Chapt. 20 TCA cycle Ch. 20 Tricarboxylic acid cyle Student Learning Outcomes: Describe relevance of...

Chapt. 20 TCA cycle

Ch. 20 Tricarboxylic acid cyle Student Learning Outcomes:• Describe relevance of TCA cycle

• Acetyl CoA funnels products• Describe reactions of TCA cycle in cell

respiration: 2C added, oxidations, rearrangements-> NADH, FAD(2H), GTP, CO2 produced

• Explain TCA cycle intermediates are used in biosynthetic reactions

• Describe how TCA cycle is regulated by ATP demand: ADP levels, NADH/NAD+ ratio

Overview TCA cycle

Fig. 1TCA cycle (Kreb’s cycle) or citric acid cycle:• Generates 2/3 of ATP

• 2C unit Acetyl CoA• Adds to 4C oxaloacetate• Forms 6C citrate• Oxidations,

rearrangements ->• Oxaloacetate again

• 2 CO2 released• 3 NADH, 1 FAD(2H)• 1 GTP

II. Reactions of TCA cycle

Reactions of TCA cycle:

• 2 C of Acetyl CoA are oxidized to CO2 (not the same 2 that enter)

• Electrons conserved through NAD+, FAD -> go to electron transport chain

• 1 GTP substrate level phosphorylation:

• 2.5 ATP/NADH; 1.5 ATP/FAD(2H)• Net 10 high-energy P/Acetyl group

Fig. 2

TCA cycle reactions

TCA cycle Reactions.A. Formation, oxidation of

isocitrate:2C onto oxaloacetate (synthase C-C synthetases need ~P)

Aconitrase move OH(will become C=O)

Isocitrate Dehydrogenase oxidizes –OH, cleaves COOH -> CO2

also get NADH

Fig. 3**

TCA cycle reactions

TCA cycle Reactions.B. -ketoglutarate to

Succinyl CoA:Oxidative decarboxylation

releases CO2Succinyl joins to CoANADH formed

GTP made from activated succinyl CoA

Fig. 3**

TCA cycle reactions

TCA cycle Reactions.D. Oxidation of Succinate to oxaloacetate:

2 e- from succinate to FAD-> FAD(2H)

Fumarate formedH2O added -> malate2 e- to NAD+ -> NADHOxaloacetate restored

(common series of oxidationsto C=C, add H2O -> -OH, oxidize -OH to C=O)

Fig. 3**

III. Coenzymes are critical: NAD+

• Many dehydrogenases use NAD+ coenzyme• NAD+ accepts 2 e- (hydride ion H-): -OH -> C=O• NAD+, and NADH are released from enzyme;

• Can bind and inhibit different dehydrogenases• NAD+/NADH regulatory role (e-transport rate)

Fig. 5

III. Coenzymes are critical for TCA cycle

• FAD can accept e- singly (as C=C formation)• FAD remains tightly bound to enzymes

Fig. 4

Fig. 6 membrane bound succinate dehydrogenase:FAD transfers e- to Fe-S group and to ETC

Coenzyme CoA in TCA cycle

CoASH coenzyme forms thioester bond:• High energy bond(Fig. 8.12 structure of CoASH formed from pantothenate)

Fig. 7

Coenzymes CoASH; TPP

Coenzymes CoASH, TPP(Figs. 8.11, 8.12)

Coenzymes in -ketoacid dehydrogenase complex.

Fig. 8

C. -ketoacid dehydrogenase complex:

• 3 member family (pyruvate dehydrogenase, branched-chain aa dehydrogenase)

• Ketoacid is decarboxylated• CO2 released

• Keto group activated, attached CoA

• Huge enzyme complexes • (3 enzymes E1, E2, E3)• Different coenzymes in each

Fig. 9

-ketoacid dehydrogenase enzyme complex: • 3 enzymes E1, E2, E3• Coenzymes: TPP(thiamine pyrophosphate). Lipoate, FAD

Lipoate is a coenzyme

Lipoate coenzyme:• Made from carbohydrate, aa• Not from vitamin precursor• Attaches to –NH2 of lysine of enzyme• Transfers acyl fragment to CoASH• Transfers e- from SH to FAD

Fig. 10

Energetics of TCA cycle

Fig. 11

Energetics of TCA cycle: overall net -G0’• Some reactions positive; • Some loss of energy as heat (-13 kcal)• Oxidation of NADH,FAD(2H) helps pull TCA cycle forward

Very efficient cycle:• Yield 207 Kcal from1 Acetyl -> CO2 • (90% theoretical 228)• Table 20.1

