INACTIVATION OF BENZOYLFORMATE DECARBOXYLASE
BY THMMIN THIAZOLONE DIPHOSPHATE
Daria Hi1 Ching Yu
A thesis submitted in conformity with the requirements
For the degree of Master of Science
Graduate Department of Chemistry
University of Toronto
Copyright by Daria Hil Ching Yu 2001
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To Daddy, M o r n y
and yenneroos
Abstract
INACTIVATION OF BENZOYLFORMATE DECARBOXYLASE
BY TEUAMIN TElIAZOLONE DIPHOSPHATE
Daria Hi1 Ching Yu
Department of Chemistry, University of Toronto
Master of Science, 200 1
In 1973, Gutowski and Lienhard designed and synthesized thiarnin thiazolone
diphosphate ('ITDP) as a transition state analogue for E. coli pyruvate dehydrogenase, an
enzyme whieh uses thiamin diphosphate as a cofactor in the conversion of pyruvate to
2-(1-hydroxyethy1)thiarnin diphosphate [Gutowski, J.A., and Lienhard, G.E. (1976)
J. Biol. Chem., 251,2863-28661. Other groups have reported different results for other
ThDP-dependent enzymes. Here, we begin the investigation of the interaction between
TTDP and P. putida benzoylformate decarboxylase (BFD). First, a recombinant form of
BFD was expressed and purified by nickel af£inity chromatography. Existing direct and
coupled assays were modified to follow the activity of the enzyme with endogenously
bound thiamin diphosphate and inhibition of enzyme activity with TTDP. Preliminary
results suggest Ki = 5 ph4 for TTDP which is on the same order as the Km of ThDP for
the enzyme. Thus, TTDP is not a transition state analogue for BFD.
Acknosvledgements
1 would Iike to thank Dr. Ron Kluger for giving me an opportunity to do research
in his lab. 1 leamed how to read and think cntically about the physical chemistry of
enzyme mechanisms. Without his constant emphasis on solving small problems, this
thesis would still be on the cornputer.
The lab group has also been supportive of m y idiosyncracies and crazy working
hours. Past or present, they are Anna, Amer, Ian, Jesse, John, Lisa, Nik, Nom, Pete, and
Steve. 1 would also like to thank Lena and Miriam of Purdue University, Indiana, Petra
and Martina of der Institut fur Enzyrntechnologie of Heninch-Heine Universitat
Dusseldorf im Forschungszentrum Jülich, Germany for their invaluable correspondence
and advice. Other people in the department have also made working here interesting.
Also my fiiends totally unrelated to chernistry have made Iiving in Toronto
enjoyable. Finally, 1 would Iike to thank my family for their love and encouragement.
Table of Contents
. . ............................................................................................................................... Abstract r i
... ............................................................................................ ........ Acknowledgements ,... - 1 1 1
............................................................................................................... Table of Contents iv
..................... List of Tables .... ..................................................................................... vi
List of Figures .................................................................................................................... vi
. J
Structures and Abbreviations .................... .. ................................................................... vii
Chapter 1: Introduction .................................................................................................. -1
....................................................................... 1.1 Benzoylformate Decarboxylase 1
....................................................................................... 1 -2 Catalytic Mechanism 1
. . ................................................................................ 1 -3 Transition State Theory 4
Chapter 2: Experimental ................................................................................................ 6
................................................................................................. 2.1 Instrumentation 6
2.2 Materials ........................................................................................................... 6
.......................................................................................................... 2.3 Syntheses -6
2.3.1 Thiarnin Disulfide ............................................................................. -7
2.3 -2 Thiarnui Thiazolone .......................................................................... -7
...................................................... 2.3 -3 Thiamui Thiazolone Diphosphate 8
2.4 Expression and Purification of Benzoylformate Decarboxylase-His6 .............. 9
2.5 Enzyme Assays .......................... ... ............................................................. 1
............................................................ 2.5.1 Direct Decarboxylase Assay 1 1
.................... ............................ 2.5.2 Coupled Decarboxylase Assay ...... 12
2.6 Inhibition with Thiamin Thiazolone Diphosphate
............. ......................*...................................... At 5 min Incubation Period ... 13
2.7 Time Dependence of Thiamui Thiazolone Diphosphate
................................................................................... on BFD-His6 Activity 1 4
Chapter 3: Results ........................................................................................................... 15 ............................................. 3.1 S ynthesis of Thiarnin Thiazolone Diphosphate 1 5
