L7 TCA Cycle

Learning Outcomes

  • Understand the reaction catalysed by PDH & its regulation
  • Describe the reactions of the TCA cycle, where they occur within the (C) & the reg of the cycle
  • Know st of pyruvate & acetyl grp of Acetyl-CoA
  • Understand E yield of glucose catabolism
  • Know the pathway of glycogen breakdown
  • Appreciate the action of arsenic as a poison

Glycolysis

  • ...occurs in cytosol
  • The next 2 steps of respiration in mitochond: Pyruvate oxidation then TCA cycle
  • Conversion of pyruvate to acetyl CoA is NOT part of the TCA cycle [need it to further catabolise]
  • Pyruvate = 3C, Acetyl CoA = 2C ∴ lose 1C of CO2

Acetyl CoA

  • Pivotal molec in metabolism (acetyl intermediate)
  • DRAW pyruvate & acetyl grp
  • presence of high E thioester grp on aceCoA

Pyruvate Dehydrogenase *

  • Complex of 3 ezms, physically linked to enable correct seq of reactions, w/o release of intermediates, each has spec coezms [each subunit has a coezm]
  • E1 = Thiamine pyrophosphate (TPP)
  • E2 = lipoic acid (acyl lipollysine) & CoA
  • E3 = FAD & NAD+
  • Pyruvate + CoA + NAD+ → CO2 + NADH + acetyl-CoA
  • ATP might inhibit the ezm
  • NADH might inhibit the prod of the ezm, & the substrate of the nxt ezm

Regulation- PDH

  • Highly regulated to respond to E charge in (C)
  • ATP = ⤒ E lvl
  • AMP = ⤓ E lvl
  • the E charge = important way for the (C) to know what to activate & inactivate
    ~ ↑ vl of NADH= reduce acti. of ezm
    ~ ↑E charge= lots of ATP= turn off the activity, reduce ct of PDH

TCA Cycle

  • Tri-carboxylic acid, Citric acid, Krebs cycle
  • Sir Hans Krebs 1937
  • Occurs in the presence of O2 (has to have O2 for the TCA cycle to work)
  • Located in mitochond (compartments in the mitochond)
  • Acts as the final common pathway for oxidation of CH2O, lipids & prots (via Acetyl-CoA) (catabolic)
  • Plays major role in glucogenesis, transamination, deamination and lipogenesis (anabolic) - later lecture
  • ∴ amphibolic
  • Produces reducing equivalents (NADH & FADH2) tht enter respiratory chain → ATP [ L9 ]

Mitochondrion

  • Pyruvate passively enters mitochond & before the TCA cycle, oxidation of pyruvate to acetyl-CoA & NADH (must be converted to aceCoA before entering the TCA cycle)
  • Acetyl-CoA then combines w/ 4C oxaloacetate & CoA is released
  • The 6 carbons from 2 pyruvates oxidised to CO2
  • NADH, FADH2 & ATP produces
  • Oxaloacetate is reformed

Steps

Energy

Acetyl-CoA Formation

  • The oxidation (oxidative decarboxylation) of Pyruvate to Acetyl-CoA is the irreversible route from Glycolysis to TCA cycle (can't use the carbons in the acetyl CoA to make pyruvate from Glycolysis to TCA Cycle - (mitochond)
  • Catalysed by sev diff ezms working sequentially in a multi-ezm complex
    = Pyruvate dehydrogenase PDH
  • 5 rxns catabolised
  • Yellow= substrates, Pink = products

Reg

  • Also subject to cov modification by PDH kinase & PDH phosphatase
  • 3 subunits: needs to be dephosphorylated by ezm phosphatase to be activated
  • ↑lvls of pyruvate= stop it frm being phosphorylated & pyruvate ⇈ in its activity
  • ↑lvls NADH = activate PDH
  • having a Pgrp @ spec site makes it inactive

Clinical Perspective

  • Tissues w/ little or no mitochond depend on glycolysis for ATP- pyruvate can't enter TCA cycle
    ~ RBC, cornea, lens, retina, kidney medulla, testic, leukocytes & white muscle
  • PDH deficiency (usually E1 ⇒ lactic acidosis ⇒) neurological defects & death, but can be managed in some instances by low CH2O diet (ketogenic- ↑fat & prot)
  • Lactate formation - later lecture

TCA Cycle Overview

  • Net 1 turn of cycle= 3 NADH, 1 GTP, 1 FADH2, releases 2 CO2
  1. 4C Oxaloacetate + 2C Acetyl CoA -6C citrate
  2. Citrate converted to isocitrate (move OH grp). Aconitase reqs Fe2+

  1. Oxidative decarboxylation of isocitrate to α-ketoglutarate. Release of first CO2 & generation of first NADH. Isocitrate DH is rate-limiting ezm & tightly controlled
  2. Oxidative decarboxylation of 𝞪-ketoglutarate DH another multi ezm complex (similar to PDH) reqs coenz: TPP, lipoic acid, FAD, NAD+, CoA. Releases CO2 & second
  1. Succinyl CoA converted to succinate, release of GTP (substrate lvl phosphorylation). Enz succinyl CoA synthatase AKA succinate thiokinase
  2. Succinate oxidised to fumarate, generates FADH2. Succinate DH is embedded in inner mitoch membrane
  1. FUmarate is hydrated to form malate- fumarase also called fumarate hydratase
  2. Malate to oxaloacetate, final NADH generated. Endergonic, but citrate synthase pulls reaction in direction of OAA

Regulation

Clinical Perspective

  • Liver only tissue in which all metabolic processes occur significantly- profound effect during liver damage (hepatitis, cirrhosis)
  • TCA v few genetic abnormalities- incompatible w/ life
  • Fumarase deficiency- fumarate → malate
    ~ Autosomal rec disorder
    ~ Severe neurological impairment, encephalopathy, dystonia [twith: can't control muscle mvt]
  • TCA is aerobic process, anoxia or hypoxia leads total or partial inhibition of cycle
  • Four soluble B vit have precise roles TCA cycle- Riboflavin (FAD), Niacin (NAD), Thiamine (𝜶-KG- DH) & Panthothenic acid (Coezm A)

Importance of NADH & FADH2

  • Main products of Glycolysis & TCA cycle = NADH & FADH2
  • Process tht utilises these e- carriers to produce ATP = Oxidative Phosphorylation
  • Occurs via Electron Transport Chain
  • E- transferred from NADH & FADH2 to O2 to drive the formation of ATP
    ~ NADH ⇒ 3ATP
    ~ FADH2 ⇒ 2ATP

Glucose (C6 H12 O6) catabolism E Yield

  • Glycolysis:
    2 ATP (net)
    2 NADH (2 x 2ATP)
  • Pyruvate to acetyl CoA
    2 NADH (2 x 3ATP)
    2 Acetyl CoA ⇒
  • TCA Cycle
    ⇒ 3 NADH (3 x 3ATP)
    ⇒1 FADH2 (2ATP)
    ⇒ 1 GTP

Glycogen

  • Glycogen → Glucose = Glycogenolysis
  • Glycogen phosphorylase regulation?

Arsenic poisoning

  • Inhbition of Glycolysis (arsenate)
  • & TCA cycle (main reason it's toxic)
  • ARsenite (trivalent arsenic)- binds to lipoic acid SH grp forming a stable complex (unable to bind to ezms: PDH & 𝜶-ketoglutarate DH -coezm)
  • Causes a spontaneous conversion = don't get ATP