CHAPTER 18 Glycolysis

18-1 What are the essential features of glycolysis? (Outline)

1. A molecule of glucose is converted to two molecules of pyruvate (3C), two ATP and two NADH
2. Essentially all cells carry out glycolysis (ten reactions)
 kinase：磷酸化反應；phosphatase：去磷酸化反應
 mutase：將一官能基從同一分子的 A 位置移到 B 位置
 isomerase：異構酵素，結構轉換為另一異構物

 Phase I phosphorylation of glucose and conversion to 2 molecules of G3P; 2 ATPs are used to
prime these reactions
 Phase II conversion of G3P to pyruvate and coupled formation of 4 ATPs
 two high energy phosphate intermediates：1,3 BPG、PEP
3. The product of glycolysis (pyruvate) is a metabolite that can be used in several ways
4. 3 possible fates for pyruvate：acetyl-coenzyme A (which is then oxidized in the TCA cycle to produce
CO2)、Lactic acid fermentation、Alcoholic fermentation
18-2 Why are coupled reactions important in glycolysis?
1. Coupled reactions convert some of the metabolic energy of glucose into ATP
2. Coupled reactions involving ATP hydrolysis are also used to drive the glycolytic pathway
18-3 What are the chemical principles and features of the first phase pf glycolysis?
1. Reaction 1 ：glucose is phosphorylated by hexokinase or glucokinase (the first priming reaction)
ℎ𝑒𝑥𝑜𝑘𝑖𝑛𝑎𝑠𝑒 + 𝐴𝑇𝑃
 glucose → glucose 6-phosphate (G6P)

 the hexokinase or glucokinase reaction is one of two priming reactions in the pathway
  the glycolysis pathway requires two priming ATP molecules to start the sequence of reactions
 ATP makes the phosphorylation of glucose spontaneous
 the cellular advantage of phosphorylating glucose：葡萄糖原本是中性分子，經由 kinase 作用後
變成帶負電的分子，因此 G6P 就會無法通過細胞膜，而 G6P 會在細胞內馬上被用掉，保持濃度
梯度 → G6P 會抑制 hexokinase

2. Reaction 2 ：phosphoglucoisomerase catalyzes the isomerization of glucose-6-phoaphate

𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑔𝑙𝑢𝑐𝑜𝑖𝑠𝑜𝑚𝑒𝑟𝑎𝑠𝑒
 the isomerization of a sugar：glucose 6-phosphate → fructose 6-phosphate

 the reaction is necessary for two reasons

A. 第三步要磷酸化時會在 C1 位置磷酸化，但是 glucose-6-phosphate 的 C1 是 hemiacetal-OH
的結構較難進行磷酸化，但是轉成 fructose-6-phosphate 則是 OH 的構型，好磷酸化
B. 第四部會進行 cleavage in aldose，會比較容易
 phosphoglucoisomerase proceeds through an enediol intermediate
 moving C1 to C2, create a new C1

3. Reaction 3 ：ATP drives a second phosphorylation by phosphofructokinase (second priming reaction)

𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑓𝑟𝑢𝑐𝑡𝑜𝑘𝑖𝑛𝑎𝑠𝑒(𝑃𝐹𝐾) + 𝐴𝑇𝑃
 fructose 6-phosphate → fructose 1,6-bisphosphate

 the second priming reaction of glycolysis → coupled (by phosphofructokinase) with phosphoryl
transfer from ATP
  PFK is value controlling the rate of glycolysis (with large, negative free energy→highly regulated)
 Phosphofructokinase activity is increased when the energy status falls (ATP decreasing ; AMP
increasing) and is decreased when the energy status is high (ATP increasing ; AMP decreasing)
 citrate is allosteric inhibitor

4. Reaction 4 ：cleavage by fructose biphosphate aldolase creates two 3-carbon intermediates

𝑎𝑙𝑑𝑜𝑠𝑒
 fructose 1,6-bisphosphate → DHAP + G3P

 class I aldolase found in animal (do not require mental ions)

 class II aldolase found in bacterial and fungi (contain active site Zn2+)
 Cyanobacteria and some other simple organisms possess both classes of aldolase
 會進行 aldol cleavage 與 aldol condensation 的雙向反應
5. Reaction 5 ：triose phosphate isomerase completes the first phase of glycolysis
 the two products of the aldolase reaction, only G3P goes into the second phase of glycolysis
𝑡𝑟𝑖𝑜𝑠𝑒 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 𝑖𝑠𝑜𝑚𝑒𝑟𝑎𝑠𝑒
 DHAP→ G3P

