SLIDE 1
Conjugation of Bile Acids Conjugation of Bile Acids
+ hBAT
+NH3CH2CH2SO3
- O
H
(Amidate, N-Cholyltaurine)
OH HO C O
NHCH2CH2SO3
- O
H OH OH C O
SCoA
O H (CA) O H OH C O OH
CoA-SH
Post-translational Post-translational Modifications to Human Bile - - PowerPoint PPT Presentation
Post-translational Post-translational Modifications to Human Bile - - PowerPoint PPT Presentation
Post-translational Post-translational Modifications to Human Bile Modifications to Human Bile Acid CoA:Amino Acid N- Acid CoA:Amino Acid N- acyltransferase acyltransferase Erin Shonsey UAB Graduate Student September 12, 2006 Conjugation
SLIDE 2
SLIDE 3
Catalytic Triad of BAT Catalytic Triad of BAT
- Various mutation and
- ther studies
performed previously led to the hypothesis that BAT works through a catalytic triad
- The members of this
catalytic triad are believed to be Cys235, His362, and Asp328
SLIDE 4
H Cys235 S
- NH
His362 N
- Asp328
C O O
- …
… . .
Bile acid C O CoA S
Nucleophilic attack Michaelis complex 1
NH His362 N H Cys235 S Bile acid C CoA S O
- +
- Asp328
C O O
- …
… Tetrahedral intermediate 2
Cys235 CH2 S C O R
Acyl-enzyme intermediate
- NH
His362 N CoA S H
- Asp328
C O O
- …
…
- N
Gly or Tau Cys235 CH2 S C O R
4
Bile acid C O N Gly or Tau
5 Bile acid conjugate
H Cys235 S NH His362 N
- +
Asp328 C O O
- …
… Active enzyme
Charge Relay Mechanism shared by hBAT, Charge Relay Mechanism shared by hBAT, thioesterases, and a large group of hydrolases thioesterases, and a large group of hydrolases
Cys235 CH2 S C O R H N Gly or Tau
CoA Gly or Tau 3
(Ser235) (H2O)
SLIDE 5
45000 48000 51000 54000 57000 60000 49516.04 50549.36 57314.61 45000 48000 51000 54000 57000 60000 Mass (m/z) 50430.89 49729.45 51477.16
MALDI-TOF MS Analysis of hBAT-Avi/cholic acid intermediate MALDI-TOF MS Analysis of hBAT-Avi/cholic acid intermediate
SLIDE 6
49000 49100 49200 49300 49400 49500 49600 49700 49800 49900 50000 50100 50200 mass
49504 49894 49504
Deconvoluted Q-TOF MS Analysis of hBAT- Deconvoluted Q-TOF MS Analysis of hBAT- Avi/cholic acid intermediate Avi/cholic acid intermediate
+ 390 Da
SLIDE 7
4HNE-Modified Cysteine Michael Addition 4HNE-Modified Histidine Michael Addition 4HNE-Modified Lysine Michael Addition 4HNE-Modified Lysine Schiff Base Adduct
S N H O CHO OH N N CHO N H O OH N H N H OH CHO O N H O N OH
4HNE-Modified Lysine 2-Pentylpyrrole Adduct
N N H O 120 Pentylpyrrole 138 Schiff base 156
Michael
Mol wt Adduct J Am Soc Mass Spectrom. 2004 Aug;15(8):1136-47.
Amino Acids Modified by 4HNE Amino Acids Modified by 4HNE
SLIDE 8
MIQLTATPVSALVDEPVHIRATGLIPFQMVSFQASLEDENGDMF YSQAHYRANEFGEVDLNHASSLGGDYMGVHPMGLFWSLKPEKLL TRLLKRDVMNRPFQVQVKLYDLELIVNNKVASAPKASLTLERWY VAPGVTRIKVREGRLRGALFLPPGEGLFPGVIDLFGGLGGLLEF RASLLASRGFASLALAYHNYEDLPRKPEVTDLEYFEEAANFLLR HPKVFGSGVGVVSVCQGVQIGLSMAIYLKQVTATVLINGTNFPF GIPQVYHGQIHQPLPHSAQLISTNALGLLELYRTFETTQVGASQ YLFPIEEAQGQFLFIVGEGDKTINSKAHAEQAIGQLKRHGKNNW TLLSYPGAGHLIEPPYSPLCCASTTHDLRLHWGGEVIPHAAAQE HAWKEIQRFLRKHLIPDVTSQL
Potential 4HNE targets in hBAT Potential 4HNE targets in hBAT
17 His 19 Arg 16 Lys 3 Cys
SLIDE 9
Mix with matrix and spot
- n MALDI plate
HNE treated hBAT
Incubate at 4°C for 1 hour
900 1520 2140 2760 3380 4000 Mass (m/z) 100 % Intensity
+ +
hBAT HNE HNE treated hBAT
