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BIOCHEMISTRY COURSE – PHARMACY BIOMEDICAL PREVIEW PROGRAM, SUMMER 2016 Ginika Ezeude, B.A. Biochemistry Barnard College of Columbia University 2nd Year Pharmacy Student Contact: [email protected] METABOLIC PATHWAYS 1. Glycolysis 2. Gluconeogenesis 3. Pentose Phosphate Pathway In animals and vascular plants, glucose has three major fates: 1. It may be stored (as a polysaccharide or as sucrose); 2. Oxidized to a three-carbon compound (pyruvate) via glycolysis to provide ATP and metabolic intermediates; 3. Or oxidized via the pentose phosphate (phosphogluconate) pathway to yield ribose 5-phosphate for nucleic acid synthesis and NADPH for reductive biosynthetic processes 1. GLUCONEOGENESIS - Comparison between Glycolysis & Gluconeogenesis - Regulation of Gluconeogenesis GLUCONEOGENESIS The important precursors of glucose in animals are three-carbon compounds such as lactate, pyruvate, and glycerol, as well as certain amino acids. Organisms need a method for synthesizing glucose from non-carbohydrate precursors; this is accomplished by gluconeogenesis. Cori Cycle CHECK POINT QUESTION What enzyme(s) are used to convert pyruvate to phosphoenolpyruvate in gluconeogenesis? A.) Pyruvate kinase B.) Pyruvate kinase and pyruvate carboxylase C.) Pyruvate Carboxylase and PEP carboxykinase D.) PEP carboxykinase and pyruvate kinase GLUCONEOGENESIS Step 10. The first of the bypass reactions in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvate (PEP). Pyruvate carboxylase, a mitochondrial enzyme, converts the pyruvate to oxaloacetate. Mitochondrial malate dehydrogenase reduces oxaloacetate formed from pyruvate to malate. Malate is then re-oxidized to oxalate. Then mitrochondrial PEP carboxykinase converts Oxalate to PEP. GLUCONEOGENESIS Step. 3 The second glycolytic reaction that cannot participate in gluconeogenesis is the phosphorylation of fructose 6-phosphate by PFK-1. The reaction is catalyzed by a different enzyme, Mg2-dependent fructose 1,6-bisphosphatase (FBPase-1), which promotes the essentially irreversible hydrolysis of the C-1 phosphate. CHECK POINT QUESTION Mitochondrial malate dehydrogenase utilizes which of the following cofactors when converting oxaloacetate to malate? A.) NAD+ B.) NADH C.) FADH2 D.) CO2 GLUCONEOGENESIS Step. 1. The third bypass is the final reaction of gluconeogenesis, the dephosphorylation of glucose 6-phosphate to yield glucose. Reversal of the hexokinase reaction would require phosphoryl group transfer from glucose 6-phosphate to ADP, forming ATP, an energetically unfavorable reaction. The reaction catalyzed by glucose 6-phosphatase does not require synthesis of ATP; it is a simple hydrolysis of a phosphate ester. REGULATION OF GLUCONEOGENESIS Pyruvate can be converted either to acetyl-CoA (by the pyruvate dehydrogenase complex; to fuel the citric acid cycle, or to oxaloacetate (by pyruvate carboxylase) to start the process of gluconeogenesis. 