Supporting Information

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1 Supporting Information Mechanism of inactivation of -aminobutyric acid aminotransferase by (1S,3S)-3-amino-4-difluoromethylenyl-1- cyclopentanoic acid (CPP-115) Hyunbeom Lee, 1, Emma H. Doud, 1,2 Rui Wu, 3 Ruslan Sanishvili, 4 Jose I. Juncosa, 1, Dali Liu, 3 Neil L. Kelleher, 1,2 and Richard B. Silverman *,1,2 1 Department of Chemistry, Chemistry of Life Processes Institute, and Center for Molecular Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, IL , United States 2 Department of Molecular Biosciences, Northwestern University, Evanston, IL 628, United States 3 Department of Chemistry and Biochemistry, Loyola University Chicago, 168 W Sheridan Road, Chicago, IL X-ray Science Division, Argonne National Laboratory, Argonne, IL 6439 S1

2 Table of Contents Figure S1-S2: Reactivation of GABA-AT after inactivation Figure S3-S4: Fluoride ion release during GABA-AT inactivation Figure S5-S7: Cofactor release using [ 3 H]PLP-reconstituted GABA-AT Figure S8: High resolution mass spectrum of metabolite 2, 21, and 22 Figure S9: Mass spectral fragmentation data for 22 Figure S1: Mass spectral fragmentation data for 21 Figure S11: Mass spectral fragmentation data for 2 Figure S12-S14: Middle down peptide proteomics Figure S15-S16: Intact mass spectrometry of GABA-AT Figure S17: Omit map for GABA-AT inactivated by CPP-115 Table S1: Crystallography data processing statistics Page S3 S4 S4-S5 S6 S7 S7 S8 S8-S1 S1-S11 S12 S13 S2

3 Enzyme Activity (%) Enzyme Activity (%) eq 1eq 1.8eq 4eq 4eq 8eq Dialyzed Time (h) Figure S1. Reactivation of GABA-AT after inactivation by - 8 equiv of CPP-115 after incubation of 3 h eq 1eq 1eq 1eq 2eq Dialyzed Time (h) Figure S2. Reactivation of the inactivated GABA-AT by - 2 equiv of CPP-115 after incubation of 24 h S3

4 Abs, mau (254 nm) ( ----) Radioactivity, dpm (----) Figure S3. Fluoride ion release and simultaneous measurement of GABA-AT activity with 2 mm CPP-115. Conversion factor of the concentration of GABA-AT to equiv of fluoride ions is Figure S4. Fluoride ion release from CPP-115 and GABA-AT activity as a function of time in the absence of -KG. Conversion factor of the concentration of GABA-AT to equiv of fluoride ions is PMP PLP Time (min) Figure S5. HPLC trace of the inactivation of [ 3 H]PLP-reconstituted GABA-AT by CPP-115 (2.5 mm) S4

5 Abs, mau (254 nm) ( ----) Abs, mau (254 nm) ( ----) Abs, mau (254 nm) ( ----) Radioactivity, dpm (----) Radioactivity, dpm (----) Radioactivity, dpm (----) 2 PMP PLP Time (min) 2 PMP PLP Time (min) Figure S6. Controls for the HPLC trace in Figure S5. The negative control (top) was run with the same concentrations of each reagent, excluding CPP-115. The positive control (bottom) was run with 4 mm GABA and no inactivator or -ketoglutarate PMP PLP Time (min) Figure S7. HPLC trace of the inactivation of [ 3 H]PLP-reconstituted GABA aminotransferase by CPP-115 after subtracting tritium generated from inactive enzyme. S5

6 Figure S8. High resolution mass spectrum of metabolite 22(A), 21(B), and 2(C) (Scheme 5) released from a reaction incubation of CPP-115 and GABA-AT. Fragmentation peaks are available in the Supporting Information (Figures S11, S12, and S13, respectively). S6

7 Figure S9. Fragmentation and assigned structures of peak m/z of 127 (22) Figure S1. Fragmentation and assigned structures of peak m/z of 171 (21) S7