 1 glucose has been converted to 2 molecule of G3P

18-4 What are the chemical principles and features of thee second phase of glycolysis?
1. Reaction 6 ：glyceraldehyde-3-phosphate dehydrogenase creates a high-energy intermediate
𝐺3𝑃 𝑑𝑒ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛𝑎𝑠𝑒
 G3P + NAD+ → 1,3-BPG + NADH + H+
 該反應為收益階段的第一個氧化還原反應，讓 G3P 氧化成 1,3-BPG；NAD+還原成 NADH
 dehydrogenase 為一種 oxireductase(氧化還原酵素)
  The reaction mechanism involves nucleophilic attack by a cysteine -SH group on the
 G3P dehydrogenase reaction is the site of action of arsenate (an anion analogous to phosphate)
 Arsenate form 1-arseno-3-phosphoglycerate but acyl arsenates are quite unstable
當砷中毒時，會產生 1-arseno-3-phosphoglycerate (unstable)，但是在下一步的糖解步驟時會導
致 ATP 不能產生的狀況，讓接下來的反應出狀況，導致細胞死亡

2. Reaction 7 ：phosphoglycerate kinase is the break-even reaction

𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑔𝑙𝑦𝑐𝑒𝑟𝑎𝑡𝑒 𝑘𝑖𝑛𝑎𝑠𝑒 (𝑃𝐺𝐾)

 1,3-BPG + ADP → 3-PG + 2*ATP

 It is appropriate to view the sixth and seventh reactions of glycolysis as a coupled pair, with 1,3-
BPG as an intermediate
 substrate-level phosphorylation：為高能量的反應物將 Pi 及能量轉移給 ADP，合成 ATP 的方式
 PGK reaction is sufficiently exogenic at standard state

 2,3-BPG, which stabilizes the deoxy form of hemoglobin and is primarily responsible for the
cooperative nature of oxygen binding by hemoglobin is formed from 1,3-bisphosphoglycerate by
bisphosphoglycerate mutase
 most cells contain only a trace of 2,3-BPG, but erythrocytes contain more 2,3-BPG (allosteric
effector for Hb → bind to Hb promotes the release of oxygen)
 the mutase that forms 2,3-BPG from 1,3-BPG requires 3PG (intermolecular phosphoryl transfer)

3. Reaction 8 ：phosphoglycerate mutase catalyzes a phosphoryl transfer

𝑚𝑢𝑡𝑎𝑠𝑒
 3PG → 2PG
4. Reaction 9 ：dehydration by enolase creates PEP
𝑒𝑛𝑜𝑙𝑎𝑠𝑒
 2PG → phosphoenolpyruvate (PEP)

 makes a high-energy phosphate in preparation for ATP synthesis

 The reaction involves the removal of a water molecule to form the enol structure of PEP
 enolase reaction is rearrange the substrate into a form from which more of this potential energy
can be released upon hydrolysis
 inhibited by fluoride ion in the presence of phosphate
5. Reaction 10：pyruvate kinase yield more ATP
 The second ATP-synthesizing reaction of glycolysis
𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 𝑘𝑖𝑛𝑎𝑠𝑒
 PEP + ADP → pyruvate + ATP

 The reaction requires Mg2+ ion and is stimulated by K+ and certain other monovalent cations.
 Pyruvate kinase possesses allosteric sites for numerous effectors：activated by AMP and F1,6-BP
and inhibited by ATP, acetyl-CoA, alanine, and higher Km for PEP
 liver pyruvate kinase is regulated by covalent modification→glucagon activate cAMP-dependent
protein kinase, which transfers a phosphoryl group from ATP to the enzyme.
 Then PEP is used as a substrate for glucose synthesis in the gluconeogenesis pathway (生理濃度)