1. Acetyl-CoA is a positive allosteric modulator of pyruvate carboxylase and a negative modulator of pyruvate dehydrogenase. REGULATION OF GLUCONEOGENESIS 2. The second control point in gluconeogenesis is the reaction catalyzed by FBPase-1 which is strongly inhibited by AMP CHECK POINT QUESTION Acetyl CoA stimulates which enzyme in the regulation of gluconeogenesis? A.) pyruvate carboxylase B.) pyruvate kinase C.) pyruvate dehydrogenase complex D.) All of the Above REGULATION OF GLUCONEOGENESIS Fructose 2,6-Bisphosphate Is a Potent Regulator of Glycolysis and Gluconeogenesis The hormonal regulation of glycolysis and gluconeogenesis is mediated by fructose 2,6bisphosphate, an allosteric effector for the enzymes PFK-1 and FBPase-1. Fructose 2,6bisphosphate activates PFK-1 and stimulates glycolysis in liver and, at the same time, inhibits FBPase-1, thereby slowing gluconeogenesis. REGULATION OF FRUCTOSE 2,6-BISPHOSPHATE LEVEL Fructose 2,6-bisphophate is formed by phosphorylation of fructose 6-phosphate, catalyzed by phosphofructokinase- 2 (PFK-2), and is broken down by fructose 2,6bisphosphatase (FBPase-2). CHECK POINT QUESTION Glucagon and Insulin regulate the balance of which enzymes? A.) FBPase-1 and PFK-1 B.) FBPase-2 and PFK-2 C.) PFK-1 and PFK-2 D.) FBPase-1 and FBPase-2 WHEN IS GLUCONEOGENESIS HAPPENING? ● Gluconeogenesis creates glucose from non-carbohydrate precursors ● GNG occurs when there are no sources of sugar o No sugar in the blood o All glycogen (glucose polymer, stored in liver/muscles) has been used up o Body can use waste materials from other cycles/other food metabolism (lipids mostly) ● In fasting or starvation states, all generated glucose is first transported to the brain 2. TCA CYCLE - Products obtained from citric acid cycle PYRUVATE -- NOT JUST LEFTOVERS ● Pyruvate is an important molecule in its own right ● From glycolysis, it is “charged” with CoA (resulting in 1 molecule CO2, 1 NADH and 1 Acetyl-CoA) and moves onto the Citric Acid Cycle, or the Kreb’s Cycle ● This cycle takes AcetylCoA and generates 1 GTP, 3 NADH, 1 FADH2 per Pyruvate ● How many Pyruvates result from Glycolysis? CITRIC ACID CYCLE ● Acetyl-CoA (2C) joined with Oxaloacetate (4C) to make Citrate (6C) o “Citric acid cycle” name is deserved! ● Carbons are swapped around and bonds are oxidized and reduced, until… ● The two extra C’s are chopped off as 2 CO2 ● Another Acetyl-CoA is added, cycle resumes o o Each molecule of glucose produces how many pyruvates? How many times must the TCA turn to process one glucose molecule? CITRIC ACID CYCLE COMPONENTS ARE IMPORTANT BIOSYNTHETIC INTERMEDIATES THINGS TO LEARN FROM TCA ● TCA has 8 steps and plenty of intermediates o these ~8-6 carbon molecules are used for many structures and functions in the body Ex: alpha-ketoglutarate is important for the synthesis and maintenance of amino acid pathway; it is a nitrogen shuttle and supplies aminos for biosynth of AA o However, while it is convenient to snatch pre-made molecules from an ongoing cycle, the cycle still needs to keep enough intermediates to function! Anaplerosis is the process of replenishing TCA cycle intermediates that have been extracted for biosynthesis What does this word look like? (HINT: ANA) o Biochemical pathways MUST BE BALANCED or else someone is going to run out of energy and/or necessary molecules TCA = END OF THE ROAD ● Glucose goes through glycolysis then the end-products, 2 pyruvate, go through the TCA cycle ● What do we get out of this? ENERGETIC YIELD OF GLUCOSE MONOMER ● Glycolysis o 2 ATP o 2 NADH 2*3 = 6 ATP o 2 Pyruvate Citric Acid Cycle ● 2 NADH from 2 transition rxns o 6 ATP ● 6 NADH (2 turns) o 18 ATP ● 2 FADH2 (2 turns) o 4 ATP ● 2 GTP (2 turns) Final Count: ● 8 ATP from glycolysis ● 30 ATP from 2 pyruvates in the citric acid cycle ● 38 ATP from 1 molecule of glucose CITRIC ACID: INS AND OUTS ● What goes in: o o o o o Acetyl-CoA H2O 3NAD+ 1 FAD 1 GDP ● What comes out: ○ ○ ○ ○ 2 CO2 3 