Question

1.  You have acetate that is isotopically labelled (¹³C) at carbon 1 and radiolabeled (¹⁴C) at carbon 2.  Assume all the acetate gets incorporated into Acetyl-CoA.  Calculate the % of each labelled carbon that gets released in the first five rounds of the TCA cycle and at what enzymatic step they get released.  

2.  Document (on extra pages attached to this sheet) your work.  Note:  you don’t have to include every step if you are clever.

1. You have acetate that is isotopically labelled (c) at carbon 1 and radiolabeled (c) at carbon 2. Assume all the acetate gets incorporated into Acetyl-CoA. Calculate the % of each labelled carbon that gets released in the first five rounds of the TCA cycle and at what enzymatic step they get released. Example: OAA labelled at CI with 13C and C4 labelled with 14C. Rnd of TCA Rnd 13c 100% Enz Step Isocitrate DH 140 100% Enz Step KG DH And now, strating with the labelled acetate: 13C 100% Enz Step Isocitrate DH 140 100% Enz Step KG DH Rnd of TCA Rnd Rnd 2 Rnd 3 Rnd 4 Rnd 5 2. Document (on extra pages attached to this sheet) your work. Note: you don't have to include every step if you are clever.

Answer 1

When pyruvate is converted to Acetyl CoA, the reaction catalyzed by the pyruvate dehydrogenase complex is an oxidative decarbonylation, an irreversible oxidation process in which the carboxyl group is removed . pyruvate as a molecule of co2 and the a remaining carbons carsons two become become the the ace acetyl group of Acetyl coA.

- 2- CoA جدا و 11 و ع 2 دان

The table given in the figure is uploaded hereby-

And now, strating with the labelled acetate: Rnd of TCA Rnd I Rnd 2 Rnd 3 Rnd 4 Rnd 5 13C 100% 100 % 100% 0% % Enz Step 140 Enz Step Citrate synthase 100% Citrate synthase Aconitase 100 % Aconitase Tocitrate dehydrogenase 0% Isocifrate dehydrogenade alpha-KG dehydro complex 0% a-KG dehydro suec COA syn0 % SUC COA syn 0

Complete enzyme names could not be given in the figure for space constraints, however, I have depicted the details in complete figure

STEP 1 - Formation of citrate va CH3 -C2-S-CoA. CITRATE ACETYL COA 120 COA-SHF GH₂-C2- + D HOLC - coon O = e-coo SYNTHASE CH2- COD CH₂ - cool OXALOACETATE CITRATE

STEP 2 - Citrate to Procitrate GH₂ - Soo Ho-c-coo CH2– 200 H₂O - H-Cucos H-C-COD A CONITASE HO-C-H CITRATE cos 0 ISOGITRATE

STEP 3 - Isocitrate to a- ketoglutarate CH₂-600 Varaf 140 RELEASED ELEASED cool CH₂-and ertz H - 2 - C- lo Vog, eso Hà - c – H 150CSTRATE DEHYD'ROGENASE

to x-ketoglutarate C.H2 - Code Succinyl CoA NADH 1. eta-cool COA-SH NAD+ 1 CH₂ + CO2 CH2 e-S-CoA 130 c-cool RELEASED - KETOGLUTARATE DEHYDROGENASE COMPLEX SUCCINYL COA К- KЕTouTARATE

COA-SH CH, - coo- Coo GDP+Pi CH₂ CH2 SCOA SUCCINYL COA SYNTHETASE 200- SUCCINATE O SUCCINYL COA