Lecture Connections 14 | Glucose Utilization and Biosynthesis © 2009 Jim-Tong Horng 15 | Principles of Metabolic Regulation 16 | The Citric Acid Cycle 19 | Oxidative Phosphorylation

CHAPTER 14 Glucose Utilization and Biosynthesis Harnessing energy from glucose via glycolysis Fermentation under anaerobic conditions Synthesis of glucose from simpler compounds: gluconeogenesis Oxidation of glucose in pentose phosphate pathway Key topics:

Four Major Pathways of Glucose Utilization When there’s plenty of excess energy, glucose can be stored in the polymeric form (starch, glycogen) Short-term energy needs are met by oxidation of glucose via glycolysis Pentose phosphate pathway generates NADPH that is used for detoxification, and for the biosynthesis of lipids and nucleotides Structural polysaccharides (e.g. in cell walls of bacteria, fungi, and plants) are derived from glucose

Glycolysis: Importance Glycolysis is a sequence of enzyme-catalyzed reaction by which glucose is converted into pyruvate Pyruvate can be further aerobically oxidized Pyruvate can be used as a precursor in biosynthesis In the process, some of the oxidation free energy in captured by the synthesis of ATP and NADH Research of glycolysis played a large role in the development of modern biochemistry –Understanding the role of coenzymes –Discovery of the pivotal role of ATP –Development of methods for enzyme purification –Inspiration for the next generations of biochemists

Glycolysis: Overview In the evolution of life, glycolysis probably was one of the earliest energy-yielding pathways It developed before photosynthesis, when the atmosphere was still anaerobic Thus, the task upon early organisms was how to extract free energy from glucose anaerobically? The solution –Activate it first by transferring couple of phosphates to it –Collect energy later form the high-energy metabolites of the activated glucose

Glycolysis: The Preparatory Phase

Glycolysis: The Payoff Phase

Glycolytic enzyme 命名 (1) Kinase  與 ATP 水解 or 合成相關之 enzyme (2) Mutase( 轉換 )  Transfer functional group from one position to another in the same molecule, 指～ P 由 C3 位置  C2 位置 (3) Isomerase  指 Aldose Ketose (4) Aldolase  指產物各為 -Aldose 分子及 ketose 分子 (5) Enolase  產生 enol form 分子 -C=C- alcohol ※ Mutase is a subclass of isomerase

The Hexokinase Reaction The first step, phosphorylation of glucose, is catalyzed by hexokinase in eukaryotes, and by glucokinase in prokaryotes Nucleophilic oxygen at C6 of glucose attacks the last (  ) phosphorous of ATP Bound Mg ++ facilitates this process by stabilizing the negative charge in the transition state This process uses the energy of ATP

Phosphohexose Isomerization

The Second Priming Reaction; The First Commitment

ATP is the donor of the second phosphate group This is an irreversible step The product, fructose 1,6-bisphosphate is committed to become pyruvate and yield energy Phosphofructokinase-1 is negatively regulated by ATP –Do not burn glucose if there is plenty of ATP

Aldolases Cleave 6-Carbon Sugars

Triose Phosphate Interconversion Aldolase creates two triose phosphates: DAP and GAP Only GAP is the substrate for the next enzyme DAP is converted enzymatically to GAP

To remind you….. Class schedule Exam Class schedule Exam University of Cambridge University of California, San Francisco

Glyceraldehyde 3-Phosphate Dehydrogenase Reaction First energy-yielding step in glycolysis Oxidation of aldehyde with NAD + gives NADH Phosphorylation yields an high-energy reaction product

First Substrate-Level Phosphorylation 1,3-bisphosphoglycerate is a high-energy compound that can donate the phosphate group to ADP to make ATP The reaction is reversible, the reverse process transfer of phosphate from ATP to phosphoglycerate Kinases are enzymes that transfer phosphate groups from molecules like ATP to various substrates

Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate This is a reversible isomerization reaction Enzymes that shift functional groups around are called mutases

Mechanism of the Phosphoglycerate Mutase Reaction Phosphoglycerate mutase employs covalent catalysis One of the active site histidines is post- translationally modified to phosphohistidine Phosphohistidine donates its phosphate to O2 before retrieving another phosphate from O3

Mechanism of the Phosphoglycerate Mutase Reaction Notice that the phosphate from the substrate ends up bound to the enzyme at the end of the reaction The two negative charges in the product are fairly close now but 2-phosphoglycerate is not good enough phosphate donor

