Gluconeogenesis  is not a reversal of glycolysis  noncarbohydrate precursors of Glc, carbon skeleton  take place in liver, minor in kidney, brain, skeletal and heart muscle, to maintain the Glc level in the blood  Glc is the primary fuel of brain, and the only fuel of red blood cells active skeletal muscle  protein breakdown  Triacylglycerol hydrolysis 

- 7.5 kcal/mol  G°´

Glycolysis vs. Gluconeogenesis ¤ Three irreversible reactions, irrespective Glycolysis: hexokinase, phosphofructokinase, pyruvate kinase Gluconeogenesis: glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase

The stoichiometry of Glycolysis vs. Gluconeogenesis ¤ Glycolysis: Glucose + 2 ADP + 2 P i + 2 NAD +  2 Pyr + 2 ATP + 2 NADH + 2H H 2 O  G 0’ = - 20 kcal / mol if reverse? ¤ Gluconeogenesis: 2 Pyr + 4 ATP + 2 GTP + 2 NADH + 6 H 2 O  Glucose + 4 ADP + 2 GDP + 6 P i + 2 NAD + + 2H +  G 0’ = - 9 kcal / mol NTP hydrolysis is used to power an energetically unfavorable reaction Both reactions are exergonic

Compartmental cooperation - mitochondrial NADH-malate dehydrogenase NAD + -malate dehydrogenase Specific transporter PEP + CO 2 PEP carboxykinase GTP Pyruvate carboxylase Mito  G 0’ decarboxylation

Pyruvate carboxylase (Pyr + CO 2 + ATP + H 2 O  OAA + ADP + P i + 2 H + ) The only mitochondrial enzymes among the enzymes of gluconeogenesis S (PCase) HCO ATP  HOCO 2 -PO ADP carboxyphosphate: activated form of CO 2 Biotin-Enz + HOCO 2 - PO 3 2-  CO 2 -biotin-Enz + Pi is activated by acetyl CoA (p. 493) CO 2 -biotin-Enz + Pyr  biotin-Enz + OAA (ATP-activating domain, p. 711)  -amino group of Lys Carbonic anhydrase

Free glucose generation F1,6bisP  F6P  G6P  Glc The endpoint of gluconeogenesis in most tissues, can keep Glc or G6P is converted into glycogen. In liver and to a lesser extent the kidney, five proteins are involved SP: a calcium-binding stabilizing protein (Does not take place in cytoplasm) Gluconeogenesis 

Reciprocal control: Glycolysis and gluconeogenesis are not highly active at the same time – Energy state – Intermedia: allosteric effectors – Regulators: hormones  Amounts and activities of distinctive enzymes Fed state: insulin low energy state   Starvation: glucagon rich in precursors high energy state p. 465

Biofunctional of phosphofructokinase 2 phosphofructokinase / fructose bisphosphatase 2 F6P  F2,6BisP L (liver) / M (muscle) isoforms a single 55-kd polypeptide chain Janus

Fructose 2,6-bisphosphate: synthesis and degradation In liver: PEP carbokinase  F 1,6-bisphosphatase  Glycolytic enzymes  (pyruvate kinase)

The first irreversible reaction of glycolysis: Glc  G6P ¤ Hexokinase: is inhibited by G6P K m of sugars: 0.01 ~ 0.1 mM Glucokinase: not inhibited by G6P K m of glucose: ~10 mM present in liver, to monitor blood-glucose level. ¤ Committed step the most important control step in the pathway G6P  glycogen biosynthesis  fatty acid biosynthesis  pentose phosphate pathway

Hormones ¤ Affect the expression of the gene of the essential enzymes – change the rate of transcription – regulate the degradation of mRNA ¤ allosteric control (~ms); phosphorylation control (~ s); transcription control (~ h to d) The promoter of the PEP carboxykinase (OAA  PEP) gene IRE: insulin response element; GRE: glucocorticoid response element TRE: thyroid response element CRE: cAMP response element

Substrate cycle (futile cycle) Biological significances Simultaneously fully active (1) Amplify metabolic signals (2) Generate heat bumblebees: PFKase F1,6-bisPTase: is not inhibited by AMP honeybees: only PFKase (02) If  10  malignant hyperthermia

Cori cycle: carriers + NADH + NAD + Ala Ala metabolism: maintain nitrogen balance transaminase Contracting skeletal muscle supplies lactate to the liver, which uses it to synthesize and release glucose PyrLactate Absence of O 2 Well-oxygenated TCA cycle

Integration of glycolysis and gluconeogenesis during a sprint

Lactate dehydrogenase ¤ a tetramer of two kinds of 35-kd subunits encoded by similar genes ¤ H type: in heart (muscle) M type: in skeletal muscle and liver ¤ H 4 isozyme (type 1): high affinity for lactate, lactate  pyruvate, under aerobic condition H 3 M 1 isozyme (type 2) H 2 M 2 isozyme (type 3) H 1 M 3 isozyme (type 4) M 4 isozyme (type 5): pyruvate  lactate under anaerobic condition  a series of homologous enzymes, foster metabolic cooperation between organs.

Biotin: abundant in some foods and is synthesized by intestinal bacteria Avidin (Mr 70,000): rich in raw egg whites/a defense function The Biotin-Avidin System can improve sensitivity because of the potential for amplification due to multiple site binding. Purification Ex. 11

96T2 96T3 97T

98T

96C 97C