.................. .............. 3.2 Purification of Benzoylformate Decarboxylase-His6 ..... 15
..............*....................... ................................... 3.3 Direct Decarboxylase Assay .. 16
.......................................... ................... 3.4 Coupled Decarboxylase Assay ..... 18
........................................ 3.5 Inhibition with Thiamin Thiazolone Diphosphate .. 20
Chapter 4: Discussion ..................................................................................................... 22
4.1 BFD-His6 Purification ..................................................................................... 22
4.2 Cornparison of Direct and Coupled Decarboxylase Assays ........................... 22
4.3 Inhibition with Thiarnin Thiazolone Diphosphate ......................................... 24
Chapter 5: Conclusions and Future Direction ............................*............................... .31
........................................................... References ................... .. .. .. .. ..... .= .......................... 32
List of Tables
Components of direct decarboxylase assay
Components of coupled decarboxylase assay
Effect of TTDP on ThDP-BFD-His6
Incubation mixtures of BFD-His6 and TTDP
Reaction mixture for tirne dependence of TTDP on BFD-His6 activity
List of Figures
Catalytic mechanism for benzoylformate decarboxylase
Calibration cuve for Bradford assay
Direct assay to follow activity of BFD-His6
Rate of reaction is proportional to amount of BFD-His6
Absorbance of NADH at 340 nrn
Following oxidation of NADH in the coupled decarboxylase assay
Checkhg for coupling of decarboxylation to NADH oxidation
Following reduction in activity of 0.02 U BFD-His6 by TTDP
BFD-His6 activity as a function of 'TTDP concentration
Time dependence of TTDP on BFD activity
Reactions in the direct and coupled decarboxylase assays
A potential transition state analog for BFD
Possible compounds that could lead to inhibiton of BFD
Free energy-reaction progress profiles for the non-enzyrnic decarboxylation of pymvate by ThDP and the reactions catalyzed by yeast pyruvate decarboxylase (SCPDC) and Zymmonas pyruvate decarboxylase (ZMPDC)
vii
Structures and Abbreviations
Abbreviation Mo Iecule
Thi amin,
ThDP R = P2O4H2, Thiarnin diphosphate
BFD-His6 Benzoylformate decarboxylase- His6
(9-2-HPP (5)-2-hydroxy- 1 -phenyl-propanone
MThDP 2-(mande1yl)thiamin diphosphate,
R = ~ ~ 0 6 ' ~
HBzThDP 2-(1-hydroxybenzyl)thiamin
diphosphate, R = pz02
TT R = H, Thiarnin thiazolone,
TTMP R = POsH, TT monophosphate,
TTDP R = PZO6H3, TT diphosphate,
TTTP R = P3O9&, TT triphosphate
LB Luria-B ertani
HLADH Horse liver alcohol dehydrogenase
NADH Dihydronicotinamide adenine
dinucleo tide
Structure
Structures and Ab b reviations (continued)
Abbreviation Molecule
Ni-NTA Nickel-nitrilotriacetic acid
BSA Bovine serum albumin
SDS-PAGE Sodium dodecyl suljphate
polyacrylamide gel electrophoresis
IPTG Isopropyl-P-thiogalactopyranoside
PMSF Phenylrnethanesulfonyl fluonde
LThDP 2-(a-1actyl)thiamin dip hosphate,
R = pzosJ
HEThDP 2-(1 -hydroxyethyl) thiamin
diphosphate, R = P ~ Q ~ - ~
HBP
methyl acetylphosphonate
hydroxybenzylphosphonate
Structure
Chapter 1 - Introduction
1 . Benzoylformate Decarboxylase
Benzoylfonnate decarboxylase (Bm), an enzyme in the mandelate pathway of
Pseudomonas and Actinobacter species ', uses thiarnin diphosphate (ThDP) to catalyze the
decarboxylation of benzoylformate. Studies involving isotope effects 2, substrate and
substrate anaiogs are consistent with Breslow's covalent catalytic mechanism for ThDP-
dependent decarboxylases 4. The crystal structure of BFD was solved to 1.6 A resolution
and spectroscopie studies helped clarie the nature of the intermediates along the catalytic
cycle. BFD also catalyzes a side reaction, carboligation, and has been used in the
asyrnmetnc synthesis of chiral 2-hydroxy ketones '-Io. For example, the "activated
benzaldehyde", 3, on the enzyme can add to acetaldehyde to yield (5')-2-hydroxy-1-phenyl-
propanone, ((S)-2-HPP, Scheme 1). The gene for the enzyme has been isolated, cloned ' and rnodified to include a C-terminal hexahistidhe extension mis6) that permits easy
purification of the enzyme using nickel affinity chromatography 'O. The activity of BFD-
His6 is the same as the native enzyme.
1.2 CataIytic Mechanism of Benzoylformate Decarboxylase
The catalytic mechanism of ThDP-dependent decarboxylases was proposed by
Breslow and McNellis and is now widely accepted I l . A detailed review of the reaction
intermediates for the decarboxylation of pymvate to acetaldehyde by pynivate
12.13 decarboxylase has been published . BFD catalyzes an analogous reaction using
benzoylfonnate as the substrate.
The enzyme environment favours the deprotonation of C-2 on the thiazolium ring.
The resulting ylide, 1, attacks C-2 of benzoylformate, giving a covalent intermediate,
2-(rnande1yl)thiami.n diphosphate, 2. Loss of carbon dioxide and subsequent proton
transfer gives 2-(1-hydroxybenzyl)thiamin diphosphate, 4. The proposed transition state
for this step involves a species that has both carbanionic and enamine character, 3. The
final step is the regeneration of the ylide and release of benzaldehyde. The decarboxylation
cycle and carboligation side reaction for BFD are depicted in Scheme 1.