Dehydration of 2-Phosphoglycerate The goal here is to create a better phosphoryl donor Loss of phosphate from 2-phosphoglycerate would merely give a secondary alcohol with no further stabilization … Enolase  產生 enol form 分子 -C=C- alcohol

Second Substrate-Level Phosphorylation … but loss of phosphate from phosphoenolpyruvate yields an enol that tautomerizes into ketone The tautomerization effectively lowers the concentration of the reaction product and drives the reaction toward ATP formation

Pyruvate Kinase is Subject to Regulation Pyruvate kinase requires divalent metals (Mg ++ or Mn ++ ) for activity Under physiological conditions, the activity of pyruvate kinase is limited by the level of Mg ++ When there is plenty of ATP, the Mg ions are sequestered by ATP; this slows down pyruvate kinase Increased concentration of metabolites in the glycolytic pathway slows down glucose utilization

SUMMARY Glycolysis Glucose+2NAD + +2ADP+2Pi  2pyruvate+2NADH+2H + +2ATP+2H 2 O 4e-

Glycolysis Occurs at Elevated Rates in Tumor Cells

BOX 14-1 FIGURE 1 The anaerobic metabolism of glucose in tumor cells yields far less ATP (2 per glucose) than the complete oxidation to CO2 that takes place in healthy cells under aerobic conditions (~30 ATP per glucose), so a tumor cell must consume much more glucose to produce the same amount of ATP. Glucose transporters and most of the glycolytic enzymes are overproduced in tumors. Compounds that inhibit hexokinase, glucose 6-phosphate dehydrogenase, or transketolase block ATP production by glycolysis, thus depriving the cancer cell of energy and killing it.

Feeder Pathways for Glycolysis

MutationSymptoms Galactokinase- deficient (high galactose conc. is found in the blood and urine) Cataracts ( 白內障 ) caused by deposition of galactose metabolite galactitol in the lens Transferase- deficient Poor growth of children, speech abnormality, mental deficiency, and liver damage even when galactose is withheld from the diet (more severe!) Epimerase-deficient Same as above but is less severe when dietary galactose is carefully controlled. Galactosemia

Under Anaerobic Conditions, Animals Reduce Pyruvate to Lactate During strenuous exercise, lactate builds up in the muscle The acidification of muscle prevents its continuous strenuous work The lactate can be transported to liver and converted to glucose there

Under Anaerobic Conditions, Yeast Ferments Glucose to Ethanol Both steps require cofactors – Mg ++ and thiamine pyrophosphate in pyruvate decarboxylase – Zn ++ and NAD + in alcohol dehydrogenase

就一個國家而言，我們終究得到的是哪一種醫師就是我 們自己的報應。 (Kenneth Ludmerer, Time to heal, 1999) University of Cambridge University of California, San Francisco 就一個國家而言，我們終究教出的是哪一種醫師就是我 們自己的報應 ( 福報 ?) 。

Gluconeogenesis: Precursors for Carbohydrates Notice that mammals cannot convert fatty acids to sugars

Glycolysis vs. Gluconeogenesis Glycolysis occurs mainly in the muscle and brain Gluconeogenesis occurs mainly in the liver

The Cory Cycle

Synthesis of Oxaloacetate Conversion of pyruvate to energy-rich phosphoenolpyruvate requires two energy- consuming steps In the first step, pyruvate is transported into mitochondria and converted into oxaloacetate by pyruvate carboxylase

Oxaloacetate Picks Up Phosphate from GTP The phosphoenolpyruvate carboxykinase reaction occurs either in the cytosol or the mitochondria

From Pyruvate to Phosphoenolpyruvate

PEP carboxykinase- Rabbit liver : mitochondria Rat liver : cytosolic Human liver : both

Pentose Phosphate Pathway The main goals are to produce NADPH for anabolic reactions and ribose 5-phosphate for nucleotides

NADPH Regulates Partitioning into Glycolysis vs. Pentose Phosphate Pathway NADPH inhibits glucose-6-phosphate dehydrogenase Whether glucose 6-phosphate enters glycolysis or the pentose phosphate pathway depends on the current needs of the cell and on the concentration of NADP in the cytosol.

蠶豆症

Chapter 14: Summary Glycolysis, a process by which cells can extract a limited amount of energy from glucose under anaerobic conditions Gluconeogenesis, a process by which cells can use a variety of metabolites for the synthesis of glucose Pentose phosphate pathway, a process by which cells can generate reducing power (NADPH) that is needed for the biosynthesis of various compounds In this chapter, we learned about: