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

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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".

I would like to thank the members of my supervisory committee, Dr. Vanya Ewart, Dr. Cathy Too, and Dr. David Waisman for their help and advice. I would like to thank Dr. John Belise for providing the genomic DNA of Mycobacterium tuberculosis, Dr. Mark Nachtigal for providing the RNA extract from rat liver, and Dr. Christopher McMaster for providing the pGEX-3X vector. I would like to thank the Cancer Research Training Program (CRTP) for the financial support that it provided me, and the Prostate Cancer Foundation for funding the AMACR project.

Of all the colorful people that have been part of the Bearne lab during the last 3 years, I would specifically like to thank Faylene Lunn, Craig Steeves, and Jennifer Bourque for being the best of friends, on top of being great colleagues. I would like to thank Dr. Joanna Potrykus, Dr. Srinath Thirumalairajan, and Dr. Ariun Narmandakh for their help.and advice. I would also like to thank Alexander Roy for lending me his textbooks and for occasionally bringing me pop from McDonald's.

I would like to thank my family for their perpetual support and motivation, especially my mother Hamida, my late father Ali, my brothers Boualem, Kamel and Mohamed, my sisters Sabina and Dalila, and my nieces Karina, Katia, Amel, and Imen; and my nephew Walim. I would also like to thank my adoptive Nova Scotian family, the Lunns/Greenes, for their hospitality and for making me feel as one of their own.

Finally, I want to add that my living experience in Halifax has been plagued with extreme highs and lows; but as an Ontarian I will mostly remember the warmth that characterize the inhabitants of this region of Canada.

"One of the symptoms of an approaching nervous breakdown is the belief that one's work is terribly important." - Bertrand Russell (1872-1970)

xv Chapter 1

Introduction

1.1 Overview. Alpha-methylacyl coenzyme A racemase (AMACR, EC 5.1.99.4) catalyzes the epimerization of (2R)- and (25)-methyl branched thioesters (Scheme 1.1). The enzyme plays an important role in the metabolism of C27 bile acid intermediates and methyl-branched fatty acids as shown in Scheme 1.2. In the early 2000s, the enzyme was identified as a biomarker for prostate cancer and, over the past years, considerable interest has focused on developing a detailed understanding of the mechanism of the enzyme, its role in prostate and other cancers, and developing inhibitors of the enzyme. To facilitate these studies, an economical, accurate, and convenient enzymatic assay is required. The present work describes the development of a continuous circular dichroism (CD)-based assay of AMACR activity.

1.2 AMACR. AMACR is an enzyme thus far found in the bacterium Mycobacterium tuberculosis (Savolainen et al, 2005) but widely in mammals including mice (Schmitz et al, 1997), rats (Schmitz et al, 1994), and humans (Schmitz et al, 1995). The enzyme is predicted to exist in other organisms such as Aspergillus niger (Pel et al, 2007) and Bradyrhizobium japonicum (Kaneko et al, 2002) through the sequencing of their respective genomes. AMACR is located in both mitochondria and peroxisomes in eukaryotic cells (Kotti et al, 2000). The forms of the enzyme found in both organelles are derived from a single transcript (Amery et al, 2000; Kotti et al, 2000). The primary amino acid sequences of the mammalian forms of the enzyme have an N-terminal mitochondrial targeting signal and a C-terminal peroxisomal targeting sequence type 1 (- KASL in humans, -KANL in rat) (Ferdinandusse et al, 2000). Recently, it has been- shown that minor splice variants of the enzyme co-exist with the major form of the enzyme referred to as AMACR 1A (Shen-Ong et al, 2003).

1 SCoA

pristanoyl-CoA 6

SCoA

ibuprofenoyl-CoA

SCoA

HCf ^ i ^ "OH H 3,7,12-trihydroxycoprostanoyl-CoA(THCA-CoA)

a-methyl-branched CoA thioester O O O /CX^G. ogo g^ ° e^ O=P—o 4

-SCoA

Scheme 1.1 Typical substrates of AMACR. AMACR only acts on the CoA thioester form of 2-methyl-branched substrates. The acyl group of the substrates varies greatly in size. The structure of the CoA moiety is shown in detail at the bottom of the scheme.

2 oxidation oxidation + + (£/Z)-phytol activation activation (4 reactions) (4 reactions)

'SCoA 'SCoA

H CH3 Q (3S)-phytanoyl-CoA (3K)-phytanoyl-CoA

a-oxidation a-oxidation + + activation activation

AMACR SCoA SCoA H3r-H (2S)-pristanoyI-CoA (2/?)-pristanoyl-CoA

p-oxidation

Scheme 1.2 Metabolism of branched-chain fatty acids. Free phytol from animal-based food is converted to phytanoyl-CoA, which undergoes a-oxidation in the peroxisome leading to the two diastereomers of pristanoyl-CoA. The stereospecificity of the P- oxidation pathway only allows for the (25)-pristanoyl-CoA to be catabolized. AMACR converts the (2i?)-prostanoyl-CoA to the (25)-pristanoyl-CoA. The study of the minor splice variants revealed a common N-terminus containing the mitochondrial targeting sequence. The C-terminal peroxisomal targeting sequence type 1 signal is missing in all minor splice variants, which implies that they all have an exclusive mitochondrial localization (Mubiru et al, 2004; Mubiru et al, 2005).

The AMACR-catalyzed reaction is a cofactor-independent, 1,1-proton transfer

(Hasson et al, 1998), in which a general basic residue (Bi:) in the active site abstracts a proton from the (i?)-thioester leading to the formation of a planar enolate intermediate

(Scheme 1.3). The conjugated acid of a general basic residue (B2H(+)) donates a proton to the opposite face concomitant with rotation of the acyl group. The AMACR-catalyzed reaction is carried out by two conserved residues, histidine (^-specific) and aspartate (R- specific), in the active site of the enzyme (Bhaumik et al, 2007) (Figure 1.1). Although the AMACR-catalyzed reaction is reversible, the equilibrium lies in favour of the (S)- thioester in vivo (Schmitz et al., 1994). The endogenously synthesized C27 bile acid intermediates (oxidized cholesterol derivatives) have exclusively an (i?)-stereochemistry

(Ferdinandusse et al, 2001; Van Veldhoven et al, 2001) (Scheme 1.4), whereas the methyl-branched fatty acids available in the diet and the 2-arylpropionic acids ingested usually have both the (S)- and (^-configurations (Shieh & Chen, 1993; Thornburg et al,

2006). Only the resulting (5)-thioester of the C27 bile acid intermediates and of the methyl-branched fatty acids are catabolized via peroxisomal (3-oxidation (Schmitz &

Conzelmann, 1997).

1.3 AMACR Catalysis. Racemases and epimerases are isomerase enzymes that catalyze the inversion of stereochemistry in biological molecules (Tanner, 2002).

4 E-B, E-BJH+ i?-pocket CoA-S CoA-S ^ " \ u"*^-—-oS-pockeR t E-Biir

Scheme 1.3 Mechanism of the AMACR-catalyzed reaction. The mechanism is a 1,1- proton transfer leading to inversion of chirality at the a-carbon formally resulting in exchanging the positions of the a-proton and the acyl chain (R group).

5 Rat ARADVLLEPFRCGVMEKLQLGPETLQQDNPKLIYARLSGFGQSGIF 95 Human KRSDVLLEPFRRGVMEKLQLGPEILQRENPRLIYARLSGFGQSGSF 116 MCR AKADVLIEGYRPGVTERLGLGPEEGAKVNDRLIYARMTGWGQTGPR 120 • ••4f^4r«4r**4f ,fc'Jemic*'ic»'ftfefcft •• 'fc^f'fc'fc'ft'fcfcmm'fcm'fc'^c*'^ • • •• • • • ••• •• •• ••• • • 12 3 Rat SKVAGHDINYVALSGVLSKIGRSGENPYPPLNLLADFGGGGLMCTL 141 Human CRLAGHDINYLALSGVLSKIGRSGENPYAPLNLLADFAGGGLMCAL 162 MCR SQQAGHDINYISLNGILHAIGRGDERPVPPLNLVGDFGGGSMFLLV 166 . ******* .*.*.*..***..*.*..****..**.**....

Figure 1.1 Amino acid sequence alignments of the regions containing the catalytic residues in TBmcr, rat, and human AMACR. His 126, Asp 156, and Asp 127 are annotated by the numbers 1, 2, and 3, respectively, above the letters corresponding to the residues. Identical residues in all three forms of the enzyme (*) and, identical residues in only 2 forms of the enzyme (:) are shown. The sequence alignment was generated using ClustalW (Pearson &Lipman, 1988). -

6 initiation (1 step)

HO ^ ^ OH

5-cholesten-3p-ol 5-cholesten-3p,7a-diol (cholesterol)

ring structure modification (4 steps)

CSCoA side chain oxidation (4 steps)

v HO ^-" ^ "OH HO" ^ "OH 25(R) 3a, 7a, 12a-trixhydroxy-5p- 5p-cholestane-3a,7a,i2a-triol cholestanoyl-CoA

side chain oxidation (3 steps)

25(S) 3a, 7a, 12a-trixhydroxy-5p- 3a, 7a, 12a-trihydroxy-5p- cholestanoyl-CoA cholan-24 one-CoA

Conjugation (1 step)

HO v ^"" OH 5p-cholanic acid-3a,7a, 12a-triol N-(carboxymethyl)-am ide (glycocholic ascid)

Scheme 1.4. The role of AMACR in the synthesis of the bile acid glycocholic acid. AMACR catalyzes the 13th step of the pathway (indicated by the arrow).

7 Racemases catalyze the stereochemical inversion around the asymmetric carbon atom in

a substrate having only one center of asymmetry. Epimerases catalyze the stereochemical

inversion of the configuration about an asymmetric carbon atom in a substrate having

. more than one center of asymmetry; thus interconverting epimers. Strictly speaking,

AMACR is not a racemase, but an epimerase considering the multiple stereogenic centers

on the Co A moiety (Scheme 1.1). In order for the reaction to proceed, the racemase or

epimerase must be able to break and reform a bond in an apparently non-stereospecific

manner. The majority of racemases and epimerases act at a chiral centre adjacent to a

carbonyl group and reversibly cleave a C-H bond. In the case of cofactor-independent

racemases, the pisTa of the carbon acid is lowered as the result of resonance stabilization of

the resulting anion (Tanner, 2002), thereby promoting deprotonation and reprotonation of

the substrate. In the case of AMACR, it is the formation of the CoA thioester that is

responsible for lowering the pKa of the a-proton from ~ 34 to 21 (Richard & Amyes,

2001). In general, the deprotonation/reprotonation reactions catalyzed by racemases

occur via one of two mechanisms: a one-base mechanism or a two-base mechanism

(Scheme 1.5).

In the one-base mechanism, a single base in the active site of the enzyme

deprotonates the substrate and then reprotonates the resulting anionic intermediate. In the

two-base mechanism, one-base in the active site deprotonates the substrate and the

conjugate acid of a second base reprotonates the planar intermediate (Tanner, 2002).

8 VUVXAAAA/WAAATUVl VUVA/WAAAA/JAAAA/l

B: © R c H3N

CO, C02 e e

•\j\f\f\s\r\r\j\s\i\f\f\s\r\r\n -irwvv/XA/v\n/vvriru\rui

B2 *) © R

•^NH3

CO, C02 e e

Scheme 1.5 Mechanisms of 1,1-proton transfer in a racemization reaction: (A) one- base mechanism and (B) two-base mechanism. Scheme was adapted from Tanner, 2002.

9 Experimentally, the two mechanisms are distinguished by the use of isotope-containing solvents (i.e., D2O). The incorporation of solvent isotope (i.e., D) must be observed in the two-base mechanism due to the proton exchange between the protic residues and the solvent. However, it can also be observed in a one-base mechanism if the a-proton can be exchanged with the solvent or if the base in the active site is polyprotic (e.g., lysine).

Therefore, these solvent isotope experiments must be conducted under initial velocity conditions (Choi et al., 1992; Gallo et al, 1993). Under such conditions, if the formed product molecules contained solvent-derived deuterium and the starting substrate did not, it could only be explained by a two-base mechanism as the anionic intermediate in a one base mechanism would partition both forward and backward, resulting in solvent isotope incorporation into both the product and the substrate.