It is also interesting to note that the non-enzyrnatic reaction between
benzoylformate and thiarnin is pH dependent 14. In acidic and neutral solutions, 4
undergoes fragmentation to give 2,s-dimethyl-4-amino-pyrimidine and
2-benzoyl-5-(2-hydroxyethyl)-4-rnethylthile ' In basic solution, the products are
thiarnin and benzaldehyde. BFD, having optimal activity at pH 6.0, circurnvents the
hgmentation reaction to give the desired elimination products.
SA* o z - / z
1.3 Transition State Theory
In 1973, Lienhard presented the concept that transition state analogues could be
potent enzyme inhibitors 16. The ideal transition state analogue could bind to the enzyme
with the same affinity as would the transition state. Gutowski and Lienhard applied this
theory to E. coli pynivate dehydrogenase, which initially catalyzes the conversion of
pymvate to 1 -hydroxyethylthiamin diphosphate and carbon dioxide. They designed thiarnin
thiazolone diphosphate, 5, so that its carbonyl fünctionality at C-2 of the thiazokun ring
would mimic the enamine character of 3. They found that TTDP binds at least 20 000
times more tightly than does ThDP 17, consistent with their expectations for a transition
state analogue.
NH2 I
thiamin thiazolone diphosphate or TTDP, 5
However, the results have been subject to other interpretations since the affinity can
be explained on other factors, including hydrophobicity of the active site 18*'9. TTDP has
been tested with other TDP-dependent enzymes, where high affinity has not been observed.
Gish (1984) showed TTDP to be an irreversible inhibitor for wheat germ pymvate
decarboxylase, binding only three times more tightly than ThDP lgv2*. Neither is TTDP an
effective transition state analogue for acetohydroxyacid synthase 21, nor is it for
transketolase 22. TTDP is a weak competitive inhibitor for these two enzymes. TTDP is a
non-competitive inhibitor of rat brain pyruvate dehydrogenase 23. These results indicate
that Lienhard's theory requires a more cornplex set of citena than TTDP c m
accommodate.
Since we are interested in the mechanism of Pseudomona puriaa benzoylfcrmate
decarboxylase with respect to ThDP, its interactions with TTDP need to be established.
Chapter 2 - Experimental
2.1 Instrumentation
Proton NMR spectra were obtained at 400 MHz. Phosphorus NMR spectra were
obtained at 121.4 MHz. Enzyme assays and inhibition kinetics with TTDP were foI1owed
by UV on a Perkin Elmer Lambda 2 or Lambda 19 spectrophotometer.
2.2 Materials
The plasmid containing the BFD-His6 insert was a gift fiom the Iab of Dr. Martina
Pohl of der Institut für Enzymtechnologie of Heinrich-Heine Universit 3t Dusseldorf irn
Forschungszentnim Jülich, Germany. Thiamin hydrochloride was a gift fkom Novopharm.
Competent E.coli BL2 1 cells were obtained fiom Novagen. The Ni-NTA agarose product
was fiom Qiagen. HLADH was a crystallïne suspension, 33 U/ml, obtained fkom FIuka.
Al1 other reagents and solvents were obtained in the highest purtty possible.
2.3 Syntheses
Thiamin was converted to thiamin disulfide, 6, and then recyclized to forrn thiarnin
thiazolone, 7. Phosphorylation of 7 gave a mixture of the mono-, di-, and
triphosphorylated species of thiamin thiazolone which were then purified by
chromatography and characterized by NMR.
Thiamin disulfide, 6 Thiamin thiazolone, 7, R = OH Thiarnin thiazolone diphosphate, 5, R =
2.3.1 Thiamin DisuEde
Thiamin disulfide was synthesized by the method described in Gish's thesis 18.
Thiamin hydrochlonde (20 g) was dissolved in 100 ml distilled water, which was adjusted
to pH 11.75 with 6 N sodium hydroxide until the colour of the solution was very pale
yellow (45 min). Then 100 ml of 23 % potassium femcyanide was added dropwise with
continuous stirring. The solution became green and yellow crystals formed near the end of
addition. Further cooling, filtration and recrystallization fiom water yielded pale yellow
product (80%).
1 H NMR in DtO/DSSlDCI, p H 1: 6 2.110 (6H, s, CH3-pyr), 2.50-2.80 (lOH, m, CH3C(4),
CHzC(S)), 3.620 (4H, t, 'J = 6.6 Hz, CHzOD), 4.725 (4H, s, CH2N>, 7.962 (2H, s, H-pyr),
8.03 1 (2H, s, CHO).
2.3.2 Thiamin Thiazolone
Thiamin disulfide was synthesized fiom the method of Todd and Sykes (19(10 g)
was suspended in 3-methyl-2-propanol(200 ml) and refluxed for 2 h. Leaving the solution
to cool at 4OC gave a pale yeilow solid. Recrystallization fkom ethanol and water (2:7 v/v)
gave white needles (60%) 24.
