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Download Chapter05, 06 代谢引论糖代谢
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教 案 ~ 2007 学年 第一 学期 2006 学 院 教 名 称 研 生命科学学院 室 课 程 名 称 授 课 对 象 授 课 教 师 陈文利 称 副教授 职 教 材 名 称 2006 年 9 月 生物化学 2005 级生物技术专业 现代生物化学 日 授课题目(教学章、节或主题) : 教学器材 与工具 第四、五章 代谢引论 糖代谢 授课时间 多媒体设施、黑板与 笔 第 8,9 周一第 21-26 节 教学目的、要求(例如识记、理解、简单应用、综合应用等层次) : 掌握糖 酵 解 途 径 ,三 羧 酸 循 环 ,掌握戊 糖 磷 酸 途 径 及 糖 异 生 途 径 教学内容(包括基本内容、重点、难点) : 第四章 代谢引论 第一节 通论 中间代谢 第二节 新陈代谢研究方法 第五章 糖与糖代谢 (carbohydrate metabolism) Overview of Glycolysis The Embden-Meyerhof (Warburg) Pathway Essentially all cells carry out glycolysis Cellular location: cytosol Ten reactions - same in all cells - but rates differ Two phases: – 1st phase- investing phase: glucose →F-1,6-2P →2G-3-P – 2nd phase-harvesting phase: produces two pyruvates, ATPs and NADH Three possible fates for pyruvate Glycolysis A. Energy Investment Phase: Glycolysis B. Energy Yielding Phase First Phase of Glycolysis The first reaction - phosphorylation of glucose Hexokinase or glucokinase This is a priming reaction - ATP is consumed here in order to get more later ATP makes the phosphorylation of glucose spontaneous Hexokinase 1st step in glycolysis; G large, negative Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell Km for glucose is 0.1 mM; cell has 4 mM glucose So hexokinase is normally active! Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in glucose Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P Glycolysis - Second Phase Metabolic energy produces 4 ATP Net ATP yield for glycolysis is two ATP Second phase involves two very high energy phosphate intermediates – 1,3 BPG – Phosphoenolpyruvate Glycolysis - Second Phase Metabolic energy produces 4 ATP Net ATP yield for glycolysis is two ATP Second phase involves two very high energy phosphate intermediates – 1,3 BPG – Phosphoenolpyruvate Substrate-Level Phosphorylation ATP is formed when an enzyme transfers a phosphate group from a substrate to ADP. Rx 8: Phosphoglycerate Mutase Phosphoryl group from C-3 to C-2 Rationale for this enzyme - repositions the phosphate to make PEP Note the phospho-histidine intermediates! Zelda Rose showed that a bit of 2,3-BPG is required to phosphorylate His Rx 9: Enolase 2-P-Gly to PEP How can such a reaction create a PEP? "Energy content" of 2-PG and PEP are similar Enolase just rearranges to a form from which more energy can be released in hydrolysis Rx 10: Pyruvate Kinase PEP to Pyruvate makes ATP These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis Large, negative G - regulation! The Fate of NADH and Pyruvate Aerobic or anaerobic?? NADH is energy - two possible fates: If O2 is available, NADH enters into Mitochondria by two ways, where it is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation. – In anaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis Significance of Glycolysis Produce ATPs Provide biosynthetic materials Glycolysis and Cancer Hypoxia and hyoxia-inducible factor The TCA Cycle A common metabolic pathway for glucose, aa and fatty acid aka Citric Acid Cycle, Krebs Cycle Pyruvate (actually acetate) from glycolysis is degraded to CO2 Some ATP is produced More NADH is made NADH goes on to make more ATP in electron transport and oxidative phosphorylation Entry into the TCA Cycle Pyruvate Dehydrogenase Complex Pyruvate is oxidatively decarboxylated to form acetyl-CoA Pyruvate dehydrogenase uses TPP, CoASH, lipoic acid, FAD and NAD+ Pyruvate dehydrogenase (E1) Dihydrolipoamide transacetylase (E2) Dihydrolipoamide dehydrogenase (E3) Citrate Synthase Formation of citrate Another example for the induced fit model OAA, the first substrate to bind to the enzyme, induce a large conformational change, creating a binding site for the second substrate, acetyl-CoA. When citroyl-CoA forms on the enzyme surface, another conformational change brings the side of a crucial Asp residue into position to cleavage the thioester. This mechanism decreases the likelihood of premature and unproductive cleavage of the thioester bond of acetyl-CoA Aconitase Isomerization of Citrate to Isocitrate Citrate is a poor substrate for oxidation So aconitase isomerizes citrate to yield isocitrate which has a secondary -OH, which can be oxidized Note the stereochemistry of the Rxn: aconitase removes the pro-R H of the pro-R arm of citrate! Aconitase uses an iron-sulfur cluster Succinyl-CoA Synthetase A substrate-level phosphorylation A nucleoside triphosphate is made Its synthesis is driven by hydrolysis of a CoA ester The mechanism involves a phosphohistidine Succinate Dehydrogenase An oxidation involving FAD This enzyme is actually part of the electron transport pathway in the inner mitochondrial membrane The electrons transferred from succinate to FAD (to form FADH2) are passed directly to ubiquinone (UQ) in the electron