The use of a radiolabeled substrate containing a 3H in the a-position leading to the exchange of the radiolabel into the solvent is often used to demonstrate that a racemase functions via a C-H cleavage reaction. For AMACR, the incubation of [2-3H]- pristanoyl-CoA with purified rat AMACR led to the formation of 3HaO due to the very rapid exchange of the H atom in the a-position with solvent water. This information, along with sequence information, suggested that the AMACR-catalyzed reaction proceeded through a two-base proton transfer mechanism. The fact that iodoacetamide, which alkylates sulfhydryl groups, did not inhibit the proton transfer suggested that the two bases were not cysteine residues (Schmitz et al, 1994) as is the case for the "classic" cofactor-independent racemases, glutamate racemase (Gallo et al, 1993) and proline racemase (Cardinale & Abeles, 1968). However, the specific catalytic residues were not identified until the crystal structure of AMACR from Mycobacterium tuberculosis

10 (TBmcr) was solved. TBmcr was studied because Mycobacterium tuberculosis is the

causative agent of tuberculosis, and responsible for approximately 2 million human

deaths every year (Snider et al, 1994). TBmcr represents a potential target for the

development of anti-tuberculosis drugs since the enzyme plays a critical role in the J3-

oxidation of methyl-branched alkanes (Sakai et al, 2004).

TBmcr is 43% and 44% identical to human and rat AMACR, respectively (Figure

1.1). The crystal structure of the unliganded TBmcr has been solved and revealed that

TBmcr is a homodimer (Figure 1.2, Panel A) with each subunit composed of two domains, the N-terminal large domain and the C-terminal small domain. The active site is located at the interface between the large and small domains of two monomers of the dimeric enzyme. Site-directed mutagenesis studies showed that the His 126Ala and

Asp 156Ala enzymes were inactive, while keeping the native conformation, thereby indicating that the two catalytic residues were His 126 and Asp 156 (Figure 1.3) and confirming that AMACR functioned by a two-base mechanism (Savolainen et al, 2005).

These two catalytic residues are conserved in AMACR from different species making

TBmcr a good enzyme to be used as a model for mammalian AMACRs.

The crystal structures of five TBmcr ligand-complexes (TBmcr with acetyl-CoA, acetoacetyl-CoA, (2i?,25)-ibuprofenoyl-CoA, (25)- and (2i?)-methylmyristoyl-CoA) further confirmed that His 126 and Asp 156 were the catalytic residues since the a-carbon was precisely located between His 126 (at 3.3 A) and Asp 156 (at 3.7 A) (Bhaumik et al,

2007).

11 Figure 1.2 Overall view of the X-ray crystal structure of the unliganded form of TBmcr. (A) TBmcr is a homodimer [pdb access code = 1X74 (Savolainen et al, 2005)]. The two subunits forming the dimer are shown in blue and magenta. The residues involved in catalysis (His 126, Asp 127, and Asp 156) are shown in green. (B) The active site is located at the interface of both subunits that form the dimer.

12 Figure 1.3 View of the TBmcr active site from the X-ray crystal structure of the TBmcr-Cl^l^-ibuprofenoyl-CoA complex. His 126 (red) and Asp 156 (yellow) are the catalytic residues. The (25)- and (2/?)-ibuprofenoyl-CoA are represented in magenta and yellow, respecively. The backbone amide (in blue) of Asp 127 stabilizes the oxyanion of the planar intermediate via hydrogen bonding with the oxygen atom (red) of the substrate. Note the different positioning of the acyl groups of the substrates confirming the movement of the acyl chain upon chiral interconversion. [pdb access code = 2GCE (Bhaumik et al, 2007)].

13 In addition, the structures suggested that Asp 127 played a role in catalysis through the stabilization of the negatively charged oxygen atom of the enolate intermediate (creating an oxyanion hole) via hydrogen bonding to its backbone amide group (Bhaumik et al,

2007). Furthermore, structural evidence suggested that both the a-methyl and Co A thioester groups remain bound to the same region of the protein after chiral inversion.

However, the acyl group interchanges positions with the hydrogen atom. The acyl group of the (5)-thioester is bound to a region called the (5)-pocket (composed of He 16, Pro 20, and Met 198 from the small domain of the adjacent subunit); whereas the acyl group of the (K)-thioester is bound to a region called the (i?)-pocket (composed of He 240 and Met

216 both from the small domain of the adjacent subunit). The chiral inversion is accompanied by a movement of the acyl group (Figure 1.3) from one pocket to the other over a hydrophobic methionine-rich surface (Bhaumik et al, 2007). The motion of a bulky hydrophobic group during catalysis has been implicated in the mandelate racemase-catalyzed racemization of mandelate (Siddiqi et al, 2005).

1.4 Role of AMACR in Metabolism. Since 2001, it has become apparent that there is a link between dietary branched chain fatty acids (especially phytanic acid), peroxisomal P- oxidation (including its "gate-keeper" AMACR), and cancer (more specifically prostate cancer) (Jiang et al, 2001). Phytanic acid is a 3-mefhyl branched dietary fatty acid

(Scheme 1.1). It is derived from the isoprenoid chain of chlorophyll A, called phytol.

Phytol can only be derived from chlorophyll A in ruminants, which explains why plant- derived foods are not a source of phytol for humans. Thus, animal-derived foods (meat and dairy products) are the only source of phytol in the form of phytanic acid (Lloyd et al, 2008). Peroxisomes are ubiquitous organelles (Seedorf, 1998) that are the site of

14 synthesis of essential lipids and of the catabolism of 'unusual' fatty acids such as the branched-chain fatty acid phytanic acid (Mukherji et al, 2003). Phytanic acid undergoes a-oxidation in the peroxisome to form formyl-CoA and pristanic acid (Mukherji et al,

2003). (2S)-Pristanoyl-CoA is shortened by three rounds of (3-oxidation producing 1 equivalent of acetyl-CoA, 2 equivalents of propionyl-CoA, and a chain-shortened intermediate that are all exported to the mitochondria via the acyl-carnitine shuttle for final catabolism. AMACR is also required for mitochondrial (3-oxidation to occur since the chain-shortened intermediates contain a-methyl groups in the (i?)-configuration

(Mukherji et al, 2003). The main difference between mitochondrial and peroxisomal (3- oxidation is that the latter is not linked to ATP production, and hence leads to the generation of hydrogen peroxide (H2O2). The H2O2 generated by peroxisomal P-oxidation is usually converted to water and oxygen by the catalase (Wanders et al, 2001).

However, up-regulation of peroxisomal P-oxidation due to a high intake of dietary methyl branched fatty acids (Giovannucci et al, 1993) is thought to generate high amounts of

H2O2 that may cause DNA damage thereby leading to tumourgenesis (Tamatani et al,

1999). In fact, high throughput cDNA analysis showed that AMACR is over-expressed in prostate carcinoma cells but not in normal or benign prostate cells (Dhanasekaran et al,

2001). This finding was later confirmed by immunohistochemistry (IHC) (Jiang et al,

2004) and Western blotting (Luo et al, 2002).

1.5 AMACR and Prostate Cancer. Prostate cancer is the most common type of cancer

t among Canadian men. In 2008, an estimated 24,700 Canadian men will be diagnosed with prostate cancer and 4,300 will die of the disease (Canadian Cancer Society, National

Cancer Institute of Canada, 2008). Prostate cancer is characterized by the absence of any

15 symptoms during early stages of the disease, which makes detection at a localized stage of the disease difficult. Most cases of prostate cancer are diagnosed at an advanced stage of the disease when symptoms associated with urinary function appear. In addition, current detection methods are not very specific (Stamey et al., 2004). The main screening method for prostate cancer is the measurement of prostate-specific antigen (PSA) levels.

PSA is a serine protease that is thought to liquiefy the seminal fluid (Lilja, 1985) when excreted from the prostate epithelium where it is produced (Lilja et al, 2008). PSA release in the blood stream is associated with prostate cancer. However, this PSA

'leakage' is not prostate cancer-specific because this phenomenon is also influenced by benign prostatic hypertrophy (BPH), prostatitis, increased age, body mass index, and race. Current management protocols suggest that patients with high PSA levels (> 4 ng/mL) undergo biopsy (Lilja et al, 2008). The biopsy tissue is subjected to a morphological observation that is time consuming and somewhat subjective. Also, the treatments that are available for prostate cancer patients (radical prostatectomy, radiotherapy, hormone-deprivation therapy, etc.) are associated with major side effects that include impotence (O'Rourke, 2000) and incontinence (Hollenbeck et al, 2003).

Both side effects cause psychological distress for the recovering patients and have major repercussions on the health care system. Therefore, new strategies for treating prostate cancer are desirable. One of the exploitable particularities of prostate cancer is that it does not rely on the upregulation of glycolysis for the energy requirements of rapid cell proliferation, which is the case for most malignencies (Warburg, 1956). Prostate cancer cells primarily rely on fatty acid metabolism for generating the energy that is necessary for its progression (Liu, 2006). Therefore, many enzymes involved in fatty acid

16 metabolism are up-regulated as is the case for AMACR, which generates elevated amounts of acetyl-CoA (Kumar-Sinha et al, 2004). This leads to an up-regulation of citrate oxidation, which is an important source of energy for the tumour (Costello et al,

1999).

AMACR over-expression is linked to human prostate cancer and also kidney tumours (Chen et al, 2005; Gupta et al, 2004) and colon carcinomas (Jiang et al, 2001).

Furthermore, it has been shown that inhibition of AMACR expression using siRNA slowed cell growth in the androgen-responsive prostate cancer cell line LAPC-4 by causing a G2-M cell cycle arrest (Zha et al, 2003). The same study showed that the expression and function of AMACR are independent of androgen-mediated signaling.

Thus, AMACR is a potential anti-cancer drug target in addition to serving as a biomarker for prostate cancer.

Targeting AMACR activity for inhibition may disrupt the energy requirements of prostate cancer cells leading to inhibition of tumour growth. The design of small molecules capable of inhibiting AMACR activity in vivo may provide a new class of anti­ cancer drugs. The inhibitory activity of these designed small molecules needs to be tested in vitro using an appropriate assay.

•1.6 Current AMACR Assays. Currently, AMACR is detected in prostate tissue sections using IHC which, although semi-quantitative for total AMACR protein, does not provide any measure of AMACR activity (Varma & Jasani, 2005). Several methods of measuring

AMACR activity have been described, all employing a fixed-time assay. A high performance liquid chromatography (HPLC)-based method has been reported wherein

AMACR activity was assessed using (25)-, (2i?)-ibuprofenoyl-CoA (Shieh & Chen,

17 1993), and (25i<:)-trihydroxycoprostanoyl-CoA (THC-CoA) (Ferdinandusse et al., 2000) as substrates. In the case of the ibuprofenoyl-CoA, the AMACR-catalyzed reaction was terminated with hydroxylamine and the resulting hydroxamate was treated with acid, followed by extraction of the free ibuprofen, conversion of the ibuprofen to its corresponding acyl chloride by treatment with thionyl chloride, and finally reacted with

(1R, 2S, 5i?)-menthol to give the (IR, IS, 5i?)-menthyl ester (Scheme 1.6). The diastereomeric composition was determined by separation of the diastereomeric esters using reversed-phase HPLC. When (25i?)-THCA-CoA was the substrate, the diastereomeric composition of the unmodified thioesters was determined directly using reversed-phase HPLC. Gas liquid chromatography (GLC)-based techniques were later developed to assay AMACR activity with (2R)- and (2£)-methylmyristoyl-CoA as the substrates (Schmitz et al, 1994). Following the quenching of the reaction with HC1 (6 M) and the extraction of the methylmyristoyl-CoA thioesters, the latter were coverted to the amides of (i?)-l-phenylethylamine via activation by l,l'-carbonyldiimidazole (Scheme

1.7). The derivatized enantiomers were separated on a 25-m SE-30 GLC column.