I H NMR in D20/DSS/DCl, pH 1: 6 2.12 (3H, s, CH3-pyr), 2.57 (3H, s, CH3C(4)), 2.8 1
(2H, t, 'J = 4.2 Hz, CHzC(5)), 3.75 (ZH, t, 2~ = 4.2 Hz, CH20D), 7.83 (lH, s, H-pyr).
2.3.3 Thiamin Thiazolone Diphosphate
The modified phosphorylation procedure of Viscontini et al., (1949) was used to
phosphorylate thiarnin thiazolone 25. Phosphoric acid (85 %, 3 ml) was heated over a
flarne until the solution became white and syrupy. Upon cooling to room temperature, the
symp hardened to a glassy solid. Thiarnin thiazolone (1.0 g) was added to this glassy solid
and this mixture was heated again to 1 15OC in an oil bath for 20 min. with occasional
stimng with a glass rod. Upon cooling, the mixture resolidified. In an ice bath, the
mixture was dissolved in water (2 rnL) with stimng ovemight. 3 L ~ NMR (in DzO, DCI,
DSS) indicated that phosphorylation did take place (peaks are significantly srnaller than
inorganic phosphate and pyrophosphate), but 'H NMR clearly shows the solution to be a
mixture of the monophosphorylated, diphosphorylated and triphosphorylated species.
Undissoived solids were removed by vacuum filtration.
Upon addition of 200 rnL 1: 1 95 % ethanol: diethyl ether, the solution went cloudy
and a light yellow residue formed. Once the solution was allowed to settie, the liquid was
carefully decanted and the oily residue was taken up again in 5 rnL water and triturated
twice more. The "P NMR spectrum of this mixture better shows the three phosphorylated
species,
3 1 ~ N-MR @20/DSS/DCI): 6 0.60 1 (s, inorganic phosphate and TTMP), -9.5 17 (m,
diphosphate and TTDP), -23 -958 (m, triphosphate and T?TP).
The mixture was then carefully titrated to pH 4.0 and appiied to a cellulose plate
with fluorescent indicator (Eastman). The solvent system, 10: 1:6 95% ethanol: n-butanol:
0.15 sodium citrate p H 4, was used to separate the components 24. Each band was then
scraped off the pIate and extracted fiom the adsorbent with water, filtered and lyophilized.
NMR was perforrned to identiQ each band. TTDP had Rf = 0.50.
2.4 Expression and Purification of Benzoylformate Decarboxylase
Competent BL2 l(DE3) cells (20 pl) were transformed with the plasmid carrying an
ampicillin resistance gene and the BFD-His6 insert (2 pl) by heat shock (5 min at 37OC).
The cells were grown in LB media (250 pL) at 30°C for L hr, plated onto LB plates
containing O. lmg/rnL ampicillin, and grown for 20 h in a 37°C incubator. One colony was
selected for a growth in a 30 rnL culture. When 0D600 = 1.0, glycerol was added to the 30
mL culture to a final concentration of 20 %. This stock culture was then fiozen in liquid
nitrogen and stored at -78OC. A pipette tip was used to scratch the stock culture and
transferred uito new LB culture (30 mL) and grown at 37OC at 250 rpm. The 30 mL culture
was then added to a 1 L culture and grown for 3 hrs and then transferred to a 3 L culture.
When OD = 1.2-1 -5, the culture was induced with IPTG (60 mg& 238.3 g/mol, final conc.
0.2 mM). After 24 h of growth, the cells were spun at 15 000 rpm for 40 min., fiozen in
liquid nitrogen, and stored at -78OC until fùrther purification.
Al1 purification steps were performed on ice. Once the cells (usually about 13 g)
were thawed in lysis buffer (2-5 mWg wet ce11 weight), lysozyme (1 mg/mL) and PMSF (to
give 1 rnM, 0.17 mg/mL) were added and incubated for 30 min. The cells were cracked
open by sonication with six 10 s bursts at 20 W with a IO s cooling period between each
bunt. The lysate was centrifüged at 10 000 rpm for 30 min at 4OC to remove cellular
debris. Ni-NTA agarose was added to the supernatant at 1 m u 4 mL and mixed gently by
shaking on a rotary shaker at IO rpm ovemight. The lysate-Ni-NTA agarose mixture was
then loaded into a column and the flow-through collected. The column was washed with
eight 5 mL fiactions of wash buffer (lysis buffer containing 20 mM imidazole) and then
eluted with ten 4 mL hctions of elution buffer (lysis buffer containing 250 m .
imidazo le).
SDS-PAGE anaiysis on 5 uL aliquots of the flow-through, wash fiactions and the
elution fractions was performed. The elution fractions that contained a significant band at
57 kDa were pooled, concentrated using a Centriprep centifûgal filter device with a YM-10
MW membrane and the elution buffer exchanged for 50 mM potassium phosphate buffer,
pH 7.0.
The spectrophotometric assay was used to obtain an approxirnate protein
concentration 26:
Protein concentration ( m m ) = 1.55 - 0.76 A260
An aliquot of the BFD-His6 preparation was then diluted appropriately and protein
content was determined more accurately using the Bradford assay (Bio-Rad) using bovine
senun albumin as standard. The BFD-His6 enzyme was stored in 50 rnM potassium
phosphate buffer, pH 7.0 with 0.1 rng/rnL of sodium azide as a preservative. Unused
enzyme can be stored at -20°C as a lyophilisate.