transport pathway Fumarase Hydration across the double bond trans-addition of the elements of water across the double bond The actual mechanism is not known for certain Malate Dehydrogenase An NAD+-dependent oxidation The carbon that gets oxidized is the one that received the -OH in the previous reaction This reaction is energetically expensive o' = +30 kJ/mol This and the previous two reactions form a reaction triad that we will see over and over! The Fate of Carbon in TCA Carboxyl C of acetate turns to CO2 only in the second turn of the cycle (following entry of acetate) Methyl C of acetate survives two cycles completely, but half of what's left exits the cycle on each turn after that. Function of the TCA Cycle Produces More ATPs As a source of biosynthetic precursors A common final metabolic pathway for glucose, aas and fatty acids Some Immediates act as effectors to regulate other metabolic pathways Produces CO2 The Glyoxylate Cycle A variant of TCA for plants and bacteria Acetate-based growth - net synthesis of carbohydrates and other intermediates from acetate is not possible with TCA Glyoxylate cycle offers a solution for plants and some bacteria and algae The CO2-evolving steps are bypassed and an extra acetate is utilized Isocitrate lyase and malate synthase are the short-circuiting enzymes Glyoxylate Cycle II Isocitrate lyase produces glyoxylate and succinate Malate synthase does a Claisen condensation of acetyl-CoA and the aldehyde group of glyoxylate - classic CoA chemistry! The glyoxylate cycle helps plants grow in the dark! Glyoxysomes borrow three reactions from mitochondria: succinate to oxaloacetate Pentose Phosphate Pathway Aka: Pentose shunt Hexose monophosphate shunt Phosphogluconate pathway It occurs in the cytosol. Two oxidative processes followed by five non-oxidative steps Operates active in the cytosol of liver and adipose cells Pentose Phosphate Pathway Cells need a constant supply of energy NADH, NADPH and ATP ATP - energy currency NADPH - reducing power Glucose --> NADH --> ATP Glucose --> NADPH --> biosynthesis (reductive) Oxidative Phase Glucose-6-P Dehydrogenase – Irreversible 1st step - highly regulated (inhibited by NADPH) Gluconolactonase – Uncatalyzed reaction happens too 6-Phosphogluconate Dehydrogenase – An oxidative decarboxylation The Nonoxidative Phase Transketolase transfer of two-carbon units Transaldolase transfers a three-carbon unit The use of NADPH Biosynthesis – fatty acids – photosynthesis – DNA synthesis Redox regulation of cellular processes – control of cellular redox state – transcription – disulfide bond formation – antioxidant defense Gluconeogenesis Net Synthesis of "new glucose" from non-sugar metabolites Substrates for Gluconeogenesis Pyruvate, lactate, glycerol, amino acids and all TCA intermediates can be utilized Even-number Fatty acids cannot! Most fatty acids yield only acetyl-CoA Acetyl-CoA (through TCA cycle) cannot provide for net synthesis of sugars Gluconeogenesis I Occurs mainly in liver and kidneys Not the mere reversal of glycolysis for 2 reasons: – Energetics must change to make gluconeogenesis favorable ( G of glycolysis = -74 kJ/mol ) – Reciprocal regulation must turn one on and the other off - this requires something new! Gluconeogenesis II Something Borrowed, Something New Seven steps of glycolysis are retained: – Steps 2 and 4-9 Three steps are replaced: – Steps 1, 3, and 10 (the regulated steps!) sugar synthesis), and they provide new mechanisms of regulation Fructose-1,6-bisphosphatase Hydrolysis of F-1,6-P to F-6-P Thermodynamically favorable -8.6 kJ/mol Glucose-6-Phosphatase Conversion of Glucose-6-P to Glucose Presence of G-6-Pase in ER of liver and kidney cells makes gluconeogenesis possible Muscle and brain do not do gluconeogenesis G-6-P is hydrolyzed as it passes into the ER Glycogen Metabolism Glycogen Breakdown Glycogen Synthesis Glycogen Storage Diseases Why use glycogen for energy storage? Muscles cannot mobilize fat as rapidly as glycogen. Fatty acid residues of fat cannot be metabolized anaerobically. Animals cannot convert fatty acids to Glc, so fat metabolism alone cannot adequately maintain essential blood Glc levels. Glycogen Breakdown Requires Three Enzymes A B. Glycogen debranching enzyme (1->6) glucosidase activities -> Glc-1-P and Glc C. Phosphoglucomutase to convert to usable formG Glycogen Synthesis Glucose must be activated into UDP-Glc Notice: ADP-Glc for Starch; GDP-Glc or UDP-Glc for Cellulose The primer is required Glycogenin Proceed from the reducing end to the non-reducing end Glycogen synthase and Glycogen branching enzyme 重点:重点介绍糖酵解 三羧酸循环 糖异生作用途径及简单的能量计算 难点:记忆这些代谢途径及调节 教学过程设计(要求阐明对教学基本内容的展开及教学方法与手段的应用、讨论、作业布置): 利用课件结合板书介绍糖代谢的主要代谢途径,了解糖代谢的的研究新进展,重点掌握糖酵 解 三羧酸循环 糖异生作用,引导学生记忆方法,在理解的基础上布置作业,让学生在作业中 发现问题提出问题,对于比较难理解的老师在课堂上再次强调。在教学过程中给学生介绍学习方 法及鼓励学生拓展知识。