8 Figure S11. Fragmentation and assigned structures of peak m/z of 41 (2) Middle Down GABA-AT Peptide Proteomics To determine what X is from Scheme 5, further studies were performed to identify the covalent adduct on the enzyme using middle-down proteomics. Digestion of the inactivated GABA-AT with endopeptidase Glu-C (protease V8) 1 was attempted to identify the added mass onto the peptide by a covalent modification of a residue, presumably Lys329 (Figure S12). Protease V8 cleaves at glutamate residues to obtain smaller peptides. In each of Figures S13 and S14 there are two peptide peaks without PLP (blue and red), and two peaks with PLP (yellow and green). This was the result of a missed cleavage of the glutamate residue (cleaved at Glu341 instead of Glu34). Unfortunately, we did not observe any added mass on the desired peptide of the inactivated GABA-AT. Very interestingly, however, we observed that the cofactor PLP is mostly gone from the inactivated peptide compared to that of the control. We can assume that the acidic conditions during digestion with protease V8 were too strong for the adduct to remain intact. S8

9 Figure S12. Sequence of pig brain GABA-AT (red box indicates the peptide of interest) Figure S13. Peptide of the inactivated GABA-AT with CPP-115. The inactivated sample was reduced with NaBH 4 before treating with protease. Blue indicates the peptide, red is a peptide with an extra glutamate residue (because of the missed cleavage), yellow is the peptide plus PLP, and green is the peptide with an extra glutamate residue plus PLP. S9

10 Figure S14. Peptide of the native GABA-AT (control). The enzyme sample was reduced with NaBH 4 before treating with protease. Blue indicates the peptide, red is a peptide with an extra glutamate residue (because of the missed cleavage), yellow is the peptide plus PLP, and green is the peptide with an extra glutamate residue plus PLP. Figure S15. Mass spectrometry of intact GABA-AT. Native GABA-AT was run as a control (black) and was followed by CPP-115-inactivated GABA-AT (red) and vigabatrininactivated GABA-AT (green). Results were deconvoluted using ProMass deconvolution software. M refers to the mass of PLP-free GABA-AT. S1

11 Figure S16. Mass spectrometry of sodium borohydride reduced GABA-AT reactions. Free GABA-AT was run as a control (green) and was followed by CPP-115-inactivated GABA- AT (pink) and vigabatrin-inactivated GABA-AT (cyan). Results were deconvoluted using ProMass deconvolution software. M refers to the mass of PLP-free GABA-AT. S11

Figure S17. Simulated Annealing Omit Map around Interpreted Ligand. A simulated annealing omit difference map (fo-fc) is shown around the interpreted ligand at the 2.

12 Figure S17. Simulated Annealing Omit Map around Interpreted Ligand. A simulated annealing omit difference map (fo-fc) is shown around the interpreted ligand at the 2.1 level as a grey mesh. Carbon atoms are in beige. The interpreted ligand is shown in stick form. Oxygen atoms are in red. Nitrogen atoms are in blue. The phosphorus atom is in orange. S12

13 Table S1. Data collection and refinement statistics Data set Native inactivated PDB codes 4YH 4YD Space Group P21 P21 Cell Dimensions a (Å) b (Å) c (Å) α = β ( ) 9 9 γ ( ) Resolution (Å) a R merge (%) 12.7(4.6) 7.4(4.3) I/sigma 6.6(5.6) 4.7 (3.3) Completeness (%) 99.9(99.5) 99.8(99.2) Multiplicity No. Total reflections No. Unique reflections b / R c work R free (%) 17.6/ /2.9 No. of Atoms No. of Solvent Atoms B-factors (Å 2 ) Overall Protein Inactivator N.D Water d RMSD Bond Length (A).2.4 d RMSD Bond Angles ( o ) Ramachandran Favored (%) Allowed (%) Outlier (%) Wu, C.; Tran, J. C.; Zamdborg, L.; Durbin, K. R.; Li, M.; Ahlf, D. R.; Early, B. P.; Thomas, P. M.; Sweedler, J. V.; Kelleher, N. L., A protease for 'middle-down' proteomics. Nat Methods 212, 9, S13

SUPPLEMENTARY INFORMATION FOR. (R)-Profens Are Substrate-Selective Inhibitors of Endocannabinoid Oxygenation. by COX-2

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