2.5 Enzyme Assays
2.5.1 Direct Decarboxylase Assay
The activity of the purified BFD-His6 was determined by initiating the reaction with
benzoylformate to the reaction mixture (Table 2.1) equilibrated at 30°C in a 1.7 mL
cuvette. The consumption of benzoylformate was followed at 343 MI for 40 min. for three
different arnounts (x) of BFD. The extinction coefficient at 343 nm for benzoylformate in
150 mM potassium phosphate, pH7.0, containing 2.5 mM MgS04 was determined to be 79
m ~ " cm-' (results not shown). 1 U is defined as the arnount of enzyme that
decarboxylates 1 p o l benzoylformate per minute under the above conditions.
2.5.2 Coupled Decarbo-xylase Assay
The coupled decarboxylase assay consisted of the following components (Table
2.2) incubated for 5 min. at 30°C. Three minutes pnor to the addition of benzoylformate,
HLADH (30 fi) and 0.05X BFD-Hisa (x) were added to the reaction mixture. The
reaction was followed by the decrease in absorbance of NADH at 340 nrn over 60 min.
Table 2.1. Components of Direct Decarboxylase Assay
1 10 mM thiamin diphosphate 1 50 1 0.5 mM 1
Stock solution
500 mM phosphate buffer, pH 7.0
40 mM magnesium sulfate
1 Water I 547 - x I I 1 1X BFD stock I X I I
Volume to take, @
300
63
r500 mM benzoylformate l 40 I 20 mM I
Final concentration
150 rnM
2.5 rnM
I Total volume 1 1000 I I
Table 2.2. Components of Coupled Decarboxylase Assay
--
Water I 620-x I
10 mM ThDP
20 mM NADH
Final concentration
50 mM
2.5 mM
Stock solution
250 mM phosphate buffer, pH 7.0
40 mM magnesium sulfate
-
10 rnM benzoylformate 1 40 1 0.40 mM
Volumes, &, to take
200
63
50
17
0.05X BFD
Total volume 1 1000 1
0.5 rnM
0.36 mM
I
x 0.01 - 0.08 U I
2.6 Inhibition with Thiamin Thiazolone Diphosphate at 5 min. Incubation Period
BFD-His6 solution was added to the following reaction mixture containhg TTDP
(x) and/or ThDP equilibrated at 30oC for 5 min. The reaction was then started with the
addition of benzoylformate and followed by W at 340 nm.
Table 2.3. Effect of TTDP on ThDP-bound BFD-His6
I Stock solutions I Volumes to take, pl
1 250 mM phosphate buffer, pH 7.0 1 200
1 10 pM ThDP I Y (to give 0-10 FM)
40 rnM rnagnesium sulfate
12.5 pM TTDP
1 NADH, 20 mM I 17
63
X (to give 0-5 pM)
1 benzoylformate, 10 mM, pH 7.0 1 40
I Total volume I 1000
2.7 Tirne Dependence of TTDP on Bm-Bis6 Activity
Different incubation mixtures of O. lx BFD-His6, and TTDP were made to a total
volume of 300 uL and incubated at SOC for 13 min., 1 80 min., and 23 h. An aliquot (30 pl)
was then added to the reaction mixture (Table 2.5) and the change in absorbance at 340 nm
was fol1 owed.
Table 2.4 Incubation mixtures of BFD-His6 and TTDP
1 Solution 1 Volumes to take, pl I
Table 2.5 Reaction mixture for time dependence of TTDP on BFD-His6 Activity
BFD-Hk6, O. 1X
Water
TTDP, 10 j.iM
[TTDPI, CtM
1 Phosphate buffer, 400 nM, pH 7.0 I 100 I
100
100
O
O
MgS04, 40 mM
HLADH, 33 U/ml
1 Benzoylformate, 10 rnM 1 37 1
100
80
20
10
1 O0
90
10
5
63
50
NADH, 20 mM
Incubation mixture (Table 2.4)
20
30
L O0
60
40
20
Water
Total volume
1 O0
O
1 O0
50
700
1000
Chapter 3 - Results
3.1 Synthesis of Thiamin Thiazolone Diphosphate
Synthesis of TTDP was simple, however its purification by ion-exchange
chromatography (amberlite, Dowex, etc) was extremely difficult. TLC using the Gutowski
solvent system gave reproducible results although isolation of the T'IDP band gave a very
small yield. The ratio of inorganic phosphate to TTDP in the h a I T'ï'DP sample was
relatively high (10: 1) despite repeated triturations with 1 : 1 95 % ethanol: diethyl ether.
