LEHNINGER PRINCIPLES OF BIOCHEMISTRY Sixth Edition David L. Nelson and Michael M. Cox © 2013 W. H. Freeman and Company CHAPTER 16 The Citric Acid Cycle

Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration

Overall reaction catalyzed by the pyruvate dehydrogenase (PDH) complex The pyruvate dehydrogenase complex consists of 3 enzymes The complex utilizes 5 cofactors for catalysis

Pyruvate Dehydrogenase Cofactors Nicotinamide Adenine Dinucleotide (NAD + ) an electron carrier. Vitamin required: niacin Flavin Adenine Dinucleotide (FAD) an electron carrier Vitamin required: riboflavin Coenzyme A (CoA) an acyl carrier with a reactive thiol - often acetyl-CoA Vitamin required: pantothenate Thiamine Pyrophosphate (TPP) functions in electron transfer Vitamin required: thiamine Lipoic Acid has two thiols that can form a disulfide bond

Lipoic Acid is attached to a Lysine of E2 of the PDH complex

Composition of Pyruvate Dehydrogenase Complex In E. coli: 24 copies of dihydrolipoyl transacetylase (E 2 ) E 2 contains covalently bound lipoate 24 copies of pyruvate dehydrogenase (E 1 ) E 1 contains bound TPP 12 copies of dihydrolipoyl dehydrogenase (E 3 ) E 3 contains bound FAD The pyruvate dehydrogenase complex is large enough to be seen with an electron microscope. ~45 nm in diameter

6 carbons5 carbons

4 carbons

Enzymes of the Citric Acid Cycle Structure and Structure and Mechanism of catalysis

X-ray crystal structure of citrate synthase Oxaloacetate (orange) binds to the enzyme first. This causes a conformational change from “open” to “closed” form. The conformational change creates a binding site for the second substrate acetyl-CoA (purple). Citroyl-CoA is formed and another conformational change puts an Asp side chain in position to cleave citroyl-CoA.

Aconitase catalyzes the transformation of citrate to isocitrate H 2 O can be added to cis-aconitate in two different ways. Isocitrate is normally formed due to the low concentration of isocitrate, rapidly converted to  -ketoglutarate.

Aconitase has a iron-sulfur center that acts in both substrate binding and catalysis.

Succinyl-CoA synthetase uses the energy of a thioester bond to produce GTP from GDP and Pi.

Succinyl-CoA synthetase is phosphorylated on a His residue during catalysis. This phosphoryl group is then transferred to GDP (or ADP) to form GTP (or ATP).

The citric acid cycle is a central metabolic hub

Biotin in pyruvate carboxylase is a carrier of one carbon groups as CO 2. CO 2 is passed on to pyruvate to form oxaloacetate in a carboxylation reaction. This reaction requires energy in the form of ATP.

Don’t eat raw eggs! Why? Eggs contain a protein called avidin. Avidin binds very tightly to biotin and prevents its absorption in the intestine. When eggs are cooked the avidin is denatured just like all other proteins.

REGULATION OF THE CITRIC ACID CYCLE

Fatty acids Amino Acids CO 2 Phosphoenolpyruvate Glucose PEP carboxykinase (Gluconeogenesis)

The Glyoxalate Cycle What plants can do and humans cannot

The Glyoxylate Cycle Net Production Of Oxaloacetate For Carbohydrate Synthesis (non-vertebrates)

In plants, enzymes of the glyoxylate cycle are in glyoxysomes