Use of mass spectrometry in the study of enzymes 02-21-06.pdfUse of mass spectrometry in the study...

1

Stephen Barnes BMG 744 02-21-06

Use of mass spectrometry Use of mass spectrometry in the study of enzymesin the study of enzymes

Stephen Barnes, PhDMCLM 452

[email protected]@gmail.com


Overview of classOverview of class• Modification of the enzyme to regulate its

activity• Examining the chemistry of

enzyme:substrate intermediates– Locating the site of inactivation of suicide

inhibitors• Reaction mechanism

– Measuring all substrates and products– Enzyme kinetics– Stopped flow

• BAT, my kinda ‘zyme

2


A good review of this topic

• Kelleher, NL and Hicks LM. Contemporary mass spectrometry for the direct detection of enzyme intermediates. Current Opinion in Chemical Biology 9: 424-430, 2005.


Enzymes often undergo posttranslational modifications in order to be active under the conditions in a cell

• for example, many enzymes in the signal transduction pathways are activated by phosphorylation on serine, threonine and tyrosine residues

• EGF receptor (tyrosine kinase), TGF beta type I receptor (serine kinase)

• sites of phosphorylation can be determined by mass spectrometry because of the increase in mass of m/z 80 of peptides containing each phosphate group

Mass spectrometry and the study of Mass spectrometry and the study of enzymesenzymes

3


Enzymes and Mass SpecEnzymes and Mass Spec

Enzymes may undergo changes in structure once activated (see above) or during the reaction they catalyze

- this could be probed by H-D exchange experiments

- simulation of phosphorylation may be necessary by mutating serine and threonine groups to aspartate and glutamate, respectively


MS of enzymesMS of enzymes

Enzymes can be inactivated by suicide substrates -these come into the active site and undergo a covalent reaction, thereby blocking the approach of other substrate molecules

- to locate the region of the enzyme to which the suicide substrate is bound, carry out a trypsin digest and look for a peak that has undergone a molecular weight change (consistent with the structure of the suicide inhibitor)

4


CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN 50WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTIN 100NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNAN 150TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLV 200CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAAN

CH3

S OO

NH

CH

C CH2

CH2

Cl

O

Reaction of chymotrypsin with Reaction of chymotrypsin with tosyl tosyl phenylalanyl chloromethylketone phenylalanyl chloromethylketone (TPCK) in His57(TPCK) in His57


Chymotrypsin has a catalytic triad consisting of Asp102, His57 and Ser195

His57 is the site of reaction of chymotrypsin with TPCK to form a stable covalent bond, thereby acting as a suicide inhibitor

5


TrypsinTrypsinCGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN 50WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTIN 100NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNAN 150TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLV 200CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAAN

GluGlu--CCCGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN 50WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTIN 100NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNAN 150TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLV 200CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAAN

ChymotrypsinChymotrypsinCGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN 50 WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTIN 100 NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNAN 150 TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLV 200 CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAAN

Possible proteases for locating TPCKPossible proteases for locating TPCK--peptidepeptide


Advantage of growing recombinant bacteria on 12C/14N labeled substrates - protein is close to monoisotopic mass

Kelleher & Hicks, 2005

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Mass spectrometry and enzymeMass spectrometry and enzyme--catalyzed reactionscatalyzed reactions

In the simplest case, an enzyme (E) reacts with a substrate (S) - an intermediate complex is formed (ES) and it is converted to an enzyme: product complex (E:P) before the product dissociates.

E + S ES EP E + P

First order reaction - some second order reactions behave like a first order reaction when there is an excess of one substrate and the conversion of the other is <10%.