Radioactivity-based assays have also been used to assess AMACR activity by measuring the amount of H2O resulting from the deprotonation of the substrates [2- H]- pristanoyl-CoA and [24, 25-3H]-THC-CoA and subsequent exchange with solvent H2O

(Savolainen et al, 2005; Schmitz et al, 1994). The fixed-time assays mentioned above are labour-intensive and/or require radiolabeled substrates that are expensive. Therefore, the aim of this project was to design a continuous assay that allows the direct monitoring of the AMACR-catalyzed reaction using either diastereomer of the 2-methylacyl-CoA thioester substrates.

18 -SCoA NH,OH .NHOH HC16N

CoASH ibuprofenoyl-CoA

SOCl2

ibuprofenoyl-(lif, IS, 5J?)-menthyl ester

(1R, 2S, 5ii)-menthol

Scheme 1.6 (2R)- and (25)-Ibuprofenoyl-CoA derivitization for HPLC-based assay. The generated menthyl esters were separated by reversed-phase HPLC (Shieh et al, 1993).

19 o SCoA + y^NT"N R \J UN methymyristoyl-CoA l,l'-carbonyldiimidazole

HN-^N. -CoASH

O O

,NH,

11 (K)- 1-phenylethylamine

HN^N^

R = -(CH2)nCH3

2-methylmyrisoyl-l-(i?)-phenylethyl-amide

Scheme 1.7 (22?)- and (25)-Methylmyristoyl-CoA derhdtization for GLC-based assay. The generated menthyl esters were separated using GLC (Schmitz et ah, 1994).

20 1.7 Direct Racemase Assays. In general, direct assays of racemases or epimerases employ spectroscopic methods that measure the interconversion of the enantiomeric or diastereomeric substrates, respectively. One of the most common direct methods involves the use of polarimetry (e.g., proline racemase (Reina-San-Martin et ah, 2000)).

Alternatively, coupled spectrophotometric assays have been employed (e.g., mandelate racemase) (Hegeman, 1970).

In addition, circular dichroism (CD)-based assays have been used to assay a variety of racemases including mandelate racemase (Sharp et ah, 1979) and glutamate racemase (Gallo & Knowles, 1993); and epimerases (e.g., diaminopimelate epimerase)

(Koo & Blanchard, 1999). A related technique, optical rotatory dispersion (ORD) polarimetry was used to assay hydantoin racemase (Wiese et ah, 2000).

The CD-based assay described in the present study offers a quick, inexpensive, and effective method for monitoring the AMACR-catalyzed epimerization in both directions of the reaction. The assay should provide an effective method to screen small molecules as potential inhibitors of AMACR activity.

21 Chapter 2

Materials and Methods

2.1 General. Racemic ibuprofen sodium salt, l,l'-carbonyldiimidazole, and (R)-(+)-a- methyl-benzylamine were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON).

(5)-ibuprofen was purchased from Fluka Analytical (Buchs, Switzerland). Coenzyme A

(trilithium salt) was purchased from BioShop Canada Inc. (Burlington, ON).

Mycobacterium tuberculosis genomic DNA was obtained from Dr. J. T. Belise

(University of Colorado) through the NIH, NIAID "TB Vaccine Testing and Research

Materials" contract NOl AI-75320. Acetonitrile (HPLC-grade) was purchased from

Fischer Scientific (Ottawa, ON). Deoxyoligonucleotide primers were commercially synthesized by ID Labs (London, ON). Restriction enzymes were purchased from New

England Biolabs (Mississauga, ON). His-Bind resin and thrombin cleavage capture kits were purchased from Novagen (Madison, WI) and Pfu Turbo DNA polymerase was purchased from Stratagene (La Jolla, CA). All chemicals were reagent grade or better.

THF was dried using sodium. The melting points were recorded using an Electrothermal

Melt-Temp model 120ID capillary melting point apparatus (Barnstead International,

Dubuque, IA). Optical rotations were measured using a Rudolph Instruments Digi 781

Automatic polarimeter (Denville, NJ). CD assays were conducted using a JASCO J-810 spectropolarimeter (Easton, MD).

2.2 Resolution of (Zf)-Ibuprofen. (7?)-ibuprofen was resolved using the protocol described by Trung et al. with modifications (Trung et al, 2006). Aqueous HC1 (1 M) was added to a solution of racemic ibuprofen (20 g, sodium salt) dissolved in water (100 mL) until the pH was 2. The free acid was then obtained after extraction into diethyl

22 ether and removal of the solvent under reduced pressure.. The racemic free acid (4.0 g,

19.2 mmol) was then dissolved in absolute ethanol (48 mL). (R)-(+)-a- methylbenzylamine (2.5 mL, 19.2 mmol) was added at 60 °C with stirring and the resulting solution was refluxed for 15 min. Upon cooling to room temperature, the white crystals formed which were isolated by filtration. After 3 successive recrystallizations from ethanol, the diastereomeric salt (1.0 g) was dissolved in 1 M HC1 (50 mL) to liberate the free acid of (i?)-ibuprofen. Extraction of the aqueous solution with diethyl ether followed by removal of the solvent under reduced pressure afforded (K)-ibuprofen as a white powder, 0.43 g (43 % yield), mp 50-52 °C (lit. 48-49 °C) (Aureli et al, 2005),

20 25 [a]D -56.08 ± 0.02 (c = 2.5, ethanol)(lit. [a]D -53 (c = 2, ethanol)) (Aureli et al, 2005).

2.3 Ibuprofenoyl-CoA. Synthesis of ibuprofenoyl-CoA was conducted using the protocol described by Sidenius et al with minor modifications (Sidenius et al, 2004). In a 25 mL round-bottom flask, l,l'-carbonyldiimidazole (0.0324 g, 200 (amol), dissolved in anhydrous THF (1 mL), was added to a solution of ibuprofen free acid (0.0206 g, 100

|j,mol) in anhydrous THF (1 mL). The mixture was stirred at room temperature under an argon atmosphere for 2 h. Water (0.5 mL) was then added, followed by the trilithium salt of CoA (0.0411 g, 50 pmol) dissolved in water (0.5 mL). All the reagents were added using a glass syringe through the rubber septum covering the flask. After 24 h, the reaction was quenched by the addition 1 M HC1 until the pH of the solution was 2.

Unreacted ibuprofen was removed from the reaction mixture using liquid phase extraction (2 x 20 mL heptane and 2 x 20 mL ethyl acetate). Ibuprofenoyl-CoA was purified using solid phase extraction (SPE) (Bond-Elut CI 8 columns, Varian Inc.,

Mississauga, ON). After washing the SPE columns with acetonitrile (MeCN) and water

23 (12 mL each), the aqueous phase was applied to the column and eluted consecutively with aqueous solutions containing 0, 10, 25, 50, and 100% MeCN (12 mL each).

Ibuprofenoyl-CoA was obtained in the 25% and 50% fractions, and quantified using reversed-phase HPLC.

2.4 Ibuprofenoyl-CoA Quantification. Ibuprofenoyl-CoA was identified in the SPE fractions using reversed-phase HPLC on a Gemini 5(j. C6-phenyl column (150 x 4.60 mm; Phenomenex, Torrance, CA). A Waters 510 pump and 486 controller were used for solvent delivery. Injections were made using a Rheodyne 7725i sample injector fitted with a 20-fj.L injection loop. Fractions were eluted under isocratic conditions using

MeCN/H20/ammonium acetate (1.0 M, pH 6.0) 40/60/1 v/v/v) at a flow rate of 1.0 mL/min. The eluted enantiomers of ibuprofenoyl-CoA were detected by monitoring the absorbance at 220 nm using a Waters 486 tuneable absorbance detector.

Chromatographic data were processed using the PeakSimple Chromatography Data

System (SRI Instruments, Torrance, CA). Those fractions from SPE containing ibuprofenoyl-CoA were pooled and the solvent (HiO/MeCN) was removed under pressure (< 37 °C). The resulting white paste was re-dissolved in 1.0 mL of potassium phosphate buffer (0.1 M, pH 7.4), and stored at -20 °C. The retention times for ibuprofenoyl-CoA and CoA were 4.3 and 2.6 min, respectively. Following the procedure of Chen et ah, thin-layer chromatography (TLC) was conducted using cellulose plates

(Selecto Scientific, Norcross, GA) (BuOH/acetic acid/H20 10/3/5 v/v/v). The R/ values for ibuprofenoyl-CoA and CoA were 0.75 and 0.12, respectively.

Ibuprofenoyl-CoA was quantified using an alkaline hydrolysis procedure

(Sidenius et ah, 2004). Ibuprofenoyl-CoA and the ibuprofen standards (100 uL) were

24 incubated with 5 M NaOH (450 uL) for 1 h at 60 °C. Following neutralization by the addition of 5 M HC1, reversed-phase HPLC analysis of the samples was conducted as described above. Comparison of the area of the ibuprofen peak in the chromatogram with a standard curve correlating peak areas and ibuprofen concentrations (0.5-10.0 mM) permitted calculation of the original ibuprofenoyl-CoA concentration. Solutions of (25)- and (2i?)-ibuprofenoyl-CoA in potassium phosphate buffer (0.1 M, pH 7.4) exhibited identical UV spectra between 220 and 320 nm with a single maximum at 260 nm. Nine solutions of (25)-ibuprofenoyl-CoA (from three t different preparations) with known concentrations, as determined using the HPLC method, were used to estimate the molar extinction coefficient (e) of (2R)- and (25)-ibuprofenoyl-CoA as 20 (±1) x 103 M_1cm_1.

The molar extinction coefficient was used to routinely estimate the concentration of ibuprofenoyl-CoA obtained after purification by SPE.

2.5 His6-tagged Rat AMACR Expression and Purification, RNA isolated from rat liver was provided by Dr. Mark Nachtigal. The cDNA was obtained using a reverse transcriptase (RT)-catalyzed reaction at 42 °C for 50 min. The open reading frame (ORF) encoding rat AMACR was amplified from rat cDNA using the polymerase chain reaction

(PCR) with forward (5'- GGCCGTTCTGCCATATGATCCTGGCGGACTTC-3') and reverse (5'- TGAAGGATCCTCAGAGGTTGGCTTTTAGC-3') primers containing restriction sites (underlined) for Ndel and BamHI, respectively. The ORF was sub-cloned into a pET15b vector (Novagen, Madison, WI) to generate the pET15b-rat plasmid, and the sequence was verified by automated DNA sequencing (DalGen Microbial Genomics

Centre, Dalhousie University, Halifax, NS). The pET15b-rat AMACR plasmid encodes the rat AMACR enzyme as a fusion protein bearing an N-terminal hexahistidine (Hise)

25 tag. Plasmids were propagated in E. coli DH5a cells and introduced into E. coli

BL21(DE3) cells as the host for target gene expression. Standard techniques were used for the PCR, DNA isolation, sub-cloning, and gel electrophoresis (Sambrook et al,

1989).

Expression of soluble recombinant His6-rat AMACR was induced using 1 mM isopropyl-D-P-thiogalactopyranoside (IPTG) and purified using metal ion affinity chromatography as described in the Novagen protocols. All enzyme purification procedures were conducted at 4 °C.

The same procedure has been used to examine the effect of lower growth temperatures (25 °C and 16 °C) and lower concentrations of IPTG (0.5 mM and 0.1 mM) on the soluble fraction of fusion protein produced.

2.6 GIutathione-S-Transferase (GST)-tagged Rat AMACR Expression and

Purification. The ORF encoding rat AMACR was amplified from rat cDNA using PCR with forward (5'- CGTGGGATCCCCGGGATGATCCTGGCGGACTTC-3') and reverse

(5'-AGCAGGGAATTCCTCAGAGGTTGGCTTTTAGC-3') primers containing restriction sites (underlined) for BamHl and EcoRl, respectively. The ORF was sub- cloned into a pGEX-3X vector (Amersham, Baie D'Urfe, PQ, from Dr. C. McMaster,

Dalhousie University) to generate the pGEX-3X-rat AMACR plasmid, and the sequence was verified by automated DNA sequencing (DalGen Microbial Genomics Centre,

Dalhousie University, Halifax, NS). The pGEX-3X-rat AMACR plasmid encodes the rat

AMACR enzyme as a fusion protein bearing an N-terminal GST tag. Plasmids were propagated in E. coli DH5cc cells and introduced into E. coli BL21(DE3) cells as the host

26 for target gene expression. Standard techniques were used for the PCR, DNA isolation,

sub-cloning, and gel electrophoresis (Sambrook et al, 1989).

