooperative interactions in wild-type ClpX 107 Multiple classes of ATP sites 108 Linkage between ClpX binding to the ssrA tag and to Mg++/ATP 109 ATP binding and structural changes in the ClpX pore 110 ADP substitutes for ATP in "fast" nucleotide binding sites 112 Discussion 114 Appendix One Communication between ClpX and ClpP during substrate processing and degradation 133 ClpP interaction requires more than two IGF loops 138 Substrate processing strengthens ClpX-ClpP affinity 141 Active-site communication between ClpP and ClpX 143 Discussion 149 3 LIST OF FIGURES AND TABLES Chapter One: The ATP-dependent protease, ClpXP and nucleotide utilization by AAA+ ATPases Figure 1 Architecture of the compartmentalized protease, ClpXP and a mechanical model for protein unfolding 10 Figure 2 Asymmetric docking of ClpX's six "IGF" loops with ClpP* 15 Figure 3 A general model for adaptor proteins 16 Figure 4 Architecture of a AAA+ active site 20 Figure 5 Three potential mechanisms of ATP hydrolysis by AAA+ hexamers 25 Figure 6 A postulated mechanism for ATP synthesis by F 1F ATP synthase 30 Table I Referenced crystal structures of AAA+ and related proteins 43 Chapter Two: SspB Delivery of Substrates for ClpXP Proteolysis Probed by the Design of Improved Degradation Tags Figure 1 - SspB delivery of ssrA-tagged substrates to ClpXP 59 Table I - Constants for ClpXP degradation and SspB binding to ssrA-tagged molecules 64 Figure 2 - ClpXP degradation of ssrA-tagged GFP variants in the presence of SspB 65 Figure 3 - ClpXP degrades the extended-tag substrates significantly faster than GFP-ssrA in the presence of the substrate-binding domain of SspB 68 Figure 4 - Equilibrium binding of SspB to GFP-ssrA 69 Figure 5 - Kinetics of dissociation and association of ssrA-tagged molecules with SspB 70 4 Chapter Three: Characterization of contacts with the adaptor protein, SspB mediated by the N-Domain dimer of ClpX Figure 1- Binding of the N Domain of ClpX and XB modules of SspB 88 Figure 2 - Binding stoichiometry of the N Domain of ClpX to XB modules of SspB 89 Figure 3 - Cartoon representation of SspB / ClpX interaction 90 Figure 4 - Perpendicular views of the N-domain dimer of ClpX 94 Chapter Four: Asymmetric interactions of ATP with the AAA+ C1pX6 unfoldase: allosteric control of a protein machine Figure 1 - A ClpX variant defective in ATP-hydrolysis 103 Figure 2 - ATP binding by various methods 105 Figure 3 - Dissociation kinetics reveals two classes of sites 109 Figure 4- Nucleotide dependence of ClpX substrate binding and conformation 111 Figure 5- ADP and substrate binding 113 Figure 6 - Models for ClpX binding to ATP and the ssrA tag of substrates 117 5 Appendix: Communication between CIpX and CIpP during substrate processing and degradation Figure 1- Symmetry mismatch between ClpX and ClpP* 137 Figure 2 - Properties of ClpX loopless 139 Figure 3 - An assay for ClpX-ClpP interaction in solution* 141 Figure 4 - ClpX-ClpP affinity changes in an ATPase-dependent fashion during substrate denaturation and translocation* 143 Figure 5 - Modification of the ClpP active sites strengthens ClpX binding* 144 Figure 6- ClpP rescues the unfolding defects of ClpX mutants* 146 Figure 7- The "ATP" state of ClpX is required for strong ClpP interactions* 147 Figure 8 - Model for the interaction of ClpX and ClpP 151 * work of Shilpa A. Joshi 6 CHAPTER ONE An introduction to ClpX, ClpP, SspB, and the AAA+ superfamily of ATPases 7 Part I: Introduction The roles of energy dependent proteolysis Many cytosolic proteins ultimately meet their fate at the hands of ATP-dependent proteases. This destruction is required to remove and recycle proteins that have become damaged by oxidation, heat unfolding and aggregation, or were simply translated from damaged mRNA (Gottesman, 1996). Energy-dependent proteases also destroy folded, native proteins as a signaling event. Examples of this occur as a response to DNA damage. environmental cues, and during the cell cycle (Jenal and Stephens, 2002; Gottesman, 2003; Jenal and Hengge-Aronis, 2003). Some of these same proteases function in quality assurance for the cell. Without quality control, translation of aberrant mRNA messages could result in partial protein products that aggregate or even actively interfere with vital cell processes. Instead, these proteins are recognized and removed by ATP-dependent proteases. Thus, energy dependent proteolysis allows the cell to dynamically control its proteome and respond rapidly to environmental cues. Energy- dependent proteases are all closely related, and belong to a much larger class of proteins called the AAA+ (ATPases associated with a variety of cellular activities) superfamily whose members all function in macromolecular disassembly (Neuwald et al., 1999; Vale, 2000; Glover and Tkach, 2001; Sauer et al., 2004).