By measuring the molecular weights of the forms of the enzyme:substrate (product) complexes, mass spectrometry can throw enormous light on the mechanism

E

E.S1

E.S2

E.S1.S2 E.P.Q

E.Q

E.P

E

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More typical reactions involve two substrates (S1 and S2) and two products (P1 and P2). The problem in this case is the order of addition

- is it a random mechanism? If so, both E.S1 and E.S2 exist

- is it an ordered mechanism? In this case, S1 has to bind first. So, there will be E.S1 and E.S1.S2, but no E.S2

- is it a Ping-Pong mechanism? In this case, E.S1 E.P1 before S2 binds to form E.P1.S2


[E]

[E.S1][E.S1.S2]

[E.S2]

[E.S1]

[E.S1.S2]

[E]

[E]

[E.S1][E.P1] [E.P1.S2]


Mass spectrometry and substrates Mass spectrometry and substrates and products of enzyme reactionsand products of enzyme reactions

• Most enzyme reactions are studied by measuring the appearance of a product or (more rarely) the disappearance of a substrate

• If the substrate or product has a unique absorbance or fluorescence, the reaction can be followed in real time

• Some substrates have no usable absorbance or fluorescence - these can be measured using a radiolabeled substrate - the product is isolated by a solvent extraction procedure, or by HPLC or TLC. These reactions cannot be observed in real time

• Mass spectrometry has the advantage that it is capable of measuring all substrates and products, as well as the enzyme itself

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Sulfotransferase Sulfotransferase -- a reaction with no a reaction with no absorbance or fluorescence to followabsorbance or fluorescence to follow

N

NN

N

NH2

O

OHO

CH2PO

O

O-

OS

O

O

-O

P O-O

O-

N

NN

N

NH2

O

OHO

CH2PO

O

O-

-O

P O-O

O-


Sulfation Sulfation ofof chitobiosechitobiose

m/z 503

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Set up for the ST assaySet up for the ST assay• NodST purified by Ni-affinity chromatography

– dialyzed against 100 mM Tris-HCl, pH 8.0 - 20 mM β-ME– Diluted into 10 mM NH4Ac buffer, pH 8.0

• Incubate (25 μl) quenched with 100 μl of MeOH containing internal standard

• Diluted incubate (40 μl) introduced into ESI source at 20 μl/min

• MS on a ThermoFinnigan LCQ monitoring m/z 503 and m/z 468 (internal standard)

Pi et al., Biochemistry 41:13283


Kinetics of chitobiose ST by ESI-MSPi et al., Biochemistry 41:13283

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Inhibition of ST by PAP using ESIInhibition of ST by PAP using ESI--MSMSPi et al., Biochemistry 41:13283


NonNon--covalent enzyme:substrate covalent enzyme:substrate complexescomplexes

• Shifting the enzyme from neutral pH conditions to the acidity of the spraying solution may break down the complex

• Spraying at neutral pH will increase the observed m/z values (the protein is less charged with protons)

• The larger m/z ions can be observed with an electrospray-TOF or a Qq TOF

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Schematic diagram of a stopSchematic diagram of a stop--flow system flow system

ESI / Q-Tof-MS

Injection valve(substrate)

Syringe pump Injection valve(Enzyme)

Zero dead volume tee

Syringe pump


Stopped flow set upStopped flow set up

From Kolakowski and Konermann (Anal Biochem 292:107)

Note the additional flow introduced by pump S3

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Effect of the delay between V1 and V2 Effect of the delay between V1 and V2 in a stopped flow experimentin a stopped flow experiment

0.5 msec

35 msec

400 msec

In this reaction, hydrolysis of acetylcholine in an alkaline buffer is monitored by the ion at m/z 146

Δ = x msec

Kolakowski and Konermann (Anal Biochem 292:107)


Following a reaction using substrate and Following a reaction using substrate and products ions in stopped flow ESIproducts ions in stopped flow ESI--MSMS

These data are from the conversion of chlorophyll A to pheophytin A (loss of Mg and gain of two protons).

The upper traces (A) are from the ESI-MS analysis. The lower traces (B) are from absorbance changes.


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Unfolding kinetics ofUnfolding kinetics of myoglobin myoglobin by by stoppedstopped--flow ESIflow ESI--MSMS

The upper trace (A) is the 14th charge state of holo-myoglobin [M+14]14+ (m/z = 1255.9)

The reaction is created by a pH jump from 6.0 to 3.0. The lower trace (B) is the absorbance at 441 nm.