Expression of soluble recombinant GST-rat AMACR was induced using 1 mM

IPTG and purified using affinity chromatography as described in the Amersham

protocols. All enzyme purification procedures were conducted at 4 °C.

Also, the GST-rat AMACR was expressed in Rosetta™ cells (Novagen), which

contain a plasmid encoding several rare E. coli tRNA genes. This procedure followed the

same steps and conditions used in the expression of GST-rat AMACR in B121(DE3)

cells.

2.7 His6-tagged TBmcr Expression and Purification. The ORF encoding TBmcr was

amplified from M. tuberculosis genomic DNA using PCR with forward (5'-

GCCGGTACACATATGGCGGGTCCGCTGAGCGGGTTG-3') and reverse (5'-

TACGAAGGATCCCTATCCGTCCCAGTCGGTGAGCAC-3') primers containing

restriction sites (underlined) for Ndel and BamHI, respectively. The ORF was sub-cloned

into a pET15b vector (Novagen, Madison, WI) to generate the pET15b-TBmcr plasmid,

and the sequence was verified by automated DNA sequencing (DalGen Microbial

Genomics Centre, Dalhousie University, Halifax, NS). The pET15b-TBmcr plasmid encodes the TBmcr enzyme as a fusion protein bearing an N-terminal hexahistidine

(His6) tag. Plasmids were propagated in E. coli DH5a cells and introduced into E. coli

BL21(DE3) cells as the host for target gene expression. Standard techniques were used for the PCR, DNA isolation, sub-cloning, and gel electrophoresis (Sambrook et al,

1989).

27 BL21(DE3) cell cultures (1 L) were grown overnight at 37 °C. Soluble recombinant His6-TBmcr was expressed and purified using metal ion affinity chromatography as described in the Novagen protocols except that the His6-TBmcr was eluted from the His-Bind resin using 90 mM imidazole. The enzyme was subsequently dialyzed overnight against Tris-HCl buffer (50 mM, pH 8.0), concentrated using a

Centriprep-30 concentrator, and stored at —80 °C. All enzyme purification procedures were conducted at 4 °C.

Thrombin-catalyzed cleavage of the His6-tag from the enzyme was conducted in cleavage buffer (70 mM HEPES, pH 8.0 containing 0.5 mM EGTA) with a thrombin ratio of 2 U/mg of target protein. Cleavage was complete after 24 h at 4 °C; the biotinylated thrombin was removed from the reaction mixture using streptavidin-agarose resin (Novagen) at a ratio of 16 uL of settled resin per unit of thrombin. Cleaved TBmcr free of biotinylated thrombin was then dialyzed against Tris-HCl buffer (50 mM, pH 8.0).

2.8 Enzyme Assay. The enzymatic reaction was initiated by addition of TBmcr (7.5 ng/mL final concentration) to a pre-incubated (at 37°C for 5 min) solution containing 2-

(/?)- or 2-(S)-ibuprofenoyl-CoA ranging in concentrations between 20 uM and 600 uM,

0.2% octyl-p-D-glucopyranoside, and Tris-HCl buffer (50 mM, pH 8.0). The final reaction volume was 1.5 mL. The change in ellipticity was followed at 279 nm over 5 min using a quartz cuvette with a 1.0 cm light path in a jacketed cell holder. All reactions were conducted at 37 °C.

Kinetic data were fit to the Michaelis-Menten equation (equation 1) where Vi is the initial reaction velocity, Vmax is the maximal reaction velocity (Fmax = kcat [E]T), Km is

28 the Michaelis constant, and [S] is the substrate concentration, using the program

GraphPad Prism 4 (GarphPad Software, La Jolla, CA).

Values of fccat were calculated for His6-TBmcr and TBmcr with the His6-tag removed using the molecular masses of 40,848 and 38,966 Da, respectively. Reported errors are standard deviations. Protein concentrations were determined using the Bio-Rad Protein

Assay (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin standards.

29 Chapter 3

Results

3.1 Expression and Purification of Recombinant AMACR

3.1.1 His6-rat AMACR. E. coli BL21 (DE3) cells were transformed with the pET15b-rat

AMACR plasmid, which encodes an N-terminally His6-tagged fusion protein. The expression of the fusion protein was induced with 1 mM IPTG and cells were grown at

37 °C. The molecular weight of the fusion protein is predicted to be 41.8 kDa from the cDNA sequence. The electrophoretic analysis showed that the total cell lysate contained a protein corresponding to 40.0 kDa consistent with the expected molecular weight of His6- rat AMACR. However, the fusion protein was not found in the elution fraction following metal ion affinity chromatography (Figure 3.1). This shows that a soluble form of His6- rat AMACR was not expressed under these conditions. In addition, it seems that a protein with the predicted size eluted at low imidazole concentration (lane 4 of Figure 3.1).

Different conditions were used to try and increase the amount of soluble His6-rat

AMACR including lowering the concentration of IPTG (0.5 mM and 0.1 mM) used to induce protein expression and lowering the culture growth temperature (25 °C and 16

°C). However, none of these modifications was successful in generating a soluble form of the protein.

3.1.2 GST-rat AMACR. Since His6-rat AMACR was not expressed in a soluble form, a plasmid encoding a fusion protein with a more soluble tag was constructed. E. coli

BL21(DE3) cells were transformed with the pGEX-3X-rat AMACR plasmid, which encodes an N-terminally GST-tagged recombinant protein. The expression of recombinant protein was induced with 1 mM IPTG.

30 M MW (kDa) 97.2 - 66.4 - ^0mJ 55.6 - 42.7 - 34.6 -

27.0 - •-*

20.0 -

Figure 3.1 SDS (10%)-PAGE gel of the metal ion affinity chromatography purification fractions of Hi$6-rat AMACR from BL21(DE3) cells. The lanes include: protein marker, M; total cell lysate, 1; clarified lysate, 2; flow-through,3 ; wash 1 (4 mM imidazole), 4; wash 2 (60 mM imidazole), 5; and eluate, 6.

31 The molecular weight of the GST portion of the protein is 27.0 kDa giving an overall molecular weight of 67.0 kDa for the GST-rat AMACR. The electrophoresis of the total cell lysate revealed the presence of a 56.7 kDa protein (Figure 3.2). Although the 56.7 kDa protein did not appear to be a major protein in the soluble fraction of the lysate, it was present in the fractions obtained by affinity chromatography (using immobilized glutathione), consistent with the presence of a GST moiety since it was eluted using reduced glutathione. However, the elution fraction was not totally pure but included a

27.0 kDa protein that could correspond to the molecular weight of the GST protein alone.

This could be explained by the fact that the production of GST-rat AMACR can be interrupted at the translation level because of the large size of the recombinant protein. In addition, the molecular weight of the major protein is lower than the expected molecular weight of the GST-rat AMACR, and this is also consistent with premature termination of translation. The expression of GST-rat AMACR in Rosetta™ cells, which contain a plasmid encoding several rare E. coli tRNA genes in order to counter the effect of codon bias, did not alter the expression pattern. Measurement of the AMACR activity in the elution fraction using the CD assay revealed that the preparation was enzymatically inactive.

3.1.3 His6-TBmcr. Due to the difficulties faced in expressing a soluble mammalian form of AMACR in a prokaryotic host, a bacterial source of AMACR was utilized. TBmer is the AMACR found in Mycobacterium tuberculosis and is soluble in aqueous solution

(Bhaumik et al., 2003). E, coli BL21(DE3) cells were transformed with the pET15b-

TBmcr plasmid, which encodes an N-terminally Hisg-tagged TBmcr.

32 2 3 4

27.0 -

20.0 -

Figure 3.2 SDS (10%)-PAGE gel of affinity chromatography purification fractions of GST-rat AMACR from BL21(DE3) cells. The lanes include: protein marker, M; total cell lysate, 1; clarified lysate, 2; flow-through, 3; wash (IX PBS), 4; and eluate, 5.

33 The cDNA sequence predicts the molecular weight of the tagged protein to be 40.8 kDa;

whereas the molecular weight of the tag-free enzyme should be 39.0 kDa. The elution

fraction resulting from the metal ion affinity chromatography contained one protein with

a molecular weight of 38.8 kDa. Thrombin-catalyzed cleavage of the hexahistdine tag

resulted in a decrease of the molecular weight of the protein (Figure 3.3) to 36.8 kDa.

The molecular weight of the protein of interest was similar to that predicted from the

ORF sequence, which suggests that the expression and purification of His6-TBmcr was

successful. The gel in Figure 3.3 also shows that the thrombin cleavage was complete

allowing us to compare the kinetic properties of the His6-tagged and tag-free forms of the

enzyme.

3.2 Characterization of Ibuprofenoyl-CoA

(2S)-, (2R), and (2R, 25)-Ibuprofenoyl-CoA were synthesized and purified. TLC on

cellulose plates showed a unique spot with an R/ value of 0.75, which is in agreement

with the literature value of 0.8 (Chen etal, 1991).

3.2.1 Ibuprofenoyl-CoA Quantification. For the CD-based assay, it is necessary to

quantify the thioesters. The method used was based on alkaline hydrolysis, in which

ibuprofenoyl-CoA is hydrolyzed by NaOH to yield free ibuprofen and free coenzyme A

(Sidenius et al, 2004). The area under the ibuprofen peak, obtained using reversed-phase

HPLC, was used to measure the concentration of ibuprofen liberated through hydrolysis by comparison to a standard curve of peak areas corresponding to different

concentrations of ibuprofen. Upon complete alkaline hydrolysis as indicated by reversed- phase HPLC, the concentration of free ibuprofen measured would be equal to the

concentration of ibuprofenoyl-CoA.

34 M 1

Figure 3.3 SDS (12%)-PAGE gel of purified TBmcr. The lanes include: protein marker, M; His6-TBmcr, 1; and tag-free TBmcr, 2. 6.3 ug of protein were loaded for samples in lanes 1 and 2.

35 Reversed-phase HPLC analysis of solutions of ibuprofen at varying concentrations (0.5-

10 mM) gave a linear dependence of the peak area with the concentration of ibuprofen

(Figure 3.4). The chromatogram of the solution obtained after alkaline hydrolysis (Figure

3.5) showed that there was total hydrolysis of ibuprofenoyl-CoA. It also showed that the peaks corresponding to free ibuprofen and free Co A were the sole result of the hydrolysis of the thioester. Ibuprofenoyl-CoA eluted as a single peak (tr = 4.3 min); but upon alkaline hydrolysis, two peaks were evident corresponding to coenzyme A (tr = 2.6 min) and ibuprofen (tr = 13.0 min). The peak corresponding to free ibuprofen had the same retention time as that of ibuprofen from the standard solutions. Thus quantification of ibuprofenoyl-CoA could be accomplished using this alkaline hydrolysis-HPLC method.

3.2.2 UV Spectra. The HPLC-based quantification provided a measurement of the concentration of the synthesized ibuprofenoyl-CoA. By knowing the concentration (Q of the thioester by the HPLC-based quantification, it was possible to calculate the molar extinction coefficient (e) of the thioesters from UV measurements. Using the Beer-

Lambert law (equation 2) where / is the light path length, £260 may be calculated as shown in equation 3:

Ax = sJC (2)

36 (S)-ibuprofen (tr=13min) B.

Figure 3.4 Linearity between the peak area and (S)-ibuprofen concentration shown as HPLC chromatograms (A) and as a standard curve (B). Chromatography conditions are as described in the Materials and Methods section. The ibuprofen standards were subjected to the same steps and conditions of the alkaline hydrolysis procedure as the ibuprofenoyl-CoA.

