BIOC 460 - DR. TISCHLER LECTURE 26 GLYCOLYSIS AND GLUCONEOGENESIS-2

OBJECTIVES Gluconeogenesis Pathway Key liver gluconeogenic precursors; 4 unique rxns relationship between nutritional status (i.e., fed or starved) and the amount of phosphoenolpyruvate carboxykinase in liver cells as well as the significance of these responses . Significance of the glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions in gluconeogenesis

Regulation of Glycolysis and Gluconeogenesis 1. Kinetics and allosteric regulation of glucokinase and hexokinase; logic for differences between the two kinases. 2. Regulation of PFK-1 and F-1,6-BPase: a) compare allosteric regulation. b) regulation coordinates glycolysis and gluconeogenesis c) metabolism of F-2,6-BP; role of bifunctional protein d) insulin via F-2,6-BP ensures during the fed state that liver glycolysis is active and gluconeogenesis is inactive 3. Regulation of pyruvate kinase via allosteric control and by covalent modification (phosphorylation/ dephosphorylation)

G6Pase unique Glucose 6-P Glucose precursor Fructose 6-P glycolytic enzyme also F1,6BPase F-1,6-BP G-3-P + DHAP Glycerol Lactate NADH LDH NAD+ NAD+ G-3-P DH Figure 1. Gluconeogenic pathway for lactate, ala, asp, glycerol NADH Alanine ALT ADP + Pi PGK ATP Pyruvate CYTOPLASM PEP PC PEPCK Pyruvate OAA PEP MITOCHONDRION Aspartate AST

pyruvate + ATP + CO2  oxaloacetate + ADP + Pi Unique Reaction 1: Pyruvate carboxylase catalyzes the reaction: pyruvate + ATP + CO2  oxaloacetate + ADP + Pi CO2 CO2 O=C-OH C=O CH3 CH2 O=C-OH C=O CO2 requires biotin CO2 CO2 O=C-OH

oxaloacetate + GTP  phosphoenolpyruvate + GDP + CO2 Unique Reaction 2: Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the reaction: oxaloacetate + GTP  phosphoenolpyruvate + GDP + CO2 PO3 CO2 PO3 CO2 O=C-OH C=O CH2 PO3 CO2 O=C-OH C-OPO32- CH2 PO3 PO3 CO2 CO2

Fructose-1,6-bisphosphate  Fructose-6-phosphate + Pi Unique Reaction 3: Fructose-1,6-bisphosphatase catalyzes the reaction: Fructose-1,6-bisphosphate  Fructose-6-phosphate + Pi PO3 PO3 PO3 PO3 O CH2OPO32- HO OH CH2OH O CH2OPO32- HO OH

Glucose-6-phosphate  Glucose + Pi Unique Reaction 4: Glucose-6-phosphatase catalyzes the reaction: Glucose-6-phosphate  Glucose + Pi PO3 PO3 PO3 O CH2OH OH O CH2OPO32- OH

COMPARATIVE KINETICS OF HEXOKINASE AND GLUCOKINASE has low Km (high affinity) for glucose ensures brain, red blood cells, etc can use glucose even when blood low in starvation Glucokinase: has high Km (low affinity) for glucose ensures that liver or pancreas only uses glucose in response to high blood levels after a meal

Figure 2. Regulation of glucokinase and hexokinase ADP ATP Hexose (e.g.,glucose, fructose, galactose) Glucose (liver; pancreas) Glucokinase Feedback inhibition by glucose-6-P No allosteric control Glucose-6-P Hexose-6-P (G6P most Tissues) Figure 2. Regulation of glucokinase and hexokinase

- = allosteric inhibition; + = allosteric activation Fructose-6-phosphate ATP Pi ATP - Citrate fructose-2,6-bisP AMP - PFK-1 H+ F-1,6-BPase (glycolysis) + AMP (gluconeogenesis) + citrate fructose-2,6-bisP H2O ADP Fructose-1,6-bisphosphate Figure 3 – Reciprocal regulation of glycolysis and gluconeogenesis at reactions catalyzed by phosphofructokinase-1 and fructose-1,6-bisphosphatase. - = allosteric inhibition; + = allosteric activation

Bifunctional protein – dephosphorylated (by protein phosphatase): PFK-2 - active FBPase-2 - inactive fructose-6-phosphate fructose-2,6-bisphosphate ATP ADP fructose-6-phosphate Bifunctional protein – phosphorylated (by protein kinase A): FBPase-2 - active PFK-2 - inactive fructose-2,6-bisphosphate H2O Pi Figure 4. Reactions in the metabolism of fructose-2,6-bisphosphate.

X fructose-6-P ATP fructose-6-P PFK-2 + ATP active inactivation PFK-1 fructose-2,6-bisP ATP ADP fructose-6-P + Pi fructose-1,6-bisP inactivation FBPase-1 gluconeogenesis X fructose-6-P + ATP fructose-1,6-bisP + ADP PFK-1 glycolysis activation Figure 5. Formation of F-2,6-bisP in the fed state leads to activation of PFK-1 in glycolysis and inactivation of gluconeogenesis.

- Figure 6. Regulation of liver pyruvate kinase. ATP ADP protein kinase A + Glucagon OH Pyruvate Kinase (active) + ATP Phosphoenolpyruvate + ADP OPO32- Pyruvate kinase (inactive) - citrate ATP alanine Pi protein phosphatase + Insulin Figure 6. Regulation of liver pyruvate kinase.