The estimated time constants for the bi-exponential process are 0.29/2.8 sec for A and 0.33/3.1 sec for B



Summary of the use of (real time) Summary of the use of (real time) ESIESI--MS to follow enzyme reactionsMS to follow enzyme reactions• The pros:

– All the substrates and products (as well as the enzyme itself) can be studied simultaneously

– It’s applicable to compounds with no absorbance or fluorescence

• The cons:– The buffer for the reaction has to be chosen very

carefully– Ammonium salts are the best candidates, but they may

have an effect on the reaction rates

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A practical example of use of MS A practical example of use of MS inin enzymology enzymology -- the enzyme BATthe enzyme BAT

• Johnson et al., J Biol Chem, 266: 10227-10233, 1991 (human BAT enzyme - purification)

• Falany et al., J Biol Chem, 269: 19375-19379, 1994 (human cDNA cloning and expression)

• Falany et al., J Lip Res, 38: 86-95, 1997 (mouse - cDNA cloning and expression)

• He et al., J Lip Res, 44: 2242-2249, 2003 (rat - cDNA cloning, expression and localization)

• Sfakianos et al., J Biol Chem, 277: 47270-47275, 2002 (mechanism of human BAT)

• Shonsey et al., Methods in Enzymology, Vol. 400, Chapter 21,pp 360-373, 2005.

• Shonsey et al., Methods in Enzymology, Vol. 400, Chapter 22,pp 374-394, 2005.


First letFirst let’’s remind ourselves s remind ourselves of some basic biochemistryof some basic biochemistry

Acetate (C2) to HMG CoA (C6)

HMG CoA to squalene (C30)

Squalene to cholesterol (C27)

Steroids (C18/C21)Vitamin D (C27)Bile acids (C24/C27)

20+ Nobel prizes

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HO

HO OH

OHH

HO

COOH

OHH

HO

HO

OH

HO

COOH

COOH

OHHHO

Neutral pathway7α-hydroxylase

Acidic pathway27-hydroxylase

C24 BA24-hydroxylase

24-hydroxylase7α-hydroxylase


Evolution of bile acid conjugationOH

OHH

HO

?

CONHCH2CH2SO3-

OHH

HO

Taurine C27 BA

OSO3-

OHH

HO

C27 BAlc sulfate

CONHCH2CH2SO3-

OHH

HO

Taurine C24 BA

CONHCH2CO2-

OHH

HO

Glycine C24 BA

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Bile acid Bile acid NN--acylamidate acylamidate formation formation (in hepatocytes)(in hepatocytes)

Bile acid + CoASH

Bile acid-SCoA + amino acidsBAT

Bile acid amidate

CoASH

BALBile acid-SCoA

ATP, Mg2+


Characterization of BATCharacterization of BAT

• Purified from human liver cytosol 465-fold to a single protein band - retained the same ratio of glycine:taurine activity during purification

• Partial amino acid sequence and specific polyclonal antibody led to isolation of λgt11 clone from human liver cDNA library

• hBAT is a 418-aa protein; when expressed using a pKK233-2 vector in bacteria, it makes both glycine and taurine conjugates (and FBAL)

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+ HS.CH2.hBAT

CoASH

C

OHHO

OH

SCoA

O

NH2CH2CH2SO3-

C

OHHO

OH

S.CH2.hBAT

O

+ HS.CH2.hBAT

C

OHHO

OH

NHCH2CH2SO3-

O


Sequence comparisons of mouse, rat and human BATsSequence comparisons of mouse, rat and human BATs*235