37 (2S)-lbuprofern (t • 4.3 mln;

(SHbuprofen (t,«13m!n)

Figure 3.5 HPLC chromatograms showing the products of alkaline hydrolysis of (2S)-ibuprofenoyl-CoA. Chromatography conditions are as described in the Materials and Methods section. The control sample is the alkaline hydrolysis of 100 uL of potassium phosphate buffer (0.1 M, pH 7.4). The (2S)-ibuprofenoyl-CoA was dissolved in potassium phosphate buffer (0.1 M, pH 7.4).

38 The UV spectra of (25)- and (2i?)-ibuprofenoyl-CoA (60 uM) were obtained between

220 nm and 320 nm (Figure 3.6). Both thioesters exhibit the same UV profile in the chosen wavelength (k) interval with a maximal absorbance (A) at 260 nm. The UV spectra suggest that both thioesters should have equal molar extinction coefficients at 260 nm (e26o).

The reversed-phase HPLC-based quantification was conducted on nine solutions

(from three different batches) of (25)-ibuprofenoyl-CoA. The molar extinction coefficient

(£260) obtained was 20 (±1) x 10J MT cm . The molar extinction coefficient from two different solutions of (2i?)-ibuprofenoyl-Co A were within the error of the value obtained with the (25)-ibuprofenoyl-CoA, confirming that the molar extinction coefficients for both thioesters are equal. Knowledge of the molar extinction coefficient of both ibuprofenoyl-CoA thioesters permits one to bypass the reversed-phase HPLC-based quantification and to directly measure the thioester concentration following solid phase extraction (SPE). The reversed-phase HPLC analysis is only required to demonstrate the purity of the ibuprofenoyl-CoA obtained after SPE.

3.2.3 CD Spectra. In order for the CD-based assay to be feasible, the (25)- and (2R)- ibuprofenoyl-CoA must have different ellipticities at a given wavelength so that the

AMACR-catalyzed reaction can be followed by a change in observed ellipticity. The CD spectra of (2R)-, (25)-, and (2i?,25)-ibuprofenoyl-CoA between 220 and 320 nm are shown in Figure 3.7.

39 220 230 240 250 260 270 280 290 300 310 320 wavelength (nm)

1.25-

1.00' B I 0.75H\y\ 8 0.50H (0 0.25'

0.00- 220 230 240 250 260 270 280 290 300 310 320 wavelength (nm)

Figure 3.6. UV spectra of (25)- and (21?)-ibuprofenoyl-CoA between 220 nm and 320 nm. Panels A and B represent the spectra of (25)- and (2i?)-ibuprofenoyl-CoA, respectively. A final concentration of 60-uM for each thioester was measured in potassium phosphate buffer (0.1 M, pH 7.4) in a cuvette with a 1 cm light path.

40 wavelength (nm)

Figure 3.7 CD spectra of (2S)- (•), (2R)- (•), and (25, 22?)-ibuprofenoyl-CoA (A) between 220 nm and 320 nm. Each sample had a final concentration of 120 uM in potassium phosphate buffer (0.1 M, pH 7.4) in a cuvette with a 1 cm path light. The ellipticity due to the potassium phosphate buffer (0.1 M, pH 7.4) buffer was subtracted from the ellipticity for each thioester.

41 While (2i?,2.S)-ibuprofenoyl-CoA exhibited only a weak ellipticity throughout this wavelength interval, solutions (120 uM) of (2S)- and (2/f)-ibuprofenoyl-CoA exhibited maximum ellipticities of+22 mdeg (at 279 nm) and -23 mdeg (at 277 nm), respectively.

At this wavelength, the ellipticity associated with (2i?,25)-ibuprofenoyl-CoA (120 uM) was -1.4 mdeg, which represents the ellipticity corresponding to complete epimerization.

Consequently, 279 nm was selected as the wavelength to be used for the CD-based assay.

The molar ellipticity of (2S)- and (2i?)-ibuprofenoyl-CoA at 279 nm ([6^279) is required so that the observed ellipticity at 279 nm (#279) may be converted into concentration as shown in equation 4 (where / is the light path length and C is the concentration):

0m=[0LlC (4)

The molar ellipticity values of (2S)- and (2/?)-ibuprofenoyl-CoA at 279 nm were determined to be 181 (± 9) x 103 and - 187 (± 15) x 103 deg mol-1 cm2, respectively. The values show that (25)- and (2/?)-ibuprfoenyol-CoA have opposite and equal values of molar ellipticity. While enantiomers would be expected to show equal and opposite molar ellipticity values, such is not necessarily the case for epimers (diastereomers). The molar ellipticity value for (2i?)-ibuprofenoyl-CoA was calculated from six measurements of the observed ellipticity at 279 nm.

42 3.3 Calculation of Velocities from CD Data

The rate of the TBmcr-catalyzed epimerization of either (2S)- or (2R)- ibuprofenoyl-CoA was determined by following the change in observed ellipticity at 279 nm. The observed ellipticity of a solution of ibuprofenoyl-CoA is given by equation 5.

9 = es+eR (5)

Consider the epimerization of (2,S)-ibuprofenoyl-CoA for which the change in the observed ellipticity is related to the change in concentrations of (2R)- and (25)- ibuprofenoyl-CoA as shown in equation 6.

A9^[0\sACsU[0\RACRl (6)

Since the decrease in concentration of (2

(t) is given by equation 8.

-ACS=ACR (7)

A0 _-[0]sACsl + [0]RACsl At At

Hence, the rate of disappearance of (25)-ibuprofenoyl-CoA as it is converted to (2i?)- ibuprofenoyl-CoA (i.e., the reaction velocity vs) is given by equation 9.

A^ v =^-=AC, ^ALl, (9) K5s A; r ([el-lely

43 Likewise, when (2i?)-ibuprofenoyl-CoA is the substrate, the rate of its conversion to (25)-

ibuprofenoyl-CoA (vR) is given by equation 10.

v =*£JL= '^ (10) R At del-[0^)1

3.4 Assay Development

3.4.1 Monitoring the TBmcr-catalyzed Reaction in Both Directions. In order to

demonstrate that the CD-based assay could be used to follow the AMACR-catalyzed

reaction starting with either diastereomers as the substrate, it was necessary to show that

TBmcr catalyzed the epimerization of (25)-ibuprofenoyl-CoA with a decrease in the

observed ellipticity, and an increase in observed ellipticity for the epimerization of (2R)-

ibuprofenoyl-CoA. After complete epimerization, the observed ellipticity of the resulting

(25, 2i?)-ibuprofenoyl-CoA is expected to be -0.72 mdeg.

Figure 3.8 shows the change in observed ellipticity for the TBmcr-catalyzed

epimerization starting from either (2S)- or (2i?)-ibuprofenoyl-CoA as the substrate.

Clearly, the purified His6-TBmcr is catalytically active, capable of epimerizing both (25)-

and (2/?)-ibuprofenoyl-CoA. The epimerization of the former is accompanied by a

decrease in ellipticity; whereas an increase in ellipticity is observed for the epimerization

of the latter. After a period of approximately 24 h, the observed ellipticity obtained for

the epimerization of (2R)- and (25)-ibuprofenoyl-CoA (120 uM each) was -0.45 mdeg

and -2.77 mdeg (average = -1.61 mdeg), close to the value expected for (2S,2R)- ibuprofenoyl-CoA. This shows that after 24 h, the AMACR-catalyzed reaction is almost complete under the conditions used in the experiment. .

44 20

G) 10 O -a (2S)-iburprofenoyl-CoA E o s*^' >» ;so -10 +3 Q.

el l -20 (2R)-ibuprofenoyl-CoA

-30 24.5

time (h)

Figure 3.8 Progress curve for the His6-TBmcr-cataIyzed reaction using (25)- and (2J?)-ibuprofenoyI-CoA. The conditions of the experiment are as described in Materials and Methods with an initial substrate concentration of 120 uM. The concentration of His6-TBmcr used was 34 ng/mL. The reaction was conducted at 37 °C.

45 3.4.2 Effect of Octyl-P-D-glucopyranoside. In previous published work on TBmcr,

octyl-P-D-glucopyranoside (OG) at a final concentration of 0.2% was used in the

radioactivity-based assay of AMACR activity (Bhaumik et al, 2007). OG is a detergent

widely utilized for solubilizing membrane-bound proteins in their native form (Levy et

al, 1992). OG has a critical micelle concentration (cmc) of 25 mM (0.8%). The effect of

OG on the tag-free TBmcr activity is shown in Figure 3.9. Interestingly, there is an

increase in TBmcr activity with an increased concentration of OG. However, the increase

in activity is only minor (~ 13%) with maximum activity being observed at OG

concentrations between 0.2-0.4%. This shows that TBmcr activity has little detergent

sensitivity, but the presence of OG in the reaction mixture does increase its activity

slightly. Because other authors used OG at concentrations of 0.2% (Bhaumik et al., 2007;

Savolainen et al., 2004), we decided to use this concentration because it provided a near

maximal increase in activity (see Figure 3.9) and would permit comparison with

published kinetic parameters. The interval of OG concentration used in Figure 3.9 is

below the cmc. Figure 3.10 shows the effect of higher OG concentrations (i.e., near and

above the cmc) on the velocity of the reaction. A final OG concentration of 0.2%

corresponds to the highest velocity. OG concentrations that were higher than 0.2%

showed a decrease in the velocity especially near the cmc.

3.4.3 Effect of Enzyme Concentration. The effect of enzyme concentration (0 to 10

ng/mL) on the activity of the tag-free TBmcr was studied at high (600 uM) and low (40

uM) substrate ((2i?)-ibuprofenoyl-CoA) concentrations (Figure 3.11) to identify the region of linearity between the enzyme concentration and the velocity of the reaction.

46 0.018

0.1 0.2 0.3 0.4 0.5 [octyl-p-D-glucopyranoside] (%)

Figure 3.9 Effect of octyl-p-D-glucopyranoside on the TBmcr-catalyzed reaction using (2U)-ibuprofenoyl-CoA as the substrate. The assay conditions are as described in the Materials and Methods. The concentration of (2i?)-ibuprofenoyl-CoA was 75 \iM. The concentration of tag-free TBmcr used was 10 ng/mL.

47 0.014-1 ^ 0.013 ^ 0.012- £ 0.011- & 0.010- .? 0.009H CD > 0.008-1 0.007 0.0 0.2 0.4 0.6 0.8 1.0 1.2 [octyl-p-D-glucopyranoside] (%)

Figure 3.10 Effect of high octyl-P-D-glucopyranoside concentrations on the TBmcr- catalyzed reaction using (2i?)-ibuprofenoyl-CoA as the substrate. The assay conditions are as described in the Materials and Methods. The concentration of (2R)- ibuprofenoyl-CoA was 75 jxM. The concentration of tag-free TBmcr used was 10 ng/mL. The OG concentration of 0.8% corresponds to the cmc of the detergent.

48 0.035-, ^ 0.030- w "S) 0.025H 4) "I 0.020- jfr 0.015- "o k £ 0.010- .~~-~ 0) > 0.005- 0.000-«** ^ o.o 2.5 5.0 7.5 10.0 12.5 [TBmcr] (ng/mL)

Figure 3.11 The effect of enzyme concentration on the tag-free TBmcr-catalyzed reaction at high and low concentrations of (2i?)-ibuprofenoyl-CoA. The experiment was conducted in the presence of OG at a final concentration of 0.2%. The observed velocities with the substrate (2/?)-ibuprofenoyl-CoA at 600 uM (•) and 40 uM (A) are shown.

49 The results revealed that at the high substrate concentration, the velocity shows a linear dependence on enzyme concentration for concentrations between 3.0-7.5 ng/mL. For enzyme concentrations less than 3.0 ng/mL, the velocities levelled off and did not go to zero. Above 7.5 ng/mL, there is a leveling off of the curve. The results at the low substrate concentration showed that the dependence of the velocity of the reaction on enzyme concentration was linear at all concentrations except above 7.5 ng/mL. Thus, the reaction velocities varied linearly with enzyme concentration for enzyme concentrations ranging between 3.0-7.5 ng/mL. For this reason, 7.5 ng/mL was chosen as the standard concentration of enzyme employed in the assays.