3.2 Protein Concentration of Benzoyiforrnate Decarboxylase-Efis6 Preparation
From the spectrophotometric assay 26, the protein concentration of the 1 X BFD-His6
stock is 1.62 mg/rnL. The ratio, A28~A260 was found to be 1.3 1 indicating little
contamination by nucleic acid. The calibration curve for the Bradford assay using BSA as
protein standard is shown in Figure 4. Using this method, protein concentration in the 1 X
BFD-His6 stock was found to be 4.162 mg/rnL.
mg/mL BSA
Figure 3- 1. Calibration curve for Bradford assay
3.3 Direct Decarboxylase Assay
Although there was no ThDP added to the growth mixture or during the purification
mixture, the final BFD-His6 preparation contained endogenous enzyme-bound ThDP.
Results of a Spica1 direct decarboxylase assay are shown in Figure 3.2. The activity is
linearly proportional to amount of protein as shown in Figure 3.3 and specific activity is
0.4 U/mL.
4 6 8 IO 12 14 16 18 20 22 2 4 2 6 283032
time, min
Figure 3.2. Direct assay to follow activity of BFD-His6
20 40 60
uL ZX BFD
Figure 3.3. Rate of reaction is proportional to amount of BFD-His6
3.4 CoupIed Decarboxylase Assay
Since benzaldehye is a substrate for HLADH, the coupled assay c m be used to
folIow the decarboxylation reaction with the advantage that it is more sensitive to decreases
in absorbance of NADH. Absorbance was found to be linear in the range of 0.05 to G.3
mM NADH (Figure 3 -4). The extinction coefficient for NADH at pH 7.0 in 50 mM
phosphate buffer c o n t a k g 2.5 mM MgS04 was 3.192 rdK1cxK1. Literature '*'O cites the
extinction coefficient to be 6.220 &'cm-'. We cannot account for this discrepancy.
To ensure that decarboxylation was coupled to W H oxidation, multiples of 5
pL of a 1/20 dilution of BFD-His6 stock were used for the assays (Figures 3.5 and 3.6)-
Reaction rates were determined fiom the iinear section of the spectra and expressed as
p o l of NADH consumed per minute.
O 0.05 0.1 0.15 02 025 0.3 035 0.4
Concentration of NADE& mi'vf
Figurs 3.4. Absorbance of NADH at 340 nm
O 2 4 6 8 10 12 14 16
tim e, min
19
volume of 0.05X BFD used
Figure 3.5. Following oxidation of NADH in the coupled decarboxylase assay
O 20 40 60 80
volume of 0.05X BFD, ul
Figure 3.6. Checking for coupling of decarboxylation to NADH oxidation
In Figure 3.6, it appears that the rates are coupled between 20 and 40 pL of O.OSX
BFD, since the rate at 40 pL 0.05X BFD is double that at 20 pL 0.05X BFD. Specifïc
activity for the O.05X BFD enzyme is thus 0.00 10 p o l NADH consumed per min per pL
of protein or lU/mL of protein. As previously reported, the amount of coupling enzyme
HLADH had to be at least 20 times more than BFD in order for the reactions to be coupled
3.5 Inhibition with Thiamin Thiazolone Diphosphate
The effect of TTDP was studied on 0.02 U of BFD-His6 with endogenous ThDP
(Figure 3.7). It appears that loss of activïty is linearly proportional to TTDP concentration
(Figure 3 -8).
1 O 2 0 30
timt. min
Figure 3.7. Following reduction in activity of 0.02 U BFD-His6 by TTDP at 5 min incubation penod
Figure
2 4
u M TTDP
BFD-His6 activity as a fùnction of TTDP concentration
However, when the time dependence of l"L'DP incubation with BFD-His6 with
endogenously bound ThDP was exarnined, it appears that TTDP has no effect on long-term
BFD activity:
TTDP concentration, uM
1-0-13min I ' i j -0- 180 min j
j+23h j
Figure 3 -9 Time dependence of TTDP on BFD activity
Chapter 4 - Discussion
4.1 BFD-His6 purification
Purification of BFD-Es6 by nickel chromatography was a very straightforward and
efficient procedure. SDS-PAGE showed that there were some contaminant proteins in the
final preparation but hardly significant compared to BFD-His6. The measured protein
concentration by the spectrophotometric assay was 2.5 times smaller than the value
measured by the Bradford assay. Since ThDP does not absorb at 595 nm, the protein
concentration value obiained by the Bradford assay would be more accurate.
4.2 Direct and Coupled Decarboxylase Assays
Previous direct and coupled assays '*'O had to be modified so that the decrease in
absorbance of benzoylformate or NADH was over at least a 20-minute interval. This
allows for more subtle changes in rate to be measured. The direct assay is a good estimate
of enzyme activity when the protein preparation has high active BFD-His6 content.