r 65% SLLASHGFATLALAYWGYDDLPSRLEKVDLEYFEEGVEFLLRHPKVLGPGVGILSVCIGA 238

m 63% SLLASRGFATLALAYWNYDDLPSRLEKVDLEYFEEGVEFLLRHPKVLGPGVGILSVCIGA 237

h 100% SLLASRGFASLALAYHNYEDLPRKPEVTDLEYFEEAANFLLRHPKVFGSGVGVVSVCQGV 238

r EIGLSMAINLKQITATVLINGPNFVSSNPHVYRGKVFQPTPCSEEFVTTNALGLVEFYRT 298

m EIGLSMAINLKQIRATVLINGPNFVSQSPHVYHGQVYPPVPSNEEFVVTNALGLVEFYRT 297

h QIGLSMAIYLKQVTATVLINGTNFPFGIPQVYHGQIHQPLPHSAQLISTNALGLLELYRT 288

*328

r FEETADKDSKYCFPIEKAHGHFLFVVGEDDKNLNSKVHAKQAIAQLMKSGKKNWTLLSYP 358

m FQETADKDSKYCFPIEKAHGHFLFVVGEDDKNLNSKVHANQAIAQLMKNGKKNWTLLSYP 357

h FETTQVGASQYLFPIEEAQGQFLFIVGEGDKTINSKAHAEQAIGQLKRHGKNNWTLLSYP 358

*362 *372

r GAGHLIEPPYSPLCSASRMPFVIPSINWGGEVIPH-AA 395

m GAGHLIEPPYTPLCQASRMPILIPSLSWGGEVIPHSQA 395

h GAGHLIEPPYSPLCCASTTHDLR--LHWGGEVIPH-AA 393

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SiteSite--specific Cys mutationsspecific Cys mutations

• Mutations were prepared for the two conserved Cys residues (C235 and C372) in BATs

• C235Y hBAT had no enzyme activity

• C372A hBAT had low activity

Sfakianos et al.

0

1

2

3

0 100 200 300 400 500 600Cholyl CoA concentration

(μM)Sp

ecifi

c ac

tivity

(fm

ol

TC/m

in/μ

g)

0

200

400

600

800

1000

1200

A

B


ESI-mass spectrum of hBAT products

500 600 700

m/z

100

75

50

25

0

m/z 512

m/z 514

wt-hBATm/z 514

m/z 512

100

75

50

25

0Rel

ativ

e In

tens

ity (%

)R

elat

ive

Inte

nsity

(%)

C372A hBAT

Sfakianos et al.

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LC-MS of C372A hBAT product

4.00 6.00 8.00 10.00 12.00

Time (min)

514/ 124R

elat

ive

Inte

nsity

(%)

512/ 124100

75

50

25

0

100

75

50

25

0

taurocholate

[TC-2H]-

Rel

ativ

e In

tens

ity (%

)

Sfakianos et al.


Metabolism in E. coli expression system

COOH

HO OH

OH

CONHCH2CH2SO3-

HO OH

OH

m/z 514

wt-hBAT

C372A hBAT

CONHCH2CH2SO3-

HO

OH

Om/z 512

7α-hydroxysteroiddehydrogenase

Sfakianos et al.

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hBAT Related Proteins from BLAST Search (Courtesy of Alexey Murzin, MRC lab)235

kan-1 SLLASHGFATLALAYWGYDDLPSRLEKVDLEYFEEGVEFLLRHPKVLGPGVGILSVCIGA 238mBAT SLLASRGFATLALAYWNYDDLPSRLEKVDLEYFEEGVEFLLRHPKVLGPGVGILSVCIGA 237 hBAT SLLASRGFASLALAYHNYEDLPRKPEVTDLEYFEEAANFLLRHPKVFGSGVGVVSVCQGV 238 MTE-I SLLAGKGFAVMALAYYNYDDLPKTMETMRIEYFEEAVNYLRGHPEVKGPGIGLLGISKGG 276 CTE-I SLLAGKGFAVMALAYYNYDDLPKTMETMRIEYFEEAVNYLRGHPEVKGPGIGLLGISKGG 235 CLCTE SLLAGKGFAVMALAYYNYDDLPKTMETMRIEYFEEAVNYLRGHPEVKGPGIGLLGISKGG 235 PLCTE SLLAGKGFAVMALAYYNYEDLPKTMETLHLEYFEEAMNYLLSHPEVKGPGVGLLGISKGG 235PTE-Ia SLLAGKGFAVMALAYNNYEDLPKDMDIIHLEYFEEAVTYLLSHPQVTGSGVGVLGISKGG 246DLHp KPFAEQGYAVLALSYFAAPGLPATAEELPLEYFDRAVAWLAAQPSVDPKAIGVYGVSKGA 138