NADH 1 FADH2 1 GTP METABOLIC COFACTORS: NOTE ● You’ve seen NAD+/NADH floating around, right? o They carry H’s around to reduce and oxidize molecules for energy o Also carry those electrons/H’s to the OxPhos Pathway, which we’ll talk about next class o FADH2 functions similarly, can “extract” 2 H o However, NAD+ has a cousin… ● NADH is a catabolic (energy-generating) cofactor o Whenever you see NADH in a pathway, you know the body is breaking down molecules for energy ● NADPH is an anabolic (biosynthetic) cofactor o Whenever you see NADPH in a pathway, you know that the body is conducting biosynthesis and some molecule is being made o A good example of an anabolic pathway is the PPP... 3. PENTOSE PHOSPHATE PATHWAY - Oxidative Phase - Non-Oxidative Phase PENTOSE PHOSPHATE PATHWAY Pentose Phosphate Pathway involves the oxidation of glucose 6phosphate to pentose phosphates. In this oxidative pathway, NADP is the electron acceptor, yielding NADPH. OXIDATIVE PHASE OF PENTOSE PHOSPHATES PATHWAY Two molecules of NADPH is generated NON-OXIDATIVE PHASE OF PENTOSE PHOSPHATES PATHWAY In this non-oxidative phase, ribulose 5phosphate is first epimerized to xylulose 5phosphate NON-OXIDATIVE PHASE OF PENTOSE PHOSPHATES PATHWAY CHECK POINT QUESTION How many NADPH’s are generated from the oxidative phase of the pentose phosphate pathway? A.) 1 B.) 2 C.) 4 D.) None, only NADH is generated PRINCIPLES OF METABOLIC REGULATION How do cells to maintain a balance even in the face of outside perturbation? COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS Hexokinase Isozymes of Muscle and Liver Are Affected Differently by Their Product, Glucose 6Phosphate Hexokinase, which catalyzes the entry of free glucose into the glycolytic pathway, is a regulatory enzyme. There are four isozymes (designated I to IV). Hexokinase II has a high affinity for glucose Muscle hexokinases I and II are allosteric ally inhibited by their product, glucose 6phosphate, so whenever the cellular concentration of glucose 6-phosphate rises above its normal level, these isozymes are temporarily and reversibly inhibited, bringing the rate of glucose 6-phosphate formation into balance with the rate of its utilization and re-establishing the steady state. What mechanism does this sound like? COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS Comparison of the kinetic properties of hexokinase IV (glucokinase) and hexokinase I. PHOSPHOFRUCTOKINASE-1 IS UNDER COMPLEX ALLOSTERIC REGULATION The metabolically irreversible reaction catalyzed by PFK-1 is the step that commits glucose to glycolysis. In addition to its substrate-binding sites, this complex enzyme has several regulatory sites at which allosteric activators or inhibitors bind. ATP is not only a substrate for PFK-1 but also an end product of the glycolytic pathway. When high cellular [ATP] signals that ATP is being produced faster than it is being consumed, ATP inhibits PFK-1 by binding to an allosteric site and lowering the affinity of the enzyme for fructose 6-phosphate. PYRUVATE KINASE IS ALLOSTERICALLY INHIBITED BY ATP High concentrations of ATP, acetyl-CoA, and long-chain fatty acids, allosterically inhibit all isozymes of pyruvate kinase and the accumulation of fructose 1,6-bisphosphate triggers its activation. CHECK POINT QUESTION Which of the following is NOT an allosteric inhibitor of Pyruvate Kinase? A.) Long chain fatty acids B.) ATP C.) NADH D.) Acetyl CoA THANK YOU! Next: Enzyme Kinetics