However, sornetimes the BFD-His6 preparation was not very pure or active (for unknown
reasons) and so the direct assay was not sensitive enough to detect the rate of
decarboxylation. Coupling the BFD-catalyzed reaction to HLADH-catalyzed oxidation of
NADH allows for larger differences in absorbance to be measured. It is important that
there is enough HLADH in the reaction mixture so that NADH oxidation is not the rate-
deterrnining reaction in the following reaction s equence:
direct assay
BFD HLADH benzoylformate- benzaldehyde - benzyl alco ho1 n
NADH NAD +
coupled assay
Figure 3.10. Reactions in the direct and coupled decarboxylase assays
The coupled assay reflects the rate of formation of benzaldehyde. Benzaldehyde is
only consumed by HLADH as fast as it is being made by BFD. Matters are a little more
complicated in the direct assay, wtiich follows the disappearance of benzoylformate. But
exactly at which step does benzoyiformate "lose" its absorbance? Does this reflect the rate
of formation of MThDP, the rate of decarboxylation of MThDP or the rate of protonation
of the enarnine/carbanion species (See Figure 1.1 )? Do any of the intermediates andlor
transient species between benzoylformate and benzaldehyde have significant absorbance at
340 nm? Sergienko et al., 2000 observed a transient species with hm = 400 nm that was
attributed to an enamine intemediate with ap-nitro substituent 6. Perhaps the decrease in
absorbance may not be due solely to benzoylformate but maybe to a composite of species
that absorb at 340 nrn. The specific activities fiom the direct and coupled assays were 0.4
U/ml and 1 U / d respectively. This suggests that a step prior to elimination of
benzaldehye is kinetically significant. The exact step is an exciting topic for fiirthur
research and discussion.
Specific activity is an indication of the purity of the enzyme preparation. Specific
activity for the enzyme is much lower cornpared to the values obtained by Iding et al., 2000
'O and Weiss et al., 1989 '. During purification, SDS-PAGE analysis indicate that the
protein preparation contains the 57 kDa monomers but does not reveal whether or not the
active tetrarneric form of the enzyme has formed properly. The enzyme could be fuahur
purified by phenyl Sepahrose FPLC 6.
Several problems were encountered while performing the assays making
subsequent measurements for the inhibition assays difficult and unreliable. The stability of
NADH is pH-dependent, and thus the extinction coefficient for NADH had to be measured
penodically. In addition, the enzyme slowly loses activity and the rernaining activity
during the inhibition studies has to be normalized. A significant problem is that the
coupled decarboxylase assay would sometimes become "uncoupled" giving sigmoidal
curves.
4.3 Inhibition with Thiamin Thiazolone Diphosphate
Removal of ThDP on the enzyme required dialysis in 12 x 1 L 50 mM phosphate
buffer, pH 7.0 over one week. Alteinatively, resuspension of cells in 12-15 ml lysis
buffer/g wet ce11 weight preceding purification gave a BFD-His6 preparation without any
activity. Although Iding et al.. 2000 'O stated that reconstitution of the apo-enzyme in 1
pM ThDP gave half-maximal activity in 24 hrs, reconsitution in our lab did not occur at
any concentration of ThDP, even after one week. Attempts to quanti@ the concentration
of ThDP in the BFD-His6 preparation were not successfbl because the absorption spectra of
proteins and ThDP overlap.
The effect of TTDP on the ThDP-bound BFD-His6 enzyme was investigated,
reflecting the ability of TTDP to cornpete for the two ThDP-sites in the enzyme. A plot of
activity vs. TTDP concentration (Figure 3.8) suggests that Ki is 5 W. Initially, it appears
that the added lTDP was able to reduce enzyme activity of the endogenous ThDP-bound
BFD-His6 by 20%. However, at higher concentrations of TTDP and at longer incubation
periods (Figure 3.9), it appears that TTDP does not greatly affect enzyme activity. TTDP
in the presence of bound ThDP cannot get into the active site even after 23 h. So, TTDP is
not a transition state analogue as it binds less tightly than ThDP to BFD (KM = 1 pM) Io,
nor is TTDP a competitive inhibitor for BFD. TTDP is aiso not a transition state analog
2 1-23 for other ThDP-dependent enzymes with the exception of E. coli pyruvate
dehydrogenase complex ".
However, the type of inhibition pattern exhibited by TTDP on BFD may be more
complex. An "alternating sites" reaction pathway has been proposed where
decarboxylation at the first active site may be dependent on the presence of substrate at the
other active site ? Also, decarboxylation at one single site can occur but at a much slower
rate. So, the overall observed rate of decarboxylation may be an average of the individual
rates and the "alternating site" rate. This provokes many questions: If only one molecule
of TTDP occupies one active site, cm substrate still bind at this site and allow
decarboxylation to occur at the other active site? Or could TTDP in one active site have no
effect on the other active site? Can two molecules of TTDP displace both ThDP molecules
to prevent decarboxylation? On average, how many molecules of TTDP are bound to the
enzyme? However, no one has yet measured the reaction order of inactivation by substrate
analogs such as Ip-(halomethyl)benzoyl]fomates ' nor by coenzyme anaiogs such as
TTDP, thiamin thiothiazolone diphosphate and thiochrome diphosphate.