3.4.4 Dependence of the Initial Velocity on Substrate Concentration. The dependence of the initial velocities on substrate concentration for the His6-TBmcr-catalysis catalyzed-

(Figure 3.12) and tag-free TBmcr-catalyzed (Figure 3.13) epimerizations was examined in order to determine the kinetic parameters of each enzyme. The kinetic parameters for both reaction directions and for both forms of the enzyme are summarized in Table 3.1.

Both forms of TBmcr show a greater affinity (lower Km) for (2i?)-ibuprofenoyl-CoA than for (2«S)-ibuprofenoyl-CoA as a substrate. Although, both enzymes exhibit a similar affinity for the (5)-thioester, the tag-free enzyme has a higher affinity for the (i?)-thioester than the His6-tagged enzyme. Also, both enzymes showed a higher turnover number (kcat) with (25)-ibuprofenoyl-CoA than with (2i?)-ibuprofenoyl-CoA. The His6-tagged TBmcr was more efficient (higher kcat/Km) in the S—*R direction; however, the tag-free enzyme was more efficient in the R—*S direction.

50 0.025

-a- 0.020 "3) "9- 0.015-

.ti 0.010- 8 > 0.005

0.000 100 200 300 400 500 600 700 [(2S)-ibuprofenoyl-CoA] (nM)

0.025

In 0.020 U) n 0.015 E 0.010 locit y ( 2 0.005

0.000 i 1 1 1 1 1 1 100 200 300 400 500 600 700 [(2/?)-ibuprofenoyl-CoA] (uM)

Figure 3.12 Representative Michaelis-Menten plots for His6-TBmcr. The conditions of the assay are as described in the Materials and Methods. The Km values were obtained directly from the non-linear regression analysis. The Fmax (mdeg/s) values obtained from the non-linear regression analysis were converted to (M/s) using equations 8 or 9. The kcat values were calculated from the Fmax (M/s) values. The kinetic constants are reported in Table 3.1.

51 0.030-,

> 0.005H

0.000 100 200 300 400 500 600 700 [(2S)-ibuprofenoyl-CoA] (uM) 0.030

^ 0.025

> 0.005H

0.000 1 1 1 1 1 1 1 100 200 300 400 500 600 700 [(2/?)-ibuprofenoyl-CoA] (JJ,M)

Figure 3.13 Representative Michaelis-Menten plots for tag-free TBmcr. The conditions of the assay are as described in the Materials and Methods. The Km values were obtained directly from the non-linear regression analysis. The Vmax (mdeg/s) values obtained from the non-linear regression analysis were converted to (M/s) using equations 8 or 9. The kcat values were calculated from the Vmax (M/s) values. The kinetic constants are reported in Table 3.1.

52 Table 3.1

Kinetic parameters for the TBmcr-catalyzed epimerization of ibuprofenoyl-CoA in both directions using His6-TBmcr and tag-free TBmcra

His6-TBmcr Tag-free TBmcr

substrate substrate

kinetic parameter (2S)- (2R)- (2S)- (2R)-

ibuprofenoyl- ibuprofenoyl- ibuprofenoyl- ibuprofenoyl-

CoA CoA CoA CoA

Km (uM) 87 (± 5) 71 (± 9) 86 (± 6) 48 (± 5)

-1 kcai (s ) 358 (±21) 228 (± 9) 450 (±14) 291 (±30)

1 KJKm{yiM-\- ) 4.1 (±0.2) 3.2 (± 0.3) 5.2 (± 0.3) 6.1 (±0.4)

Ke<{ 1.3 (±0.1) 0.8 (±0.1) a Assays conducted in triplicates in the presence of 0.2 % OG. The errors are the standard deviations.

53 In addition, the tag-free TBmcr exhibited a higher turnover number in both directions of the reaction than the His6-tagged enzyme. The same observation held for the efficiency since the untagged-form was ~2-fold and 1.3-fold more efficient than His6- tagged TBmcr in the £—>i? and R-+S directions, respectively. The equilibrium constants

(i^eq) calculated using the Haldane relation (equation 11) (Segel, 1993) for the His6- tagged and untagged enzymes, respectively, are close to unity.

R s K^ = {kJKmf^ l(kJKmf^ (11)

While the K^ values are experimentally equal (i.e., within two standard deviations), this is not necessarily expected since TBmcr (or AMACR) is an epimerase and not a racemase.

54 Chapter 4

Discussion

4.1 Enzyme Expression and Purification. Initially, a mammalian form of AMACR

(more specifically rat AMACR) was chosen as the enzyme to be used for developing the

CD-based assay and the expression and purification of a His6-rat AMACR from E. coli

BL21(DE3) cells was pursued. As shown in Figure 3.1, rat AMACR was present in the total cell lysate, but is not found in the elution fraction of the purification. Therefore, the expression of a soluble form of the His6-rat AMACR was unsuccessful due to the fact that the fusion protein was aggregated, possibly in inclusion bodies. Inclusion bodies are refractile intracellular protein aggregates (Fahnert et al, 2004). The formation of inclusion bodies is quite common in the expression of a eukaryotic target protein in a bacterial expression system due to differences in the process of protein synthesis in prokaryotes and eukaryotes. In general, prokaryotic organisms have a rapid translation process, with protein folding occurring post-translationally in most cases (Sorensen &

Pedersen, 1991). In eukaryotic cells, the rate of translation is slower than in E. coli with the maturation of nascent proteins often beginning co-translationally in certain domains and continuing post-translationally after the release of the protein from the ribosome

(Frydman et al, 1999). This slower rate of translation in eukaryotic cells plays an important role in the proper folding of proteins by allowing the sequential folding of individual domains during the translation process (Netzer & Haiti, 1997). Therefore, protein synthesis of a eukaryotic protein in E. coli can generate misfolded proteins that aggregate into inclusion bodies possibly due to the exposure of hydrophobic surfaces

(Oberg et al, 1994). Strategies to slow down the rate of synthesis in BL21(DE3) cells may be used to overcome these problems. For example, the culture growth temperature

55 may be lowered from 37 °C to 25 °C or 16 °C. Alternatively, IPTG concentrations lower

than 1 mM may be used (e.g., 0.5 mM and 0.1 mM). These modifications, however, did

not aid in the expression of a soluble form of His6-rat AMACR. It is possible that the

fusion protein of interest eluted at low imidazole concentration since a protein of the

expected size is present in Lane 4. This suggests that the His6-tag is buried in the N-

terminal region of rat AMACR. Further structural characterizations are needed to confirm

this speculation.

Consequently, the expression of rat AMACR in BL21(DE3) cells was attempted

using a fusion protein with an N-terminal GST tag. GST fusion proteins are, in most

cases, soluble because of the stabilizing effect that the GST tag has on the recombinant

protein (Terpe, 2003). Figure 3.2 shows the presence of a 56.7 kDa protein in the total

cell lysate and in the elution fraction that could correspond to the GST-rat AMACR

fusion protein (67.0 kDa). However, the elution fraction also showed the presence of

additional proteins of lower molecular weight that may have arisen due to premature

termination of translation or proteolytic cleavage. This observation is consistent with an

abrupt termination of protein translation due to codon bias (the unavailability of tRNAs

corresponding to rare codons) (Dong et al, 1996). The expression of GST-rat AMACR in

Rosetta™ cells, which contain a plasmid encoding several rare E. coli transfer RNAs

(AUA for He, AGG, AGA and CGG for Arg, CUA for Leu, CCC for Pro, and GGA for

Gly), did not alter the expression pattern. Furthermore, the assay of the elution fraction using the CD-based assay showed that it contained no active form of the enzyme. Despite the fact that the GST tag appeared to improve the solubility of rat AMACR, it did not generate an active enzyme. Hence, there may be other factors affecting the solubility and

56 folding of the recombinant protein. One of these factors could be the lack of post- translational modifications in prokaryotes such as glycolsylation, which could affect protein folding and solubility of the protein (Solovicova et al, 1996). Also, the cytosol of

E. coli is known to be a reducing environment that does not readily permit the formation of disulfide bonds, possibly leading to a misfolding of the protein of interest (Fahnert et al, 2004). The protein sequence of rat AMACR shows the presence of six cysteine residues that could potentially form disulfide bonds (Schmitz et al, 1997). Maltose- binding protein (MBP) is another widely used tag that has proved to be the most effective tag in promoting the solubility of recombinant mammalian proteins (Kapust & Waugh,

1999). In addition to its chaperone-like effect that allows it to interact with hydrophobic areas that are exposed on misfolded proteins (Richarme & Caldas, 1997), MBP is a periplasmic protein and directs the whole fusion protein to the periplasm. The periplasm is a cell compartment with an oxidising redox state where the formation of disulfide bonds of the recombinant proteins is favoured (Fahnert et al, 2004).

There is no experimental evidence supporting the presence of post-translational modifications or disulfide bonds in AMACR due to the lack of structural studies on any . mammalian form of AMACR. Because optimization of the conditions for expression and purification of rat AMACR could be a time-consuming procedure, and the main purpose of the present work was to develop a continuous CD-based assay, AMACR from a bacterial source (Mycobacterium tuberculosis) was expressed in E. coli BL21(DE3) cells as an N-terminal His6-tagged fusion protein. The His6-TBmcr was successfully expressed and purified using metal ion affinity chromatography (Figure 3.3). Figure 3.3 shows that the molecular weight of the protein was reduced by ~2.0 kDa after treatment with

57 thrombin that catalyzes the removal of the His6-tag. It is important to note that the His6-

TBmcr was subjected to all the conditions and steps used for the thrombin-catalyzed

cleavage without adding thrombin. Hence, the effect of the presence of the hexahisitdine

on TBmcr activity could be studied relative to TBmcr with His6-tag removed. Following

thrombin-catalyzed cleavage, three amino acid residues (GlySerHisMeti...) from the

linker are still attached to the N-terminal end of TBmcr.

The first kinetic studies conducted on AMACR used the crude mammalian

enzyme present in rat liver homogenates (Knihinicki et al, 1989). Subsequent studies

utilized various sub-cellular fractionation (Shieh & Chen, 1993), ammonium sulfate

precipitation (Schmitz et al, 1994; Schmitz et al. 1995), or immuno-precipitation (Kotti

et al, 2000) techniques to purify mammalian AMACRs from other sources including rat,

mouse, and human. Rat and mouse AMACR have been expressed in E. coli as His6-

tagged enzymes by using a pQE30 vector or a pTrcHis vector (Reichel et al, 1997;

Schmitz et al, 1997); whereas human AMACR has been expressed in E. coli as an MBP-

tagged enzyme (Ferdinandusse et al, 2000). The vector used for expression of rat

AMACR in E. coli was from a different commercial source than the one used in the

present study but identical conditions have been used for its expression and purification.

As for TBmcr, only its expression in E. coli as a tag-free enzyme using a pET3a vector has been reported. The enzyme was purified using ammonium sulfate precipitation

followed by ion-exchange chromatography (Bhaumik et al, 2003). This present study reports the first expression and purification of the His6-tagged TBmcr in E. coli.

4.2 Quantification of the Ibuprofenoyl-CoA Substrates. TLC of the products obtained from the synthesis of (2S)-, (2R)-, and (2S, 2i?)-ibuprofenoyl-CoA showed only one spot

58 with an R/value of 0.75. This value is in excellent agreement with the literature value of

0.8 (Chen et al., 1991). Reversed-phase HPLC analysis confirmed the purity of the substrate preparation as shown in Figure 3.5, in which (2S)-ibuprofenoyl-CoA elutes as a single peak. Upon alkaline hydrolysis, reversed-phase HPLC of the products gave two peaks that corresponded to CoA and (^-ibuprofen. The compound that elutes at 13 min corresponds to (^-ibuprofen because it elutes with the same retention time as the (S)- ibuprofen standard. Since alkaline hydrolysis leads to a total hydrolysis of the thioester, the concentration of the free ibuprofen is equal to the concentration of its corresponding thioester. Because the chromatogram peak areas varied linearly with the concentration of ibuprofen (Figure 3.4), the peak area of the released ibuprofen could be used to estimate the concentration of the original thioester. The alkaline hydrolysis procedure provides a reliable estimate of thioester concentration but is time-consuming. Consequently, the

Beer-Lambert equation (equation 2) was used to determine the molar extinction coefficient (e26o) for (25)- and (2i?)-ibuprofnoyl-CoA by obtaining their UV spectra at a known concentration (Figure 3.6). The UV spectra of (2S)- and (2i?)-ibuprofenoyl-CoA are identical with a maximal absorbance at 260 nm. CoA absorbs maximally at 260 nm but ibuprofen does not absorb significantly at this wavelength. This demonstrates successful incorporation of the CoA moiety. Nine solutions of (25)-ibuprofenoyl-CoA

(from three different batches) were quantified using the alkaline hydrolysis method and were used to determine the molar extinction coefficient at 260 nm. For (2S)-

j i _i ibuprofenoyl-CoA, the molar extinction coefficient was 20 (±1) x W NT cm . The molar extinction coefficients determined using two different solutions of (2R)- ibuprofenoyl-CoA were 19.5 x 103 M^cm"1 and 19.7 x 103 M^cnf1. Thus both

59 thioesters have the same extinction coefficient, which was subsequently used to directly determine the concentration of the synthesized thioester.