Load more

Recommended publications
  • Structure of SARS-Cov-2 Main Protease in the Apo State Reveals the 2 Inactive Conformation 3
    bioRxiv preprint doi: https://doi.org/10.1101/2020.05.12.092171; this version posted May 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Structure of SARS-CoV-2 main protease in the apo state reveals the 2 inactive conformation 3 4 Xuelan Zhoua,1, Fangling Zhongb,c,1, Cheng Lind,e, Xiaohui Hua, Yan Zhangf, Bing Xiongg, 5 Xiushan Yinh,i, Jinheng Fuj, Wei Heb, Jingjing Duank, Yang Ful, Huan Zhoum, Qisheng Wang 6 m,*, Jian Li b,c,*, Jin Zhanga,* 7 8 a School of Basic Medical Sciences, Nanchang University, Nanchang, Jiangxi, 330031, China. 9 b College of Pharmaceutical Sciences, Gannan Medical University, Ganzhou, 341000, Jiangxi, 10 PR, China. 11 c Laboratory of Prevention and treatment of cardiovascular and cerebrovascular diseases, 12 Ministry of Education, Gannan Medical University, Ganzhou 341000, PR China 13 d Shenzhen Crystalo Biopharmaceutical Co., Ltd, Shenzhen, Guangdong, 518118, China 14 e Jiangxi Jmerry Biopharmaceutical Co., Ltd, Ganzhou, Jiangxi, 341000, China. 15 f The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 330031, China 16 g Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese 17 Academy of Sciences, 555 Zuchongzhi Road , Shanghai 201203 , China. 18 h Applied Biology Laboratory, Shenyang University of Chemical Technology, 110142, 19 Shenyang, China 20 i Biotech & Biomedicine Science (Jiangxi）Co. Ltd, Ganzhou, 341000, China 21 j Jiangxi-OAI Joint
    [Show full text]
  • Mechanistic Insights Into the Inhibition of Prostate Specific Antigen by [Beta
    proteins STRUCTURE O FUNCTION O BIOINFORMATICS Mechanistic insights into the inhibition of prostate specific antigen by b-lactam class compounds Pratap Singh,1,2* Simon A. Williams,2 Meha H. Shah,3 Thomas Lectka,3 Gareth J. Pritchard,4 John T. Isaacs,2 and Samuel R. Denmeade1,2 1 Department of Chemical and Biomolecular Engineering, Whiting School of Engineering, The Johns Hopkins University, Baltimore, Maryland 21218 2 Chemical Therapeutics Program, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231 3 Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218 4 Department of Chemistry, Loughborough University, Loughborough, Leicestershire LE11 3TU, United Kingdom ABSTRACT for the effect of stereochemistry of the lactam ring on the in- hibitory potency was elucidated through docking of b-lactam Prostate Specific Antigen (PSA) is a biomarker used in the enantiomers. As a validation of our docking methodology, diagnosis of prostate cancer and to monitor therapeutic two novel enantiomers were synthesized and evaluated for response. However, its precise role in prostate carcinogenesis their inhibitory potency using fluorogenic substrate based ac- and metastasis remains largely unknown. A number of stud- tivity assays. Additionally, cis enantiomers of eight b-lactam ies arguing in the favor of an active role of PSA in prostate compounds reported in a previous study were docked and cancer development and progression have implicated this their GOLD scores
    [Show full text]
  • Determining the Catalytic Role of Remote Substrate Binding Interactions in Ketosteroid Isomerase
    Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase Jason P. Schwans1, Daniel A. Kraut1, and Daniel Herschlag2 Department of Biochemistry, Stanford University, Stanford, CA 94305 Edited by Vern L. Schramm, Albert Einstein College of Medicine, Bronx, NY, and approved June 24, 2009 (received for review February 2, 2009) A fundamental difference between enzymes and small chemical with substrate binding solvent is displaced and excluded from the catalysts is the ability of enzymes to use binding interactions with active site and solvent exclusion may be important in shaping the nonreactive portions of substrates to accelerate chemical reactions. electrostatic environment within the active site (26, 27). Indeed, Remote binding interactions can localize substrates to the active solvent exclusion by substrate binding has been suggested to be site, position substrates relative to enzymatic functional groups important for catalysis in numerous enzymes (e.g., refs. 26–32). and other substrates, trigger conformational changes, induce local Jencks and others realized that remote binding interactions destabilization, and modulate an active site environment by sol- can do more than provide for tight binding between substrate vent exclusion. We investigated the role of remote substrate and enzyme. Reactions of bound substrates can be facilitated by binding interactions in the reaction catalyzed by the enzyme use of so-called ‘‘intrinsic binding energy’’, which can pay for ketosteroid isomerase (KSI), which catalyzes a double bond migra- substrate desolvation, distortion, electrostatic destabilization, tion of steroid substrates through a dienolate intermediate that is and entropy loss (3, 7, 33, 34). The term intrinsic binding energy stabilized in an oxyanion hole.