328kan-1 ALGLVEFYR--TFEETAD-KDSKYCFPIEKAHGHFLFVVGEDDKNLNSKVHAKQAIAQLM 345mBAT ALGLVEFYR--TFQETAD-KDSKYCFPIEKAHGHFLFVVGEDDKNLNSKVHANQAIAQLM 344hBAT ALGLLELYR--TFETTQV-GASQYLFPIEEAQGQFLFIVGEGDKTINSKAHAEQAIGQLK 345MTE-I KDGLLDVVE--ALQSPL--VDKKSFIPVERSDTTFLFLVGQDDHNWKSEFYAREASKRLQ 382CTE-I KDGLKDVVD--ALQSPL--VEQKSFIPVERSDTTFLFLVGQDDHNWKSEFYANEISKRLQ 341CLCTE KDGLKDVVD--ALQSPL--VEQKSFIPVERSDTTFLFLVGQDDHNWKSEFYANEISKRLQ 341PLCTE KDGYADIVD--VLNSPLEGPDQKSFIPVERAESTFLFLVGQDDHNWKSEFYANEACKRLQ 343PTE-Ia KDGLKDIVD--LLNNPLEGPDQKSLIPVERSDTAFLFLVGQDDHNWKSEFYAREASKRLQ 354DLHp SNYMAFIYGLYDTGLKAADAHPQAAIPVEKIHGPVMLISGRADAMWSSSAMSDAVVARLK 258

362kan-1 KSGKK-NWTLLSYPGAGHLIEPPYSPLCSASRMPFVIPSINWGGEVIPH-AA 395mBAT KNGKK-NWTLLSYPGAGHLIEPPYTPLCQASRMPILIPSLSWGGEVIPHSQA 395hBAT RHGKN-NWTLLSYPGAGHLIEPPYSPLCCASTTHDLR--LHWGGEVIPH-AA 393MTE-I AHGKE-KPQIICYPEAGHYIEPPYFPLCSAGMHLLVGANITFGGEPKPH-SV 432CTE-I AHGKE-KPQIICYPEAGHYIEPPYFPLCSAGMHLLVGANITFGGEPKPH-SV 391CLCTE AHGKE-KPQIICYPEAGHYIEPPYFPLCSAGMHLLVGANITFGGEPKPH-SV 391PLCTE AHGRR-KPQIICYPETGHYIEPPYFPLCRASLHALVGSPIIWGGEPRAH-AM 393PTE-Ia AHGKE-KPQIICYPETGHHIEPPYFPLCKASLNSLVGGPVIWGGEPRAH-AM 404DLHp AKGFAHKVSHLAYPDAGHTAGMPALMGGSDK-----GADEAVGGTVEGN-RF 304


The Protein Structure Modeling of hBAT

Courtesy of Alexey Murzin, Center for Protein Engineering, MRC Cambridge, UK

Ollis D.L., Protein Engineering 5(3): pp.197-221.

His362Cys235

Asp328

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Cys235→Ser, what will C235S-BAT be, transferase or thioesterase?

235kan-1 SLLASHGFATLALAYWGYDDLPSRLEKVDLEYFEEGVEFLLRHPKVLGPGVGILSVCIGA 238mBAT SLLASRGFATLALAYWNYDDLPSRLEKVDLEYFEEGVEFLLRHPKVLGPGVGILSVCIGA 237 hBAT SLLASRGFASLALAYHNYEDLPRKPEVTDLEYFEEAANFLLRHPKVFGSGVGVVSVCQGV 238 MTE-I SLLAGKGFAVMALAYYNYDDLPKTMETMRIEYFEEAVNYLRGHPEVKGPGIGLLGISKGG 276 CTE-I SLLAGKGFAVMALAYYNYDDLPKTMETMRIEYFEEAVNYLRGHPEVKGPGIGLLGISKGG 235 CLCTE SLLAGKGFAVMALAYYNYDDLPKTMETMRIEYFEEAVNYLRGHPEVKGPGIGLLGISKGG 235 PLCTE SLLAGKGFAVMALAYYNYEDLPKTMETLHLEYFEEAMNYLLSHPEVKGPGVGLLGISKGG 235PTE-Ia SLLAGKGFAVMALAYNNYEDLPKDMDIIHLEYFEEAVTYLLSHPQVTGSGVGVLGISKGG 246DLHp KPFAEQGYAVLALSYFAAPGLPATAEELPLEYFDRAVAWLAAQPSVDPKAIGVYGVSKGA 138

? Nucleophile Cys235→Ser


H

Cys235

S----

NH

His362

N

---Asp328C

O

O

-……

..