4.3 Circular Dichroism of the Ibuprofenoyl-CoA Substrates. CD is based on the use of circularly polarized light, which is composed of left- and right-handed circularly polarized light. An optically active molecule absorbs one component more than the other resulting in an elliptical polarized light, which is characterized by its ellipticity (9)

(Fasman, 1996). Enantiomers absorb either the left-handed or the right-handed component with an equal intensity leading to opposite and equal CD spectra, while such may or may not be the case for diastereomers which contain more than one chiral centre

(e.g., ibuprofenoyl-CoA). (25)-Ibuprofenoyl-CoA has a maximal ellipticity at 279 nm; whereas the maximal ellipticity of (2i?)-ibuprofenoyl-CoA is at 277 nm. (2S,2R)- ibuprofenoyl-CoA showed only a weak ellipticity over the wavelength interval examined.

Consequently, a wavelength of 279 nm was chosen for the assay so that the epimerization of either (25)- or (2i?)-ibuprofenoyl-CoA would show the greatest change in observed ellipticity. The molar ellipticities of (25)- and (2i?)-ibuprofenoyl-CoA were determined to be 181 (± 9) x 103 and -187 (± 15) x 103 deg mof1 cm2, respectively, revealing that (2R)- ibuprofenoyl-CoA absorbs left-handed circularly polarized light to the same magnitude as

(25)-ibuprofenoyl-CoA absorbs the right-handed component. It is important to note that a low ellipticity due to the potassium phosphate buffer (0.1 M, pH 7.4) was subtracted from the observed ellipticity for each thioester in order to determine the molar ellipticity due to the thioester only.

4.4 AMACR Assay. The AMACR-catalyzed epimerization can be monitored using CD spectroscopy as shown in Figure 3.8. Incubation of (25)- and (2i?)-ibuprofenoyl-CoA

60 with TBmcr leads to a gradual, time-dependent decrease and increase in observed ellipticity, respectively. The change in ellipticity with time over the first 5 min of the reaction is rapid and then gradually slows down. This is expected since initially the reaction in the forward direction predominates and the rate of the reverse reaction is negligible (i.e., initial rate conditions). With time, the rate of the reverse reaction increases due to an increase in the concentration of the product leading to a lower rate of change in the observed ellipticity. After approximately 24 h, the observed ellipticities for the reactions in both directions were very close to the observed ellipticity of (2S, 2R)- ibuprofneoyl-CoA showing not only that His6-TBmcr is active, but that it is also able to catalyze the complete epimerization of its two substrates. Note that the initial observed ellipticities of (25)- and (2i?)-ibuprofenoyl-CoA were 17 mdeg and -26 mdeg, respectively. These values are lower than the observed ellipticity values obtained from the CD spectra (Figure 3.7) at the same wavelength and at the same concentration (i.e.,

22 mdeg and -29 mdeg, for (2S)- and (2R)- ibuprofenoyl-CoA, respectively). This difference arises because of the rapid rate of reaction early on during the time course, such that after addition of the enzyme, mixing, and initiation of the CD measurements, some of the substrate has already reacted. This reaction was conducted using a relatively high concentration of enzyme so that the reaction would be essentially complete by 24 h.

However, the results also show that it is important to use an enzyme concentration that is below 34 ng/mL so that initial rates may be accurately measured.

Previously reported assays of TBmcr activity used a non-ionic detergent (OG) at a final concentration of 0.2% (Bhaumik et al, 2007). Figure 3.9 shows the effect of OG on the velocity of the reaction. It is apparent that there is an increase in reaction velocity

61 with the increased concentration of OG, although this increase is minor. Figure 3.10

shows that at OG concentrations higher than 0.2%, there is a decrease in velocity. The

decrease in velocity is accentuated at the OG concentration corresponding to the cmc

(0.8%). OG is a detergent with excellent solubilizing properties toward biological

membranes with a cmc of 25 mM (Bernat et al, 2008). It functions by forming micelles

in water and allowing separation and purification of membrane proteins without a

denaturing effect (Bernat et al, 2008). This observation suggests that AMACR (and

TBmcr) might be a membrane-associated protein, even though tagged and tag-free forms

of the enzyme have been purified without the use of a detergent (Bhaumik et al, 2003;

Schmitz et al, 1995). AMACR and TBmcr may be associated with a membrane through

a small hydrophobic domain. The exposure of this domain to the aqueous environment might cause the enzyme.to adopt a "non-optimal" conformation. The presence of OG in

solution may help the enzyme adopt a more optimal conformation for catalysis. The hydrophobicity profile of TBmcr (Figure 4.1) shows the presence of disperse hydrophobic segments throughout the enzyme. Some of these segments seem to be exposed to the solvent which might lead to partial misfolding of the enzyme in the absence of OG. The lack of sub-organellar localization studies makes it very difficult to speculate on whether TBmcr is associated with membranes either in mitochondria or in peroxisomes. One similar example is that of enzymes of the mammalian diacylglycerol kinase (DKG; EC 2.7.1.107) family which contains at least eight isoforms. These freely soluble enzymes have to translocate to the membrane of cells where they catalyze the

ATP-dependent phosphorylation of the diacylglycerol (DAG) (hnai et al, 2005; Luo et al, 2004).

62 Figure 4.1 Hydrophobicity profile of the TBmcr dimer. Two dimers are shown in the figure. The hydrophibicity is represented as a gradient with hydrophilic and hydrophobic regions shown as blue and red, respectively. Regions with medium hydrophobicity/hydrophilicity are shown as purple. The figure was prepared from the X- ray crystal structure of the unliganded TBmcr [pdb access code = 1X74 (Savolainen et al, 2005)] using the MBT protein workshop software http://www.rcsb.0rg/pdb/home/home.dohttp://www.rcsb.org/pdb/home/home.do (Moreland et al, 2005).

63 Because DKG is soluble, it is purified without the use of any detergent, but it does

require the use of OG for its in vitro assay (Bregoli et al, 2001). This is explained by the

fact that OG facilitates the interaction between DKG (soluble enzyme) and DAG

(insoluble substrate) (Verger, 1976). This particular example offers an alternative

explanation for the effect of OG on TBmcr activity. The fact that the substrates of TBmcr

are soluble due to the Co A moiety may explain the limited dependence of TBmcr activity

on OG concentration. Moreover, this could be verified by studying the effect of OG on

TBmcr activity using more hydrophobic substrates such as pristanoyl-CoA or THCA-

CoA, in which case the dependence of catalysis on OG concentration might be expected

to increase. Since the OG concentrations between 0.2-0.4% gave maximal activity with

ibuprofenoyl-CoA as the substrate, TBmcr assays in the present study used OG at a final

concentration of 0.2%.

The initial velocity of an enzyme-catalyzed reaction is expected to be proportional

to the total enzyme concentration (see equation 1). Figure 3.10 shows the dependence of

the initial velocity of the reaction on the concentration of TBmcr at both low and high

substrate concentrations. While the dependence is primarily linear, there are values of

TBmcr concentrations for which upward and/or downward curvatures occur. At high

substrate concentration (600 uM), an upward curvature is observed at low enzyme

concentration (< 3.0 ng/mL) so that at zero enzyme concentration the velocity is not zero.

In general, an upward curvature of initial velocity with increasing enzyme

concentration may arise from a variety of factors. For example, the presence of a small

amount of an irreversible inhibitor of the enzyme in the assay mixture will lead to low velocities since small amounts of enzyme are completely inhibited and enzyme activity

64 will only be detected after a certain amount of enzyme is added to the assay solution

(Tipton, 1992).

These irreversible inhibitors can be heavy metal ions that contaminate the different buffers and solutions used in the assay. In the case of the present assay, the upward curvature corresponds to a certain level of activity and therefore can not be explained by the presence of irreversible inhibitors (Figure 4.2). A second explanation involves the presence of dissociable activator in the enzyme solution (Tipton, 1992). The presence of such an activator (A) in the enzyme solution will lead to the formation of new enzyme species (EA). This complex does not increase linearly with the increase of enzyme concentration, leading to a non-linear curve. However, this is not the case for

TBmcr since the graph is a straight line for enzyme concentrations between 3.0-7.5 ng/mL. The most plausible explanation for the observed upward curvature might be the inability of the CD spectropolarimeter to accurately measure the initial velocity at low enzyme concentrations as they are very small values. The presence of an apparent reaction in the absence of enzyme can be explained by the high and unstable photomultiplier voltage (-616 mV) in the presence of the high substrate concentration. In accord with this explanation is the fact that this upward curvature does not appear at low substrate concentrations where photomultiplier voltages are much lower (~283 mV). In the future, this problem might be circumvented by using a cuvette with a shorter light path.

65 ^ 12.5n "E IO.OH 3

>_

>» •4->

>

[enzyme] (arbitrary units)

Figure 4.2 Upwardly-curving dependence of initial velocity on enzyme concentration. Curve (•) shows the normally-expected relationship; curve (A) represents the case where there is an irreversible inhibitor contaminating the assay mixture. Figure adapted from Tipton, 1992.

66 At both low and high substrate concentrations, a slight downward curvature is

observed at high enzyme concentration (> 7.5 ng/mL). This downward curvature could be

due to the rapid reaction and the failure to measure the true initial rate of the reaction, or

due to the presence of a dissociable inhibitor in the enzyme solution (Tipton, 1992). In

the second case, the increase of the enzyme concentration results in a proportional

increase of the inhibitor (I) and of the inactive form of the enzyme (EI). This leads to a

downward curvature of the line on the graph to an apparent maximum value. The

presence of the inhibitor can arise from contamination of the solutions and reagents used

in the experiments. However, this seems unlikely. The more likely explanation for the

downward curvature observed in Figure 3.10 is that at high enzyme concentrations, a

portion of the reaction has occurred so that the observed velocity is less than the true

initial velocity. This explanation is in accord with the findings obtained for the

epimerization of (25)- and (2i?)-ibuprofenoyl-CoA shown in Figure 3.8. Thus the

dependence of initial velocity on enzyme concentration was only linear for enzyme

concentrations between 3.0 ng/mL and 7.5 ng/mL. Consequently, 7.5 ng/mL was the

enzyme concentration used in the subsequent kinetic studies. Also, it is important to note that the effect of erizyme concentration may be different at higher OG concentrations since it has only been studied at an OG concentration of 0.2%.

Michaelis-Menten plots were constructed to determine the kinetic parameters of the His6-tagged and tag-free TBmcr-catalyzed reaction. As mentioned in the Introduction, it has been suggested that during catalysis, the 2-methyl group and the CoA moiety do not move and are essentially anchored to their respective binding pockets on the enzyme

(Bhaumik et al, 2007). However, the a-proton and the acyl group move and interchange

67 positions to achieve epimerization. In the case of the tag-free TBmcr, the enzyme has a higher turnover number (kcat) (1.5-fold) with (25)-ibuprofenoyl-CoA relative to that observed for (2i?)-ibuprofenoyl-CoA. This greater value of kcat may be due to the (S)- specific general base His 126 being closer to the a-carbon (3.3 A) compared to the (R)- specific general base Asp 156 (3.7 A) (Bhaumik et al, 2007). This assumes that the a- proton abstraction is rate-limiting which often is the case for cofactor-independent racemases (Tanner, 2002). The movement of the acyl group during chiral interconversion might not be expected to affect the turnover number since it is a process that occurs along the same loop in opposite directions. However, it is possible that conformational changes may affect &cat differently in the R—>S and S—>R reaction directions.