V. Regulation of TCA cycle

Fig. 12

Many points of regulation of TCA cycle:• PO4 state of ATP (ATP:ADP)• Reduction state of NAD+ (ratio NADH:NAD+)• NADH must enter ETC

Table 20.2 general regulatory mechanisms

Table 20.2 general regulation metabolic paths

• Regulation matches function (tissue-specific differences)• Often at rate-limiting step, slowest step • Often first committed step of pathway, or branchpoint• Regulatory enzymes often catalyze physiological irreversible

reactions (differ in catabolic, biosynthetic paths)• Often feedback regulation by end product• Compartmentalization also helps control access to enzymes• Hormonal regulation integrates responses among tissues:

• Phosphorylation state of enyzmes• Amount of enzyme• Concentration of activator or inhibitor

Citrate synthase simple regulation

Citrate synthase simple regulation:• Concentration of oxaloacetate, the substrate• Citrate is product inhibitor, competitive with S• Malate -> oxoaloacetate favors malate

• If NADH/NAD+ ratio decreases, more oxaloacetate• If isocitrate dehydrogenase activated, less citrate

Allosteric regulation of isocitrate Dehydrogenase

Isocitrate dehydrogenase (ICDH):• Rate-limiting step• Allosteric activation by ADP

• Small inc ADP -> large change rate• Allosteric inhibition by NADH

• Reflect function of ETC

Fig. 13

Other regulation of TCA

Regulation of a-ketoglutarate dehydrogenase:• Product inhibited by NADH, succinyl CoA• May be inhibited by GTP• Like ICDH, responds to levels ADP, ETC activity

Regulation of TCA cycle intermediates:• Ensures NADH made fast enough for ATP homeostasis• Keeps concentration of intermediates appropriate

VI. Precursors of Acetyl CoA

VI. Many fuels feed directly into Acetyl CoA

• Will be completely oxidized to CO2

Fig. 14

Pyruvate Dehydrogenase complex (PDC)

Fig. 15

Pyruvate Dehydrogenase complex (PDC):• Critical step linking glycolysis to TCA• Similar to KGDH (Fig. 20.15)

• Huge complex; • Many copies each subunit:

(Beef heart 30 E1, 60 E2, 6 E3, X)

Regulation of PDC

Fig. 16

PDC regulated mostly by phosphorylation:• Both enzymes in complex

• PDC kinase add PO4 to ser on E1

• PDC phosphatase removes PO4

• PDC kinase:• inhibited by ADP, pyruvate• Activated by Ac CoA, NADH

TCA cycle intermediates and anaplerotic paths

Fig. 17

GABA

TCA cycle intermediates - biosynthesis precursors• Liver ‘open cycle’ high efflux of intermediates:• Specific transporters inner mitochondrial membrane

for pyruvate, citrate, a-KG, malate, ADP, ATP.

Anaplerotic reactions

Fig. 18

Anaplerotic reactions replenish 4-C needed to regenerate oxaloacetate and keep TCA cycling:

• Pyruvate carboxylase• Contains biotin

• Forms intermediate with CO2

• Requires ATP, Mg2+ (Fig. 8.12)

• Found in many tissues

Amino acid degradation forms TCA cycle intermediates

Fig. 19

Amino acid oxidation forms many TCA cycle intermediates:

• Oxidation of even-chain fatty acids and ketone body not replenish

Key concepts

• TCA cycle accounts for about 2/3 of ATP generated from fuel oxidation

• Enyzmes are all located in mitochondrial• Acetyl CoA is substrate for TCA cycle:

• Generates CO2, NADH, FAD(2H), GTP• e- from NADH, FAD(2H) to electron-transport chain.

• Enzymes need many cofactors• Intermediates of TCA cycle are used for

biosynthesis, replaced by anaplerotic (refilling) reactions

• TCA cycle enzymes are carefully regulated

Nuclear-encoded proteins in mitochondria

Nuclear-encoded proteins enter mitochondria via translocases:

• Proteins made on free ribosomes, bound with chaperones

• N-terminal aa presequences

• TOM complex crosses outer• TIM complex crosses inner• Final processing

• Membrane proteins similar

Fig. 20

Review question

Succinyl dehydrogenase differs from other enzymes in the TCA cycle in that it is the only enzyme that displays which of the following characteristics?

a.It is embedded in the inner mitochondrial membrane

b.It is inhibited by NADHc.It contains bound FADd.It contains fe-S centerse.It is regulated by a kinase