Incubate at 37°C overnight
HNE modified hBAT
In vitro In vitro Modification of hBAT with Modification of hBAT with HNE HNE
SLIDE 10
20 40 60 80 100 120 control 8 16 32 64 128 HNE Concentration (µM) % Residual Activity 1.6µM hBAT
Inactivation of hBAT by HNE Inactivation of hBAT by HNE
SLIDE 11
1524.83 1009.62 1163.60 1599.91 2405.38 1664.94 1029.58 1487.73 1758.86 1178.61 1395.66 970.59 1739.92 1936.13 1883.10 1563.78 1039.64 2151.00 900 1220 1540 1860 2180 2500 Mass (m/z) 1009.62 1523.82 1039.55 1163.60 1487.71 1758.86 1320.71 1563.79 1914.95 2152.00 970.62 1890.00 1719.95 1395.87 1077.57 1195.67
Native hBAT Modified hBAT
MALDI-TOF MS of Chymotrypsin Digest MALDI-TOF MS of Chymotrypsin Digest
- f HNE modified hBAT
- f HNE modified hBAT
SLIDE 12
Histidine Residues Modified by 1 mM Histidine Residues Modified by 1 mM HNE Found by QTOF HNE Found by QTOF
156 156 312 156 156 Mass Shift Modified Mass Unmodified Mass Sequence Residue Numbers 2093 1937 GFASLALAYHNYEDLPR 185-201 1719 1563 HGQIHQPLPHSAQ 271-283 2292 2136 LHWGGEVIPHAAAQEHAWK 382-400 1320 1164 AHAEQAIGQLK 336-345 1875 1563 HGQIHQPLPHSAQ 271-283
SLIDE 13
400 600 800 1000 1200 1400 1600 1800 m/z
b3 b7y5 y6 [M+2H]2+ - HNE Michael adduct b5 b6 b9 y7 y8 y9 b12 y10 b13 y11 b14 y12 b15 y13 y14 y15 b17 y16
M I Q L T A T P V S A L V D E P V H I R
MS/MS Spectra Using CID of [MH] MS/MS Spectra Using CID of [MH]+
+ 2362.3051
2362.3051
SLIDE 14
Electron Capture Dissociation Electron Capture Dissociation
Curr Opin Biotechnol. 2004 Feb;15(1):12-6.
SLIDE 15
H N H C R1 H C O N H C R2 H C O N H C R3 H C O N H C R4 H C O OH H
+
y3 b1 z3 c1 z2 c2 z1 c3
ECD Fragmentation ECD Fragmentation
SLIDE 16
200 400 600 800 1000 1200 1400 m/z
A H A E Q A I G Q L K R
[M+3H]3+ [M+2H]2+ ~ x 5
z1 z2 z3 z4 z7 z8 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11
MS/MS Spectra Using ECD of [MH]+ 1460.8408 MS/MS Spectra Using ECD of [MH]+ 1460.8408
SLIDE 17
Sequence Coverage Using FT-ICR/MS Sequence Coverage Using FT-ICR/MS
MIQLTATPVSALVDEPVHIRATGLIPFQMVSFQASL EDENGDMFYSQAHYRANEFGEVDLNHASSLGGDYMG VHPMGLFWSLKPEKLLTRLLKRDVMNRPFQVQVKLY DLELIVNNKVASAPKASLTLERWYVAPGVTRIKVRE GRLRGALFLPPGEGLFPGVIDLFGGLGGLLEFRASL LASRGFASLALAYHNYEDLPRKPEVTDLEYFEEAAN FLLRHPKVFGSGVGVVSVCQGVQIGLSMAIYLKQVT ATVLINGTNFPFGIPQVYHGQIHQPLPHSAQLISTN ALGLLELYRTFETTQVGASQYLFPIEEAQGQFLFIV GEGDKTINSKAHAEQAIGQLKRHGKNNWTLLSYPGA GHLIEPPYSPLCCASTTHDLRLHWGGEVIPHAAAQE HAWKEIQRFLRKHLIPDVTSQL
70.1% Sequence Coverage
SLIDE 18
H362, C372, C373, H378 NNWTLLSYPGAGHLIEPPYSPLCCASTTHDLR 1*156 RANEFGEVDLNHASSLGGDYMGVHPMGLFWSLKPEK H18 H18 MIQLTATPVSALVDEPVHIR K329, K334 K329, K334 K329, K334 K329, K334 K329, K334 AQGQFLFIVGEGDKTINSK H397 H397 H397 H397 RLHWGGEVIPHAAAQEHAWK H336 H336 H336 H336 AHAEQAIGQLKR 8 µM HNE 16 µM HNE 32 µM HNE 64 µM HNE 128 µM HNE Peptide Modified Amino Acid
Amino Acids Modified by HNE Using Amino Acids Modified by HNE Using FT-ICR/MS FT-ICR/MS
SLIDE 19
Summary Summary
- A covalent enzyme intermediate was
identified between hBAT and cholic acid
- LC-ESI-MS/MS on the Q-tof identified 6
modifications at 1 mM HNE
- LC-ESI-FT-ICR/MS identified up to 10
modifications at 128 µM, down to 3 modifications at 8 µM
- 4HNE may first alter sites in the region of the
catalytic triad
SLIDE 20