Tag-free TBmcr exhibited a higher affinity for (2i?)-ibuprofenoyl-CoA relative to

(2S)-ibuprofenoyl-CoA (1.8-fold). This could be explained by the different environments of their respective binding pockets. The amino acid residues forming the (R)-pocket are mostly from the small domain of the other subunit; whereas the (5)-pocket is predominantly formed from residues located in the large domain of the same subunit as the active site (Bhaumik et al, 2007). In addition, the enzyme catalyzes the epimerization of (2i?)-ibuprofenoyl-CoA with a slightly greater catalytic efficiency (kcJKm) than it exhibits with (25)-ibuprofenoyl-CoA (1.2-fold).

The His6-tagged TBmcr has similar kinetic characteristics as the tag-free TBmcr, exhibiting a higher turnover number (1.6-fold) with (25)-ibuprofenoyl-CoA and a higher affinity (1.2-fold) for (2i?)-ibuprofenoyl-CoA, but a higher efficiency (1.3-fold) with the

(6)-thioester. Overall, the His6-tagged and tag-free TBmcr have similar affinity for (2S)- ibuprofenoyl-CoA but the His6-tagged enzyme has a lower affinity for (2i?)-ibuprofenoyl-

68 CoA. This shows that the N-terminal hexahistidine tag has at least a minor effect on the structure of the active site. The crystal structure of TBmcr showed that the C-terminal tail of one subunit folds back on its own N-terminal large domain (Savolainen et al, 2005).

The presence of the His6-tag in the N-terminal large domain may be responsible for modifying the positioning of the C-terminal small domain that contains the majority of the residues forming the (i?)-pocket, thereby changing the affinity of the enzyme for the

(i?)-thioester only. Overall, the presence of the His6-tag leads to a minor decrease in activity relative to the tag-free enzyme.

Calculation of the equilibrium constants for epimerization (K^ using the Haldane equation (equation 11) revealed that Keq is close to unity. This was not necessarily expected since AMACR is not a racemase but an epimerase, acting on substrates with more than one chiral centre. Diastereomers have different transition states leading to different rates of the reaction. However, AMACR only acts on one chiral centre of its substrates while the other stereogenic centers located on the CoA moiety remain unchanged. This means that AMACR might be considered as a "pseudo-racemase".

Previous studies on AMACR from rat (Reichel et al, 1997; Schmitz et al, 1997), as well as studies on TBmcr (Savolainen et al, 2005; Bhaumik et al, 2007) specifically, did not report substantial amounts of kinetic data other than relative activities. The dissociation constant (K&) for a tag-free TBmcr with (25)-ibuprofenoyl-CoA was estimated to be 36 uM by Bhaumik et al, 2007. This value is slightly different from the value obtained in this study (86 uM). The differences in the type of assay used are probably responsible for the difference in values. Using a fixed-time assay, the binding affinity was obtained by using (25)-ibuprofenoyl-CoA as a competitive inhibitor of the epimerization of [2-

69 H]pristanoyl-CoA. Fixed-time assays are prone to more errors than continuous assays due to the greater number of steps required in the procedure. Also, different purification procedures have been used for these two enzymes. No direct kinetic data were available for TBmcr with (2i?)-ibuprofenoyl-CoA as the substrate before this study. Nor is any kinetic data available for either of the mammalian forms of AMACR using ibuprofen

CoA-thioesters as, substrates. It is apparent from the lack of kinetic information that the development of a continuous assay for AMACR activity was required.

70 Chapter 5

Future Work

Multiple studies (e.g., Dhanasekaran et al, 2001; Luo et al, 2002; Zha et al,

2003) have shown that AMACR plays a central role in prostate cancer initiation and progression, which makes it a target for prostate cancer treatment. Small molecules designed to inhibit the AMACR activity in vitro and in vivo represent a potential class of anti-cancer drugs specifically aimed at prostate cancer. The assay developed in the present work provides an essential tool for assessing and screening the inhibitory effect of such small molecules.

One of the first issues to be addressed is the expression and purification of a soluble form of mammalian AMACR. The expression of a soluble from of Hisg-tagged and GST-tagged rat AMACR was unsuccessful. The use of a maltose-binding protein

(MBP)-tag, known to be more soluble and for directing the recombinant protein to the periplasmic compartment (Fahnert et al, 2004), is one potential strategy. Alternatively, one could try to express a mammalian form of AMACR in a different expression system such as in insect cells (Sf21) using a baculovirus system (Nestle & Roberts, 1969).

One strategy for designing potential AMACR inhibitors is to mimic the structure of the substrate ibuprofenoyl-CoA. The profen structure is used as a scaffold that can be modified to generate inhibitors that might reversibly or irreversibly inhibit AMACR activity both in vitro and in vivo. A potential class of reversible inhibitors would present two profen rings to the enzyme - one to block each binding pocket (Scheme 5.1).

71 (ft)-pocket

(R)-pocket SCoA SCoA

(S)-pocket (S)-pocket\^|

Scheme 5.1 Potential reversible AMACR inhibitors. The two side aryl chains of the inhibitors are expected to simultaneously occupy the (S)- and (i?)-pockets leading to the inhibition of the enzyme.

72 These putative reversible inhibitors would hamper the binding of the substrate to the enzyme resulting in the inhibition of AMACR. Irreversible inhibitors that covalently react to modify residues in the active site of the enzyme could also be designed.

However, a recent study using (3-halogen-containing ibuprofen-based compounds did not lead to the expected irreversible inhibition (Scheme 5.2) (Caraell et at, 2007). An alternative approach using a carboxylic acid compound containing an epoxide group or a j?ara-bromomethyl group could be employed (Scheme 5.3). These carboxylic acids would be converted into their corresponding CoA-thioester for the in vitro assay. The inhibitory activity of all compounds could easily be tested in vitro using recombinant

AMACR from Mycobacterium tuberculosis (TBmcr) (or from a mammalian source) and the continuous circular dichroism (CD) assay. This assay may be used to determine the mode of inhibition, the reversibility of the inhibition, and the binding affinity (K{) of each compound.

Those compounds that exhibit inhibition of either TBmcr or rat AMACR in vitro would subsequently be used in in vivo studies to determine the effect of AMACR inhibition on prostate cancer cell lines (e.g., LAPC-4, LNCaP). In order to achieve that, cell proliferation would be measured using BrDU labeling and MTT assays and apoptosis would be examined using a caspase-3 assay.

73 SCoA SCoA

R- CH3(CH2)io. CH3(CH2)n, CH3(CH2)i2 X= H, F Y= CF3> CHF2, CH3

O

'SCoA

SCoA

Scheme 5.2 Competitive inhibitors of AMACR. The K{ values of the inhibitors shown were determined by using reversed-phase HPLC to monitor the epimerization of (255*, 25i?)-THCA-CoA catalyzed by AMACR purified from rat liver. The K{ values ranged between 0.9-137 uM (Carnell et at, 2007).

74 Rs UlA "^ V^kJ^ R = H.i-Bu ^^ X^ ^SCoA ^^ X^ ^SCoA l— n v .0 E-B^ OH E-B- < -f

E-N

SCoA

Scheme 5.3 Potential irreversible AMACR inhibitors. The ibuprofen-based irreversible inhibitors contain an epoxide or a /?ara-bromomethyl group that could react to covalently modify one of the catalytic general bases of AMACR.

75 Chapter 6

Conclusion

In the present study, a CD-based kinetic assay has been developed to directly

monitor the reaction catalyzed by the prostate cancer biomarker AMACR. This assay

could be used as a starting point for the development of a more sensitive diagnostic test

for prostate cancer. Furthermore, this assay should serve as a useful tool for the

identification of compounds that inhibit AMACR. Such compounds represent potential

anti-cancer drugs. The fact that prostate carcinoma cells rely on lipid metabolism for

growth suggests that inhibition of enzymes such as AMACR that are involved in the

catabolism of a-branched fatty acids may be a valid strategy for developing drugs for prostate cancer.

Several cases of human AMACR deficiency have been reported. It is associated

with adult-onset sensory motor neuropathy due to the accumulation of pristanic acid in the brain (Ferdinandusse et al, 2000). This particular condition, which only appears in

adults, shows that the neurological damage manifests itself only after very long periods of time during which pristanic acid accumulates in the body. In addition, no obvious phenotype was associated with the Amacr_/~ mouse model. However, when the AMACR- deficient mice were fed a diet rich in phytol, liver injuries such as multivacuolar degeneration and coagulation necrosis were observed (Savolainen et al, 2004).

Consequently, an anti-cancer drug that targets AMACR activity might not have serious side effects for the patient, especially if the patient's diet is appropriately controlled.

76 Appendix

Open Reading Frame of TBmcr in pET15b vector. The Nde I and BamH I restriction Sites used for the cloning of TBmcr ORF are underlined. The translation start and stop codons are in bold. Also, the start codon of the wild-type ORF is in bold.

5'-ATG GAA AGG AGA TAT ACC ATG GGC AGC AGG CAT CAT CAT CAT CAT CAC AGC AGC GGC CTG GTG CCG CGC GGC AGC CAT ATG GCG GGT CCG CTG AGC GGG TTG CGA GTT GTC GAG CTG GCG GGC ATC GGG CCG GGC CCG CAC GCA GCG ATG ATC CTG GGG GAC CTC GGT GCC GAC GTG GTG CGC ATC GAT CGC CCG TCA AGT GTC GAC GGT ATT TCG AGA GAC GCC ATG TTG CGT AAC CGG CGT ATC GTG ACC GCC GAC CTG AAG TCC GAT CAG GGA CTC GAG CTT GCG CTC AAA CTC ATC GCC AAG GCC GAC GTG TTG ATC GAG GGT TAC CGT CCC GGC GTC ACC GAA CGG CTG GGA TTG GGT CCG GAA GAA TGT GCG AAG GTC AAC GAC CGG CTG ATC TAC GCG CGG ATG ACC GGC TGG GGC CAA ACC GGC CCG CGT AGT CAG CAG GCC GGT CAC GAC ATC AAC TAC ATC TCG CTG AAC GGC ATT TTG CAC GCC ATT GGC CGG GGC GAC GAG CGA CCG GTG CCG CCG CTG AAC CTG GTT GGT GAC TTC GGC GGC GGC TCG ATG TTC CTG CTG GTC GGC ATC CTG GCC GCG CTA TGG GAG CGG CAG AGC TCC GGC AAG GGC CAG GTC GTC GAT GCG GCG ATG GTC GAC GGG TCC AGC GTG CTG ATT CAG ATG ATG TGG GCG ATG CGA GCG ACG GGC ATG TGG ACC GAC ACA AGA GGG GCC AAC ATG CTC GAC GGC GGG GCA CCC TAC TAC GAC ACC TAC GAA TGC GCC GAC GGC CGC TAC GTC GCT GTC GGC GCC ATT GAG CCG CAG TTC TAT GCG GCC ATG CTG GCC GGA TTG GGT CTA GAC GCC GCC GAG CTG CCC CCG CAA AAC GAC CGC GCC CGT TGG CCC GAA CTG CGG GCG CTG CTG ACC GAA GCG TTC GCG AGC CAC GAC CGT GAC CAT TGG GGC GCG GTG TTC GCC AAT TCC GAT GCC TGT GTG ACG CCG GTG CTG GCG TTC GGT GAG GTG CAC AAC GAG CCG CAC ATC ATC GAG CGA AAC ACC TTT TAT GAA GCC AAC GGC GGA TGG CAA CCC ATG CCG GCT CCG CGG TTC TCC CGC ACC GCT TCG AGC CAG CCA CGC CCG CCG GCC GCC ACG ATC GAC ATC GAG GCA GTG CTC ACC GAC TGG GAC GGA TAG GGA TCC GGC TGCTAA-3'

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