Post-translational Modifications to Human Bile Acid CoA:Amino Acid N- acyltransferase

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1 Post-translational Modifications to uman Bile Acid oa:amino Acid - acyltransferase Erin Shonsey UAB Graduate Student September 12, 2006

2 onjugation of Bile Acids SoA S 3 - hbat oa-s 2 2 S - 3 (Amidate, -holyltaurine) (A)

3 atalytic Triad of BAT Various mutation and other studies performed previously led to the hypothesis that BAT works through a catalytic triad The members of this catalytic triad are believed to be ys235, is362, and Asp328

4 harge Relay Mechanism shared by hbat, thioesterases, and a large group of hydrolases ys 235 is 362 ys 235 is 362 Bile acid S S ---- oa. ucleophilic attack Michaelis complex Asp S Bile acid S oa - Tetrahedral intermediate Asp 328 ys 235 (Ser235) 2 is 362 R 2 S 4 Gly or Tau ( 2 ) Gly or Tau 3 oa ys S R S Acyl-enzyme intermediate oa Asp 328 ys 235 ys 235 is 362 R - 2 S 5 Gly or Tau Gly or Tau Bile acid Bile acid conjugate + S Active enzyme - Asp 328

5 MALDI-TF MS Analysis of hbat-avi/cholic acid intermediate Mass (m/z)

6 Deconvoluted Q-TF MS Analysis of hbat- Avi/cholic acid intermediate Da mass

7 Amino Acids Modified by 4E 4E-Modified Lysine Michael Addition 4E-Modified ysteine S Michael Addition Adduct Michael Schiff base Pentylpyrrole 4E-Modified Lysine Schiff Base Adduct Mol wt J Am Soc Mass Spectrom Aug;15(8): E-Modified Lysine 2-Pentylpyrrole Adduct 4E-Modified istidine Michael Addition

8 Potential 4E targets in hbat MIQLTATPVSALVDEPVIRATGLIPFQMVSFQASLEDEGDMF YSQAYRAEFGEVDLASSLGGDYMGVPMGLFWSLKPEKLL TRLLKRDVMRPFQVQVKLYDLELIVKVASAPKASLTLERWY VAPGVTRIKVREGRLRGALFLPPGEGLFPGVIDLFGGLGGLLEF RASLLASRGFASLALAYYEDLPRKPEVTDLEYFEEAAFLLR PKVFGSGVGVVSVQGVQIGLSMAIYLKQVTATVLIGTFPF GIPQVYGQIQPLPSAQLISTALGLLELYRTFETTQVGASQ YLFPIEEAQGQFLFIVGEGDKTISKAAEQAIGQLKRGKW TLLSYPGAGLIEPPYSPLASTTDLRLWGGEVIPAAAQE AWKEIQRFLRKLIPDVTSQL 17 is 16 Lys 19 Arg 3 ys

9 In vitro Modification of hbat with E hbat + E Incubate at 4 for 1 hour E treated hbat + E treated hbat E modified hbat Incubate at 37 overnight Mix with matrix and spot on MALDI plate 100 % Intensity Mass (m/z)

10 Inactivation of hbat by E 120 % Residual Activity control E oncentration (µm) 1.6µM hbat

11 MALDI-TF MS of hymotrypsin Digest of E modified hbat ative hbat Modified hbat Mass (m/z)

12 istidine Residues Modified by 1 mm E Found by QTF Residue umbers Sequence Unmodified Mass Modified Mass Mass Shift GFASLALAYYEDLPR GQIQPLPSAQ GQIQPLPSAQ AAEQAIGQLK LWGGEVIPAAAQEAWK

13 MS/MS Spectra Using ID of [M] M I Q L T A T P V S A L V D E P V I R [M+2] 2+ - E Michael adduct y 8 y 10 b 13 b 9 b y7 5 b 3 b y 5 7 y 6 b 6 y 9 b 12 b 14 y 11 b 15 y 13 y 12 y 14 y 15 b 17 y m/z

14 Electron apture Dissociation urr pin Biotechnol Feb;15(1):12-6.

15 ED Fragmentation y 3 z 3 z 2 z 1 R1 R2 R3 R4 + b 1 c 1 c 2 c 3

16 MS/MS Spectra Using ED of [M] A A E Q A I G Q L K R [M+3] 3+ [M+2] 2+ ~ x 5 c 5 z 8 c 3 c 4 c z 7 c z 2 8 c 11 1 z 3 z 4 c 6 z c c 7 9 c m/z

17 Sequence overage Using FT-IR/MS MIQLTATPVSALVDEPVIRATGLIPFQMVSFQASL EDEGDMFYSQAYRAEFGEVDLASSLGGDYMG VPMGLFWSLKPEKLLTRLLKRDVMRPFQVQVKLY DLELIVKVASAPKASLTLERWYVAPGVTRIKVRE GRLRGALFLPPGEGLFPGVIDLFGGLGGLLEFRASL LASRGFASLALAYYEDLPRKPEVTDLEYFEEAA FLLRPKVFGSGVGVVSVQGVQIGLSMAIYLKQVT ATVLIGTFPFGIPQVYGQIQPLPSAQLIST ALGLLELYRTFETTQVGASQYLFPIEEAQGQFLFIV GEGDKTISKAAEQAIGQLKRGKWTLLSYPGA GLIEPPYSPLASTTDLRLWGGEVIPAAAQE AWKEIQRFLRKLIPDVTSQL 70.1% Sequence overage

18 Amino Acids Modified by E Using FT-IR/MS Peptide 128 µm E Modified Amino Acid 64 µm E 32 µm E 16 µm E 8 µm E AAEQAIGQLKR RLWGGEVIPAAAQEAWK AQGQFLFIVGEGDKTISK 397 K329, K K329, K K329, K K329, K334 K329, K334 MIQLTATPVSALVDEPVIR RAEFGEVDLASSLGGDYMGVPMGLFWSLKPEK 1*156 WTLLSYPGAGLIEPPYSPLASTTDLR 362, 372, 373, 378

19 Summary A covalent enzyme intermediate was identified between hbat and cholic acid L-ESI-MS/MS on the Q-tof identified 6 modifications at 1 mm E L-ESI-FT-IR/MS identified up to 10 modifications at 128 µm, down to 3 modifications at 8 µm 4E may first alter sites in the region of the catalytic triad

20 Acknowledgements Barnes Lab: Stephen Barnes Tracy D Alessandro Kim Lab: elen Kim Shannon Eliuk FT-IR/MS Lab for Biomedical Research: Matthew Renfrow Monica Stinnett Mass Spec Shared Facility: Marion Kirk Landon Wilson