Important updates No class on Monday, Nov. 19 Lab on Monday, Nov. 19 will pick up with the 1st week of GOT Don’t forget Monday’s lab quiz Omitting the G1P lab completely Best lab weighted more Midterm 2 will be Friday, Nov. 30 Classes will go through Dec. 7 (which was the original date of our final exam) Exam date is Thurs., Dec. 13, 2-5 pm 12/5/2018 S.A. McFarland © 2007

Citric acid cycle Ch. 16

Common themes Where in the CAC do you see this common theme? Oxidation-hydration-oxidation Where in the CAC do you see this common theme? 12/5/2018 S.A. McFarland © 2007

Common themes Oxidation-hydration-oxidation 12/5/2018 Different enzymes to facilitate the same “type” of reaction sequence. In both examples, we use an oxidation-hydration-oxidation sequence to oxidize a saturated carbon to a carbonyl group. 12/5/2018 S.A. McFarland © 2007

CAC is amphibolic Amphibolic indicates both catabolic and anabolic At first glance pathways appear either catabolic (release and conservation of free energy) or anabolic (requirement for free energy) CAC catabolic Involves degradation Major free-energy conservation system in most organisms Intermediates required in only catalytic amounts to maintain degradative function of cycle CAC anabolic Several biosynthetic pathways use CAC intermediates as starting materials for anabolic reactions 12/5/2018 S.A. McFarland © 2007

CAC intermediates Use and replenishment 12/5/2018 S.A. McFarland © 2007

Pathways that use Glucose biosynthesis (gluconeogenesis) Fatty acid biosynthesis Amino acid biosynthesis 12/5/2018 S.A. McFarland © 2007

Amino acid biosynthesis Reductive amination catalyzed by glutamate DH Utilizes NADH or NADPH 12/5/2018 S.A. McFarland © 2007

12/5/2018 S.A. McFarland © 2007

Pathways that replenish Anaplerotic reactions Anaplerotic rxns- literally, “filling-up” reactions 12/5/2018 S.A. McFarland © 2007

Anaplerotic reactions Replenishing CAC intermediates 12/5/2018 S.A. McFarland © 2007

Pyruvate carboxylase Conversion of pyruvate to oxaloacetate Most important of the anaplerotic reactions Exists in mitochondria of animal cells but not in plants Provides direct link between glycolysis and the CAC Tetrameric protein of identical 1160-residue subunits Each subunit has biotin prosthetic group covalently bound to lysine and a Mg2+ site Absolute requirement for acetyl-CoA 12/5/2018 S.A. McFarland © 2007

Sample problem Acetyl-CoA is an activator of pyruvate carboxylase. When acetyl-CoA levels are high, pyruvate is converted to oxaloacetate. Why? 12/5/2018 S.A. McFarland © 2007

Biotin Coenzyme that carries CO2 Imidazoline ring that is cis-fused to a tetrahydrothiophene ring bearing valerate side chain Positions 1, 2, and 3 constitute a ureido group Covalently attached to carboxylases by an amide linkage between valeryl carboxyl and ε-amino group of Lys 12/5/2018 S.A. McFarland © 2007

Formation of biotinyl-enzyme biotinyllysine = biocytin Cleavage of ATP to form carboxyphosphate and ADP Formation of a “high-energy” carboxyphosphate intermediate (activated form of CO2) Exergonic reaction of biotin with CO2 Biotin-bound carbonyl is “activated” relative to bicarbonate (transfer does not involve further free-energy input) 12/5/2018 S.A. McFarland © 2007

Conversion of pyruvate to oxaloacetate biotinyllysine = biocytin Activated carboxyl group is transferred from carboxybiotin to pyruvate in a 3-step reaction Two reaction phases occur on different sites of same enzyme; the 14-Å arm of biocytin transfers the biotin ring between the two sites 12/5/2018 S.A. McFarland © 2007

Movement of biocytin 12/5/2018 S.A. McFarland © 2007

Sample problem Biocytin (aka, biotinyllysine) functions similar to what other coenzyme? Where is this other coenzyme found and what is its function? 12/5/2018 S.A. McFarland © 2007

All enter cells on same transporter All become covalently attached to proteins by similar mechanisms All provide flexible tether that allows bound reaction intermediates to move from one active site to another (substrate channeling) 12/5/2018 S.A. McFarland © 2007

Sample problem Biotin is a vitamin required by the human diet; it is abundant in many foods and is synthesized by intestinal bacteria. Biotin deficiency is rare, but can sometimes be caused by a diet rich in raw eggs. Considering that egg whites contain a large amount of the protein avidin, suggest a possible explanation. 12/5/2018 S.A. McFarland © 2007

Lipoic acid (thioctic acid) Universal antioxidant 12/5/2018 S.A. McFarland © 2007

Lipoic acid (thioctic acid) Universal antioxidant Involved in reactions of acyl group transfer coupled with oxidation-reduction Isolated and characterized in 1951, required 10 tons of liver to isolate 30 mg Initially considered a vitamin, but new studies show that cells synthesize it in very small quantities Additional biochemical functions Antioxidant Metal chelator Universal antioxidant, more powerful in blocking destructive oxidative processes than vitamins C or E Regenerates vitamins C and E (redox cycling) Soluble in both fatty- and water-based tissue Being examined for treatment of diabetes, cataracts, glaucoma, and heavy-metal poisoning Get it from spinach, liver, and brewer’s yeast 12/5/2018 S.A. McFarland © 2007

Sample problem Arsenic is a toxic metal. Indicate one possible mode of toxicity in terms of the pathways we’ve studied. Suggest a possible treatment. 12/5/2018 S.A. McFarland © 2007

Sample problem Arsenic is a toxic metal. Indicate one possible mode of toxicity in terms of the pathways we’ve studied. 12/5/2018 S.A. McFarland © 2007