Bile acid

C

O

CoAS

Nucleophilic attack

Michaelis complex

1NH

His362

NH

Cys235

S

Bile acid

C

CoAS

O-+

-----

-

---Asp328C

O

O

-……

Tetrahedral intermediate

2

Cys235

CH2

S

C

O

R

Acyl-enzyme intermediate

---NH

His362

N

CoAS

H ----

Asp328C

O

O

-……

-N

Gly or Tau

Cys235

CH2

S

C

O

R

4

Bile acid

C

O

NGly or Tau

5

Bile acid conjugate

H

Cys235

S

NH

His362

N

---

----+

Asp328C

O

O

-……

Active enzyme

Charge Relay Mechanism shared by hBAT, thioesterases, and a large group of hydrolases

Cys235

CH2

S

C

O

R H

NGly or Tau

CoA

Gly or Tau3

(Ser235)

(H2O)

Sfakianos, JBC 277:47270

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Purification of hBAT with 6x HisPurification of hBAT with 6x His--tagtagpQE 31

hBATContaminant (GroEL)

Contaminant (a histidine-rich protein)

• Impure hBAT

• Inactivated by imidazole in elution buffer

M C E

111 kD-73 kD-

47.5 kD-

33.9 kD-

28.8 kD-

20.5 kD-

Sfakianos et al.

15000 20000 25000 30000 35000 40000 45000 50000 55000 60000 65000mass0

100

%


Purification of hBAT with Purification of hBAT with AviAvi--tag tag pET21a(+)

M FT E

hBAT

110 kD-76 kD-

49 kD-

35.6 kD-

29 kD-

21.1 kD-

Contaminant (GroEL)

• Impure hBAT

ATP regenerating system

Sfakianos et al.Shonsey et al.

kDa

20011596

51

37

29

20

Cyt 6HisFT

AviFT

Aviwash

hBAT-avi

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(Thioesterase)

hBAT

OH

CH3

OH

CH3

CH3

COO-

HO

cholic acid m/z 407

OH

CH3

OH

CH3

CH3HO

C

O

NHCH2COO-

glycocholate m/z 464

glycine glycine (m/z 74)

OH

CH3

OH

CH3

CH3HO

C

O

NHCH2CH2SO3-

taurocholate m/z 514

taurine

taurine (m/z 124)

OH

CH3

OH

CH3

CH3HO

C

O

S CoA

(Transferase)hBAT

cholyl CoA

Sfakianos et al.


Kinetics of transferase and thioesterase activities of wild-type hBAT with glycine

100 200 300 400 500 600cholyl CoA (μM)

0

20

40

60

80

100

120

0

Spec

ific

activ

ity (μ

mol

/min

/g)

transferase activity

thioesterase

Sfakianos et al.

24


0 100 200 300 400 500 600

cholyl CoA (μM)

0

50

100

150

200

250

300

350

400Sp

ecifi

c ac

tivity

(μm

ol/m

in/g

)

Kinetics of transferase and thioesterase activities of C235S hBAT variant with glycine

transferase activity

thioesterase

Sfakianos et al.


Spec

ific

Act

ivity

(nm

ol/m

in/u

g)

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

LC-ESI-MS-MRM Analysis of Reaction Products

B. C235SA. wild type

glycine taurine glycine taurine

Transferase (glycine)

Transferase (taurine)

Thioesterase

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Bile acidBile acid CoACoA:amino acid N:amino acid N--acyltransferaseacyltransferase

• Has a ping-pong reaction mechanism

• Bile acid CoA undergoes a thioester interchange with Cys235-BAT

• Ser can replace Cys, but the complex is less stable– This can either lead to lowered activity, or increased

turnover

Use of mass spectrometry in the study of enzymes 02-21-06.pdfUse of mass spectrometry in the study...

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