    [Show full text]
  • 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.
    [Show full text]
  • 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
    [Show full text]
  • Intrinsic Evolutionary Constraints on Protease Structure, Enzyme
    Intrinsic evolutionary constraints on protease PNAS PLUS structure, enzyme acylation, and the identity of the catalytic triad Andrew R. Buller and Craig A. Townsend1 Departments of Biophysics and Chemistry, The Johns Hopkins University, Baltimore MD 21218 Edited by David Baker, University of Washington, Seattle, WA, and approved January 11, 2013 (received for review December 6, 2012) The study of proteolysis lies at the heart of our understanding of enzyme evolution remain unanswered. Because evolution oper- biocatalysis, enzyme evolution, and drug development. To un- ates through random forces, rationalizing why a particular out- derstand the degree of natural variation in protease active sites, come occurs is a difficult challenge. For example, the hydroxyl we systematically evaluated simple active site features from all nucleophile of a Ser protease was swapped for the thiol of Cys at serine, cysteine and threonine proteases of independent lineage. least twice in evolutionary history (9). However, there is not This convergent evolutionary analysis revealed several interre- a single example of Thr naturally substituting for Ser in the lated and previously unrecognized relationships. The reactive protease catalytic triad, despite its greater chemical similarity rotamer of the nucleophile determines which neighboring amide (9). Instead, the Thr proteases generate their N-terminal nu- can be used in the local oxyanion hole. Each rotamer–oxyanion cleophile through a posttranslational modification: cis-autopro- hole combination limits the location of the moiety facilitating pro- teolysis (10, 11). These facts constitute clear evidence that there ton transfer and, combined together, fixes the stereochemistry of is a strong selective pressure against Thr in the catalytic triad that catalysis.
    [Show full text]
  • Crotonase Review
    1 Crotonases – Nature’s Exceedingly Convertible Catalysts 2 3 Christopher T. Lohans†#, David Y. Wang†#, Jimmy Wang†, Refaat B. Hamed‡, and 4 Christopher J. Schofield†* 5 6 † Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, 7 OX1 3TA, United Kingdom. 8 ‡ Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, 9 Egypt 10 11 *Address correspondence to: Christopher Schofield, [email protected], 12 Tel: +44 (0)1865 275625, Fax: +44 (0)1865 285002. 13 14 # C.T.L. and D.Y.W. contributed equally to this Perspective. 15 1 16 Abstract 17 The crotonases comprise a widely-distributed enzyme superfamily that has multiple 18 roles in both primary and secondary metabolism. Many crotonases employ oxyanion hole- 19 mediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) 20 thioester substrates (e.g., malonyl-CoA, α,β-unsaturated CoA esters) with both nucleophiles 21 and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases 22 that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates 23 including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, 24 Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved 25 fold formed from a central β-sheet core surrounded by α-helices, which typically 26 oligomerizes to form a trimer, or dimer of trimers. The presence of a common structural 27 platform and mechanisms involving intermediates with diverse reactivity implies that 28 crotonases have considerable potential for biocatalysis and synthetic biology, as supported by 29 pioneering protein engineering studies on them.
    [Show full text]
  • 4.3.2 Dynamic Light Scattering (DLS)
    Thesis Tobias Krojer Crystal Structure of DegP from Escherich Ca r d i f f UNIVERSITY PRIFYSGOL C ae RD y§> 2004 UMI Number: U584668 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U584668 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Crystal Structure of DegP from Escherichia coli by Tobias Krojer submitted for the degree of Ph.D. to School of Biosciences Cardiff University Cardiff CF10 3US United Kingdom The research was performed at: Max-Planck-Institut fur Biochemie Abteilung Strukturforschung 82152 Martinsried Germany 2004 Part of the work presented here has been previously published in: Krojer T, Garrido-Franco M, Huber R, Ehrmann M, Clausen T. (2002). Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine. Nature 416(6879):455-9. Summary DegP is a heat-shock protein which is localized in the periplasm of Escherichia coli . It is a common protein quality control factor and represents the only protein that can alternate between the two antagonistic activities of a protease and a chaperone in a temperature- dependent manner.
