Supplementary Information. A study on the chiral inversion of mandelic acid in humans
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1 Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry 2014 Supplementary Information A study on the chiral inversion of mandelic acid in humans Maksims Yevglevskis, Catherine R. Bowskill, Chloe C. Y. Chan, Justin.-J. eng, Michael D. Threadgill, Timothy J. Woodman, and Matthew D. Lloyd* Medicinal Chemistry, Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, U. K. M.D.Lloyd@bath.ac.uk
2 Table of Contents Characterisation of synthetic compounds pages 3-8 Incubations of acyl-coa esters with recombinant human AMACR 1A pages 9-16 Kinetic plots for ACT1 pages Kinetic plots for ACT2 pages Supplementary Figure S1: Sequence alignment of AMACR and MCR page 37
3 S-2-ydroxyphenylacetyl-CoA 2S ( 1 NMR, Mz) S CoA
4 R-2-ydroxy-2-phenylacetyl-CoA 2R ( 1 NMR, Mz) S CoA
5 S-2-Methyl-2-phenylacetyl-CoA 8S ( 1 NMR, Mz) Me SCoA
6 R-2-Methyl-2-phenylacetyl-CoA 8R ( 1 NMR, Mz) Me SCoA
7 S-2-ydroxy-2-phenylacetyl-CoA 2S (as Na adduct) SCoA
8 S-2-Phenylpropanoyl-CoA 8S Me SCoA
9 Incubation of ±-Fenoprofenoyl-CoA with human recombinant AMACR 1A S CoA eat-inactivated enzyme Live enzyme
10 Incubation of S-2-hydroxy-2-phenylacetyl-CoA 2S with human recombinant AMACR 1A SCoA eat-inactivated enzyme Live enzyme
11 S-2-ydroxy-2-phenylacetyl-CoA 2S incubated with heat inactivated AMACR (full spectrum) S CoA S-2-ydroxy-2-phenylacetyl-CoA 2S incubated with active AMACR (full spectrum) S CoA
12 Incubation of R-2-hydroxy-2-phenylacetyl-CoA 2R with human recombinant AMACR 1A SCoA eat-inactivated enzyme Live enzyme
13 R-2-ydroxy-2-phenylacetyl-CoA 2R incubated with heat inactivated AMACR (full spectrum) S CoA R-2-ydroxy-2-phenylacetyl-CoA 2R incubated with active AMACR (full spectrum) S CoA
14 Stack plot showing incubation of 2R and 2S with live and heat-inactivated AMACR 2R, Live enzyme 2R, heat-inactivated enzyme 2S, Live enzyme 2S, heat-inactivated enzyme
15 Incubation of S-2-phenylpropanoyl-CoA 8S with human recombinant AMACR 1A Me SCoA eat-inactivated enzyme Live enzyme
16 Incubation of R-2-phenylpropanoyl-CoA 8R with human recombinant AMACR 1A Me SCoA eat-inactivate enzyme Live enzyme
17 ACT1, Myristoyl-CoA 11 S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax to Km to Ki to Goodness of Fit Degrees of Freedom 21 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 8 Number of replicates 3 Total number of values 24 Number of missing values 0 Michaelis-Menten Rate (nmol/min/mg)
18 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals 6 4 Residuals (nmol/min/mg)
19 ACT-1, S-2-hydroxy-2-phenylacetyl-CoA 2S S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax to Km to Ki to Goodness of Fit Degrees of Freedom 21 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 8 Number of replicates 3 Total number of values 24 Number of missing values 0 Michaelis-Menten Rate (nmol/min/mg)
20 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals 4 2 Residuals (nmol/min/mg)
21 ACT-1, R-2-hydroxy-2-phenylacetyl-CoA 2R S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax to Km to Goodness of Fit Degrees of Freedom 21 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 8 Number of replicates 3 Total number of values 23 Number of missing values 1 Michaelis-Menten Rate (nmol/min/mg)
22 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals 4 3 Residuals (nmol/min/mg)
23 ACT-1, S-2-Phenylpropanoyl-CoA 8S Me S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax 26, e e+7 to 1.385e+7 Km 35, e e+7 to 1.859e+7 Ki to Goodness of Fit Degrees of Freedom 21 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 8 Number of replicates 3 Total number of values 24 Number of missing values 0 Michaelis-Menten Rate (nmol/min/mg)
