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Assay of the Prostate Cancer Biomarker A-Methylacyl Coenzyme a Racemase (AMACR)

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Assay of the Prostate Cancer Biomarker A-Methylacyl Coenzyme a Racemase (AMACR) Assay of the Prostate Cancer Biomarker a-Methylacyl Coenzyme A Racemase (AMACR) by Dahmane Ouazia Submitted in partial fulfillment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia June 2008 © Copyright by Dahmane Ouazia, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-43994-4 Our file Notre reference ISBN: 978-0-494-43994-4 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada DALHOUSIE UNIVERSITY To comply with the Canadian Privacy Act the National Library of Canada has requested that the following pages be removed from this copy of the thesis: Preliminary Pages Examiners Signature Page (pii) Dalhousie Library Copyright Agreement (piii) Appendices Copyright Releases (if applicable) Dedication Page This thesis is dedicated to my brother Dr. Boualem Ouazia to whom I owe the privilege of being a Canadian citizen. Table of Contents List of Tables viii List of Figures ix List of Schemes xi Abstract xii List of Abbreviations and Symbols Used xiii Acknowledgments xv Chapter 1 Introduction 1 1.1 Overview 1 1.2AMACR 1 1.3 AMACR Catalysis 4 1.4 Role of AMACR in Metabolism 14 1.5 AMACR and Prostate Cancer 15 1.6 Current AMACR Assays 17 1.7 Direct Racemase Assays 21 Chapter 2 Materials and Methods 22 2.1 General 22 2.2 Resolution of (jR)-Ibuprofen 22 2.3 Ibuprofenoyl-CoA 23 2.4 Ibuprofenoyl-CoA Quantification 24 2.5 His6-tagged Rat AMACR Expression and Purification 25 2.6 Glutathione-S-transferase (GST)-tagged Rat AMACR Expression and Purification 26 v 2.7 His6-tagged TBmcr Expression and Purification 27 2.8 Enzyme Assay 28 Chapter 3 Results 30 3.1 Expression and Purification of Recombinant AMACR 30 3.1.1 His6-rat AMACR 30 3.1.2 GST-rat AMACR 30 3.1.3 His6-TBmcr 32 3.2 Characterization of Ibuprofenoyl-CoA 34 3.2.1 Ibuprofenoyl-CoA Quantification 34 3.2.2 UV Spectra 36 3.2.3 CD Spectra 39 3.3 Calculation of Velocities from CD Data 43 3.4 Assay Development 44 3.4.1 Monitoring the TBmcr-catalyzed Reaction in Both Directions 44 3.4.2 Effect of Octyl-p-D-glucopyranoside 46 3.4.3 Effect of Enzyme Concentration 46 3.4.4 Dependence of the Initial Velocity on Substrate Concentration 50 Chapter 4 Discussion 55 4.1 Enzyme Expression and Purification 55 4.2 Quantification of the Ibuprofenoyl-CoA Substrates 58 4.3 Circular Dichroism of the Ibuprofenoyl-CoA Substrates 60 4.4 AMACR Assay 60 VI Chapter 5 Future Work Chapter 6 Conclusion Appendix Bibliography vn List of Tables Table 3.1: Kinetic parameters of the TBmcr-catalyzed epimerization of ibuprofenoyl-CoA in both directions using His6-TBmcr and tag-free TBmcr 53 vin List of Figures Figure 1.1: Amino acid sequence alignments of the regions containing the catalytic residues in TBmcr and, rat and human AMACR 6 Figure 1.2: Overall view of the X-ray crystal structure of the unliganded form of TBmcr 12 Figure 1.3: View of the TBmcr active site from the X-ray crystal structure of the TBmcr-(25, 2i?)-ibuprofenoyl-CoA complex 13 Figure 3.1: SDS (10 %)-PAGE gel of the metal ion affinity chromatography purification fractions of His6-rat AMACR in BL21 (DE3) cells 31 Figure 3.2: SDS (10 %)-PAGE gel of affinity chromatography purification fractions of GST-rat AMACR in BL21 (DE3) cells 33 Figure 3.3: SDS (12 %)-PAGE gel of purified TBmcr 35 Figure 3.4: Linearity between the peak area and (5)-ibuprofen concentration shown as an HPLC chromatogram (A) and as a standard curve (B) 37 Figure 3.5: HPLC chromatograms showing the products of alkaline hydrolysis of (25)-ibuprofenoyl-CoA 38 Figure 3.6: UV spectra of (25)- and (2i?)-ibuprofenoyl-CoA between 220 nm and 320 nm 40 Figure 3.7: CD spectra of (25)- (•), (2R)- (•), and (2S, 2#)-ibuprofenoyl-CoA (A) between 220 nm and 320 nm 41 Figure 3.8: Progress curve of the His6-TBmcr-catalyzed reaction using (25)- and (2i?)-ibuprofenoyl-CoA 45 Figure 3.9: Effect of octyl-P-D-glucopyranoside on the TBmcr-catalyzed reaction using (2i?)