    [Show full text]
  • Role of Communication Between Subunits and Enzymes in Clpxp-Mediated Substrate Unfolding and Degradation
    Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation by Shilpa Arun Joshi A.B., Molecular and Cell Biology University of California at Berkeley, 1997 Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology at the Massachusetts Institute of Technology February 2004 2004 Shilpa A. Joshi. All rights reserved. The author hereby grants MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part. Signature of Author............................................................................................................................ Department of Biology February 2, 2004 Certified by........................................................................................................................................ Robert T. Sauer, Salvador E. Luria Professor of Biology Thesis Supervisor Accepted by....................................................................................................................................... Alan D. Grossman, Professor of Biology Co-Chair, Biology Graduate Committee Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation by Shilpa Arun Joshi Submitted to the Department of Biology on February 2, 2004 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ABSTRACT Chaperones and proteases play important roles in quality control
    [Show full text]
  • Δ3-Δ2-Enoyl-Coa Isomerase from the Yeast Saccharomyces Cerevisiae. Molecular and Structural Characterization
    ∆3-∆2-ENOYL-CoA ISOMERASE ANU FROM THE YEAST MURSULA SACCHAROMYCES CEREVISIAE Department of Biochemistry and Biocenter Oulu, Molecular and structural characterization University of Oulu OULU 2002 ANU MURSULA ∆3-∆2-ENOYL-CoA ISOMERASE FROM THE YEAST SACCHAROMYCES CEREVISIAE Molecular and structural characterization Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Kajaaninsali (Auditorium L6), Linnanmaa, on April 19th, 2002, at 2 p.m. OULUN YLIOPISTO, OULU 2002 Copyright © 2002 University of Oulu, 2002 Reviewed by Professor Juha Rouvinen Professor Reijo Lahti ISBN 951-42-6657-9 (URL: http://herkules.oulu.fi/isbn9514266579/) ALSO AVAILABLE IN PRINTED FORMAT Acta Univ. Oul. A 380, 2002 ISBN 951-42-6656-0 ISSN 0355-3191 (URL: http://herkules.oulu.fi/issn03553191/) OULU UNIVERSITY PRESS OULU 2002 Mursula, Anu, 3-2-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae Molecular and structural characterization Department of Biochemistry, University of Oulu, P.O.Box 3000, FIN-90014 University of Oulu, Finland, Biocenter Oulu, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland Oulu, Finland 2002 Abstract The hydratase/isomerase superfamily consists of enzymes having a common evolutionary origin but acting in a wide variety of metabolic pathways. Many of the superfamily members take part in β- oxidation, one of the processes of fatty acid degradation. One of these β-oxidation enzymes is the ∆3- ∆ 2-enoyl-CoA isomerase, which is required for the metabolism of unsaturated fatty acids. It catalyzes the shift of a double bond from the position C3 of the substrate to the C2 position.
    [Show full text]
  • Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis † ‡ × ⊥ × † ∥ △ # Florian Richter, , , Rebecca Blomberg, , ,+ Sagar D
    Article pubs.acs.org/JACS Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis † ‡ × ⊥ × † ∥ △ # Florian Richter, , , Rebecca Blomberg, , ,+ Sagar D. Khare, Gert Kiss, , Alexandre P. Kuzin, ∥ ◆ † ⊥ ∥ ∇ Adam J. T. Smith, , Jasmine Gallaher, Zbigniew Pianowski, Roger C. Helgeson, Alexej Grjasnow, ¶ # # # # # Rong Xiao, Jayaraman Seetharaman, Min Su, Sergey Vorobiev, Scott Lew, Farhad Forouhar, ¶ # ¶ ⊗ # ∥ Gregory J. Kornhaber, John F. Hunt, Gaetano T. Montelione, , Liang Tong, K. N. Houk, ⊥ † ‡ § Donald Hilvert,*, and David Baker*, , , † ‡ § Department of Biochemistry, Interdisciplinary Program in Biomolecular Structure and Design, and Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, United States ⊥ Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland ∥ Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States # Northeast Structural Genomics Consortium, Department of Biological Sciences, Columbia University, New York, New York 10027, ¶ and Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, United States ⊗ Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, United States ∇ Department of Biochemistry, Ludwig-Maximilians-UniversitatMü ̈nchen, 81377 München, Germany *S Supporting Information ABSTRACT:
    [Show full text]
  • Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole
    PLoS BIOLOGY Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole Daniel A. Kraut1, Paul A. Sigala1, Brandon Pybus2, Corey W. Liu3, Dagmar Ringe2, Gregory A. Petsko2, Daniel Herschlag1* 1 Department of Biochemistry, Stanford University, Stanford, California, United States of America, 2 Department of Biochemistry, Brandeis University, Waltham, Massachusetts, United States of America, 3 Stanford Magnetic Resonance Laboratory, Stanford University, Stanford, California, United States of America A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing pKa models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/pKa unit, suggesting a bond shortening of ;0.02 A˚/pKa unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (DDG ¼0.2 kcal/mol/pKa unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (DDH ¼2.0 kcal/mol/pKa unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ;300-fold.
    [Show full text]