If TTDP is not a transition state analog for BFD, then what is the transition state
and can we design one that will bind much more strongly than ThDP? TTDP was
designed for pyruvate dehydrogenase, and does not reflect the methyl group derived fiom
pymvate. This suggests that pyruvate dehydrogenase does not utilize the methyl group to
achieve the transition state. For BFD, whose substrate contains a phenyl group, it may use
the steric group for critical interactions in the transition state. A better transition state
analog for BFD might be 10 which incorporates the phenyl group into the enamine
structure:
Figure 4.1. A potential transition state analog for BFD
Another possibility is that the enamine/carbanion, 9, does not reflect the transition
state of a kinetically significant step. Whether or not the transition state looks more like
MThDP or HBzThDP has to be further clarified. Jordan et al., 1999, synthesized
HBzTHDP and used it as a probe for thc nature of the transition state of a mutant form of
pyruvate decarboxylase 19. This compound forms its conjugate base on the enzyme and
leads to recovery of enzyme activity. An analog of HBzThDP thus should have more
affinity for BFD.
No one has yet synthesized MThDP for BFD but Kluger et al., 198 1 were able to
synthesize 2-(a-1actyl)thiamin and found that the rate of decarboxylation is 1 O' times
srnaller than the dissociation step ". They suggest that the enzyme rnay have to
destabilize 2-(a-1actyl)thiamin diphosphate (LThDP) in order for decarboxylation to occur.
Also, if decarboxylation were the rate-determining step for the overall rnechanism, then
analogs of LThDP would better approxirnate the transition state. With this in mind, methyl
acetyiphosphonate (MAP) was designed to form adducts with ThDP that are analogous to
the labile LThDP. Pyruvate dehydrogenase, pyruvate oxidase and pynivate decarboxylase
al1 responded differently to the pyruvate analog. MAP is a very potent inhibitor of
pyruvate dehydrogefiase, binding to the enzyrne better than pyruvate by a factor of 104.
Together, MAP and pymvate dehydrogenase generate a reactive intermediate analog
However, MAP inhibits pyruvate oxidase only at high concentrations and appears not to
inhibit yeast pyruvate decarboxylase ". The results were rationalized by differences in
stereoelectronic alignment of the phosphonate relative to the carboxylate.
Despite the above results, phosphonate analogs of benzoylformate that could
generate the desired reactive intermediate analog in situ or phosphonate analogs of MThDP
might be potential inhibitors of BR) (Figure 4.2). Benzoylphosphonate,
benzoyhydroxamate, and the a-hydroxybenzylphosphonates (HBP) would bind to the
enzyme but they would not able to undergo decarboxylation by BFD.
benzo ylformate
0- H H O substrate analogs
phenylacetate benzyIphosphonate
< Q R N:"H
\ / analogs that would
H generate transition state analog in situ
benzoylphosphonate benzohydroxamate
(R, .S')-a-HBP (Re,$)-methyl-a-HBP transition state analog!
Figure 4.2. Possible compounds that could Iead to inhibition of BFD.
Testing these compounds with BFD would provide more information of d e
nature of the transition state of highest affinity and where it lies along the catalytic
mechanism. The compound that exhibits the greatest inhibition would best reflect the
types of interactions involved in the transition state at the kinetically significant step.
Schowen (1998) elegantly summarized the fiee energy-reaction profiles for the
nonenzymic and enzymic decarboxylation of pyruvate by ThDP 13. Figure 4.3 shows how
much pyruvate decarboxyIase fiom both yeast and Zymrnonas rnobilis stabilize the reactant
and transition states. Note also that decarboxylation and proton transfer in the nonenzymic
reaction is combined into one step for the enzymic reactions. Transition state stabilization
was used to account for the catalytic power of the two enzymes 30. Similar profiles for
pyruvate oxidase, transketolase, E. coli pyruvate dehydrogenase and acetolactate synthase
would help iden te any differences in relative energy levels of the reactant and transition
states and perhaps this would help explain why TTDP acts as a transition state analog for
E. coli pymvate dehydrogenase but not for the other enzymes. Once we have created
similar fkee energy reaction profiles for the non-enzymic and ef lz~~nic decarboxylation of
benzoylformate by ThDP, we can design a tight binding inhibitor for the most kinetically
important step.
1----1 I l I 1 I 1 1---; ; I 1 1 l
I I ; ; ; : : 1 I * 1 I 1 : I I :
; ; i f ThDP alone 1 L 1 t 1 c I I L ;
l ! ! i
cofactor binding
SCPDC 1
Figure 4.3. Free energy-reaction progress files for the non-enzyrnic decarboxylation of pyruvate by ThDP and the reactions catalyzed by yeast pyruvate decarboxylase
(SCPDC) and Zprnonas pyruvate decarboxyalse (ZMPDC). I 3
Chapter 5: Conclusions and Future Work
Preliminary results suggest that TTDP is an inhibitor but not a transition state
analog as proposed by Gutowski and Lienhard 17. The problems with the coupled assay
have to be addressed before M e r results with TTDP can be interpreted reliably. Also the
folding problem encountered during the developrnent of the assays could be further
investigated as the two ThDP-sites on the enzyme may affect enzyme activity as suggested
by Sergienko, et al., 2000 6. Obtaining the stable apo-enzyme and incubation with TTDP
would provide more details into the nature and mechanism of inhibition. Determinhg
whether MThDP or HBzThDP binds more strongly to BFD would help in the design and
synthesis of a better inhibitor that might be or lead to a potential transition state analog.
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