-ibuprofenoyl-CoA as the substrate 47 Figure 3.10 Effect of high octyl-P-D-glucopyranoside concentrations on the TBmcr-catalyzed reaction using (2i?)-ibuprofenoyl-CoA as the substrate 48 Figure 3.11: The effect of enzyme concentration on the tag-free TBmcr-catalyzed reaction at high and low concentrations of (2i?)-ibuprofenoyl-CoA 49 Figure 3.12: Representative Michaelis-Menten plots for Hisg-TBmcr 51 IX Figure 3.13: Representative Michaelis-Menten plots for tag-free TBmcr 52 Figure 4.1: Hydrophobicity profile of the TBmcr dimer 63 Figure 4.2: Upwardly-curving dependence of initial velocity on enzyme concentration 66 x List of Schemes Scheme 1.1: Typical substrates of AMACR 2 Scheme 1.2: Metabolism of branched-chain fatty acids 3 Scheme 1.3: Mechanism of the AMACR-catalyzed reaction 5 Scheme 1.4: The role of AMACR in the synthesis of the bile acid glycocholic acid 7 Scheme 1.5: Mechanisms of 1,1-proton transfer in a racemization reaction: (A) one- base mechanism and (B) two-base mechanism 9 Scheme 1.6: (2R)- and (25)-Ibuprofenoyl-CoA derivitization for HPLC-based assay 19 Scheme 1.7: (2R)- and (25)-Methylmyristoyl-CoA derivitization for GLC-based assay 20 Scheme 5.1: Potential reversible AMACR inhibitors 72 Scheme 5.2: Competitive inhibitors of AMACR 74 Scheme 5.3 Potential irreversible AMACR inhibitors 75 XI Abstract a-Methylacyl-CoA racemase (AMACR) catalyzes the epimerization of the (2i?)- and (25)-methyl branched coenzyme A thioesters. AMACR is over-expressed in prostate carcinoma cells and not in benign and normal prostate cells and is a recognized biomarker for prostate cancer and a target for the development of therapeutic agents directed against the disease. A continuous circular dichroism-based assay has been developed using (2R)- or (2S)-ibuprofenoyl-CoA as substrates. The open reading frame encoding AMACR from Mycobacterium tuberculosis (TBmcr) was sub-cloned into a pET15b vector, overexpressed, and purified using metal ion affinity chromatography. The assay showed that TBmcr catalyzes the complete epimerization of (25)-and (2R)- ibuprofenoyl-CoA. The kinetic parameters for both directions of the AMACR-catalyzed reaction were obtained using both the (His)6-tagged and untagged forms of TBmcr. Both enzyme forms exhibited a greater affinity (l/Km) for (2i?)-ibuprofenoyl-CoA than the (2S)-thioster, but a greater turnover number (^at)iWith (25)-ibuprofenoyl-CoA. Overall, both enzymes exhibited a similar catalytic efficiency {kcJK^) with both substrates. The presence of the His6-tag leads to a 22% and-47% decrease in catalytic efficiency of TBmcr with the (25)- and (2i?)-thioesters, respectively. This assay offers a novel, economical, and efficient alternative method to the existing radioactivity-based assay in assessing AMACR activity and for studying the inhibitory activity of small molecules targeting AMACR. xn List of Abbreviations and Symbols Used AMACR alpha-methylacyl coenzyme A racemase (EC 5.1.99.4) BPH benign prostatic hypertrophy BuOH butanol CD circular dichroism cmc critical micelle concentration CoA coenzyme A DAG diacylglycerol DKG diacylglycerol kinase (EC 2.7.1.107) GLC gas liquid chromatography GST glutathione-S-transferase HEPES 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid His6 hexahistidine HPLC high performance liquid chromatography IHC immunohistochemistry IPTG isopropyl-(3-D-thiogalactopyranoside MBP maltose-binding protein MeCN acetonitrile mp melting point OG octyl-P-D-glucopyranoside ORD optical rotatory dispersion ORF open reading frame PBS phosphate buffered saline xm PCR polymerase chain reaction PSA prostate-specific antigen R/ retention factor RT reverse transcriptase SDS-PAGE sodium dodecyl sulfate-polyacrylarriide gel electrophoresis SPE solid phase extraction TBmcr AMACR from Mycobacterium tuberculosis THCA-CoA trihydroxycoprostanoyl-CoA THF tetrahydrofuran TLC thin-layer chromatography tr retention time xiv Acknowledgments First, I would like to thank my supervisor, Dr. Stephen Bearne, for his guidance and supervision, for his extremely helpful editing, and for occasionally ridiculing my "extreme leftism".
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