24 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals 6 4 Residuals (nmol/min/mg)
25 ACT-1, R-2-phenylpropanoyl-CoA 8R Me S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax to Km to Goodness of Fit Degrees of Freedom 22 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 8 Number of replicates 3 Total number of values 24 Number of missing values 0 Michaelis-Menten 6 5 Rate (nmol/min/mg)
26 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals Residuals (nmol/min/mg)
27 ACT-2, Myristoyl-CoA 11 S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax to Km to Ki to Goodness of Fit Degrees of Freedom 20 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 8 Number of replicates 3 Total number of values 23 Number of missing values 1 Michaelis-Menten Rate (nmol/min/mg)
28 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals 6 4 Residuals (nmol/min/mg)
29 ACT-2, S-2-hydroxy-2-phenylacetyl-CoA 2S S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax to Km to Ki to Goodness of Fit Degrees of Freedom 21 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 8 Number of replicates 3 Total number of values 24 Number of missing values 0 Michaelis-Menten Rate (nmol/min/mg)
30 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals 4 2 Residuals (nmol/min/mg)
31 ACT-2, R-2-hydroxy-2-phenylacetyl-CoA 2R S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax to Km to Goodness of Fit Degrees of Freedom 18 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 7 Number of replicates 3 Total number of values 20 Number of missing values 1 Michaelis-Menten Rate (nmol/min/mg)
32 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals 3 2 Residuals (nmol/min/mg)
33 ACT-2, S-2-Phenylpropanoyl-CoA 8S Me S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax 33, e e+7 to 1.650e+7 Km 46, e e+7 to 2.323e+7 Ki to Goodness of Fit Degrees of Freedom 21 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 8 Number of replicates 3 Total number of values 24 Number of missing values 0 Michaelis-Menten Rate (nmol/min/mg)
34 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals 4 2 Residuals (nmol/min/mg)
35 ACT-2, R-2-Phenylpropanoyl-CoA 8R Me S CoA Parameters Value ±Std. Error 95% Conf. Interval Vmax to Km to Goodness of Fit Degrees of Freedom 22 AICc R² Sum of Squares Sy.x Runs Test p Value Data Number of x values 8 Number of replicates 3 Total number of values 24 Number of missing values 0 Michaelis-Menten 6 5 Rate (nmol/min/mg)
36 Lineweaver-Burk /Rate (nmol/min/mg) / Residuals Residuals (nmol/min/mg)
37 MCR AMACR MCR AMACR MCR AMACR MCR AMACR MCR AMACR MCR AMACR MCR AMACR MAGPLSGLRVVELAGIGPGPAAMILGDLGADVVRIDRPSSVDGISRDAMLRNRRIVTAD --MALQGISVVELSGLAPGPFCAMVLADFGARVVRVDRPGSRYDVSR--LGRGKRSLVLD LKSDQGLELALKLIAKADVLIEGYRPGVTERLGLGPEECAKVNDRLIYARMTGWGQTGPR LKQPRGAAVLRRLCKRSDVLLEPFRRGVMEKLQLGPEILQRENPRLIYARLSGFGQSGSF SQQAGDINYISLNGILAIGRGDERPVPPLNLVGDFGGGSMFLLVGILAALWERQSSGK CRLAGDINYLALSGVLSKIGRSGENPYAPLNLLADFAGGGLMCALGIIMALFDRTRTGK GQVVDAAMVDGSSVLIQMMWAMRATGMWTDTRGANMLDGGAPYYDTYECADGRYVAVGAI GQVIDANMVEGTAYLSSFLWKTQKSSLWEAPRGQNMLDGGAPFYTTYRTADGEFMAVGAI EPQFYAAMLAGLGLDAAELPPQNDRARWPELRALLTEAFASDRDWGAVFANSDACVTP EPQFYELLIKGLGLKSDELPNQMSMDDWPEMKKKFADVFAKKTKAEWCQIFDGTDACVTP VLAFGEVNEPIIERNTFYEANG---GWQPMPAPRFSRTASSQPRPPAATIDIEAVLTD VLTFEEVVDNKERGSFITSEEQDVSPRPAPLLLNTPAIPSFKRDPFIGETEEILEE WDG FGFSREEIYQLNSDKIIESNKVKASL Figure S1: Sequence alignment of MCR from M. tuberculosis (06543) and human AMACR 1A (Q9UK6), showing residues involved in methyl group binding pocket (Bold).
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