EFB325 Cell Physiology

The Citric Acid Cycle

The process of glycolysis yielded only 2 ATP and 2 NADH, as well as 2 molecules of pyruvate. Also there has been no conversion of glucose to CO₂ yet. There is still a great deal of oxidizable energy remaining in the pyruvate. That energy is extracted and the organic acids are broken down and released as CO₂ through the citric acid acid cycle.

Pyruvate is transported into the mitochondria by a carrier protein

• glycolysis occurs in the cytosol, the citric acid cycle occurs in the matrix of the mitochondria

One step occurs before the cycle starts=oxidation of pyruvate to acetyl CoA

• pyruvate (a 3 carbon organic acid) is oxidized (NAD⁺ is reduced to NADH)
• one molecule of CO₂ is released=decarboxylation
• the remaining 2 carbon molecule (acetate) is linked to coenzyme A (CoA) to form acetyl CoA
• acetyl-CoA is the molecule that starts the citric acid cycle

CoA is a large molecule, linked to the organic acid by a high energy bond (a thioester bond)

• this high energy linkage of acetate to CoA results in an activated form of acetate
• CoA is called a coenzyme because it participates in the reaction, but is released unchanged in the end

Overview of the citric acid cycle

• discovered by Hans Krebs in '30s, so also called Krebs cycle; also called the tricarboxylic acid (TCA) cycle (citric acid has three carboxylic acid groups)
• first step combines a 2-carbon organic acid with a 4-carbon organic acid (to produce a 6-carbon)
• this undergoes 4 oxidations, including 2 decarboxylations that release 2 CO₂, to generate NADH/FADH₂ and also 1 ATP
• the original 4-carbon acceptor is regenerated, completing the cycle

Key concepts:

• acetate (acetyl CoA) contributes 2 carbons and there is a release of 2 carbons as CO₂
• the O in the CO₂ comes ultimately from water, not from O₂
• there are 4 oxidation steps in the cycle; 3 reduce NAD⁺ to NADH; 1 reduces FAD to FADH₂
• 1 molecule of ATP is produced by a type of substrate-level phosphorylation
• the cycle goes around twice for each molecule of glucose
• the intermediates are organic acids (rather than phosphorylated sugars and acids as in glycolysis)
• each step is catalyzed by a different enzyme, which specifically binds its substrate
• the NADH (and FADH₂) must be oxidized back to NAD⁺ (and FAD) to allow the cycle to continue

Key steps:

• acetate (2 C) is released from CoA and combines with oxaloacetate (OAA, 4 C) to make citric acid (6 C)-the energy in the bond with CoA drives the reaction forward
• citric acid is converted to isocitrate; which is oxidized in a decarboxylation reaction-producing alpha-ketoglutarate (5 C) and CO₂ and reducing NAD⁺ to NADH
• alpha-ketoglutarate (5 C) is oxidized in a decarboxylation reaction, but also a bond is formed with CoA (remember this is a high energy bond); producing succinyl-CoA (succinate has 4 C) and CO₂ and reducing NAD⁺ to NADH
• the CoA is released from succinate and the energy released is sufficient to drive the synthesis of ATP from ADP + Pi
• succinate is then oxidized to fumarate (no release of C, so still 4 C), but in this reaction a different molecule is reduced: FAD is reduced to FADH₂
• fumarate is converted to malate; which is oxidized to oxaloacetate (no release of C, so still 4 C); again reducing NAD⁺ to NADH; oxaloacetate is regenerated so that it can combine with acetyl CoA and run through the cycle again

So far:

• glucose has been oxidized to 6 CO₂, but a net of only 4 ATP has been produced (2 ATP in glycolysis and 2 ATP through 2 cycles of the citric acid cycle)
• also produced 10 NADH (2 in glycolysis, 2 in the oxidation of 2 pyruvate to acetyl CoA, and 6 through 2 cycles of the citric acid cycle) and 2 FADH₂ (through 2 cycles of citric acid cycle)

The citric acid cycle can act in synthesis, as well as in breakdown

• some of the organic acids, which are intermediates in the citric acid cycle, are the precursors for the synthesis of other molecules
• for example, pyruvate, oxaloacetate, and alpha-ketoglutarate can be aminated (have an amino group added) to form the amino acids alanine, aspartate, and glutamate
• since the citric acid does both synthesis (anabolic) and breakdown (catabolic) activities, it is called an amphibolic pathway
• BUT, when an intermediate is removed for synthesis, then that cycle is broken (can't regenerate oxaloacetate)
• there are additional reactions that can produce oxaloacetate (4 C) by carboxylation of either pyruvate or phosphoenolpyruvate (PEP) (both 3 C)

The rate at which the citric acid cycle runs is regulated by inhibiting/activating some of the enzymes

• enzyme activity is regulated by the NAD⁺/NADH ratio (NADH inhibits some of the enzymes) and by the ADP/ATP ratio (ADP activates some of the enzymes)

Glucose is released from polymers, either in an animal's food or from storage polymers

• storage glycogen (animal cells) and starch (plant cells) are broken down by the addition of Pi=phosphorolytic cleavage
• in animals, the enzyme used is phosphorylase, in plants, starch is cleaved by starch phosphorylase
• releases glucose-1-P, which is easily converted to glucose-6-P
• energy was needed to build the polymer, but that energy is conserved when the polymer is broken down to release a sugar-phosphate
• only one ATP is used in the initial steps of glycolysis

In human food, enzymes in our digestive tract break down polymers into smaller units

• amylase in saliva cleaves starch to smaller units
• proteases cleave protein to amino acids
• disaccharides are cleaved to release monosaccharides
• sucrose is cleaved by invertase to release glucose and fructose
• lactose is cleaved by lactase to release galactose and glucose
• maltase cleaves maltose to release glucoses
• sucrose is an important transported form of carbohydrate in plants - it is cleaved by invertase at the site of use (for respiration)

Breaking down fat for energy produces acetyl CoA, which feeds directly into the citric acid cycle

storage fats=triglycerides=glycerol (3 C) bound with 3 fatty acids

• fats are excellent long-term reserves of energy, because they hold alot of energy (as reduced carbon) per volume and weight
• to break down fats, first cleave the fatty acids from glycerol
• glycerol (3 C) can be converted to a triose-phosphate to enter glycolysis
• fatty acids are broken down into units of acetyl CoA (2 C each) by the process of beta oxidation (beta refers to the second carbon in from the end of the fatty acid)
• each time an acetyl CoA is released by oxidation, 1 NADH and 1 FADH₂ are generated by that cleavage reaction
• plus then the acetyl CoA enters the citric acid cycle, producing 3 more NADH, 1 FADH₂, and 1 ATP

Proteins can also be broken down to feed into the citric acid cycle and generate energy

• under extreme starvation in animals or during senescence in plants
• polypeptides are cleaved to amino acids, amino group is cleaved off of amino acid forming an organic acid that can enter the citric acid cycle

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