 Forming 4 ATP , 2 NADH , 2 pyruvate ; he product of glycolysis is 2 ATP , 2NADH , 2pyruvate in total
18-5 What are the metabolic fates of NADH and pyruvate produced in glycolysis?
1. Aerobic metabolism
 NADH can be recycled by both aerobic and
Under aerobic conditions, pyruvate can be sent into the citric acid cycle, where it is oxidized to
CO2 with the production of additional NADH
Under aerobic conditions, the NADH produced in glycolysis and the citric acid cycle is re-oxidized
to NAD+ in the mitochondrial electron-transport chain
2. Anaerobic Metabolism of Pyruvate Leads to Lactate or Ethanol
 In yeast, it is reduced to ethanol
 fermentation：the production of ATP energy by reaction pathways in which organic molecules
function as donors and acceptors of electrons
 Pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase in an essentially
irreversible reaction
 the reduction of acetaldehyde to ethanol by NADH, is catalyzed by alcohol dehydrogenase
 The end products of alcoholic fermentation are thus ethanol and carbon dioxide
 pyruvate reduction to ethanol in yeast provides a means for regenerating NAD+ consumed in the
G3P dehydrogenase reaction (phase II)

3. Lactate Accumulates Under Anaerobic Conditions in Animal Tissues

 In animal tissues experiencing anaerobic conditions, pyruvate is reduced to lactate
 In oxygen-depleted muscle NAD+ is regenerated in the lactate dehydrogenase (LDH) reaction.
 lactate is recycled to glucose by gluconeogenesis
烏龜在冬眠時的生存方式：利用儲存的肝醣轉成葡萄糖進行糖解作用，形成乳酸，其體液具備很
高的緩衝能力，能忍受大量的乳酸累積及 PH 值降低
4. The Warburg effect and cancer
 Warburg observed in 1924 that rapidly proliferating cancer cells metabolize glucose mainly to
lactate, even when oxygen if plentiful
 this behavior arises because cells need more nucleotides, amino acids, lipids than ATP
 cancer cells divert large amounts of glucose to the pentose phosphate pathway to produce
NADPH for biosynthesis
 cancer cells route up to 90% of acquired glucose and glutamine into lactate and alanine,
producing large amounts of NADPH
  tumor diagnosis using positron emission tomography (PET)：注入氟 18 標定的 glucose，進入血
流後快速被腫瘤細胞利用分解，而後產生正子，trace 後攝影，即可作腫瘤造影
18-6 How do cells regulate glycolysis?
1. 調節的中心原則：
在放能反應中，下游永遠是 Inhibitor，上游永遠是 activator，而 ATP、NADH、FANH2 屬於 inhibitor
在需能反應中，下游永遠是 Inhibitor，上游永遠是 activator，而 ATP、NADH、FANH2 屬於 activator
2. 糖解作用中的不可逆反應：人體可通過葡萄糖新生，即從非糖化合物進行葡萄糖新生時，葡萄糖新
生中的其中七步反應是糖解作用中的逆反應，它們有相同的酵素催化。但是糖解作用中有三步反應，
是不可逆反應。在葡萄糖新生時必須繞過這三步反應，代價是更多的能量消耗
3. glycolysis 的調控常出現在於 one-way 的反應
4. hexokinase, phosphofructokinase and pyruvate kinase reactions all exhibit large negative DG values
under cellular conditions → the sites of glycolytic regulation
5. Inhibition of the three key enzymes by allosteric effectors brings glycolysis to a halt
6. G6P is the branch point for several metabolic pathway

 G6P 一共有 3 個調控路線，經由 isomerase 作用後形成 F6P 進入糖解作用

 G6P 也可由 mutase 作用形成 G1P 形成 glycogen、glucuronate 進行儲存或合成
 G6P 的另一條路線則是經由 pentose phosphate pathway(五炭糖磷酸化)來進行調控
另外 F6P 則可以形成 glucosamine-6-phosphate 進行碳水化合物的合成
7. Hexokinase：
 Hexokinase 具有 4 種 isoenzyme form (I、II、III、IV)
 hexokinase (I、II、III)in most animal, plant, microbial cells → allosteric regulation
A. Km 值小(Kmglucose=0.1mM) ，因此只需要低濃度的 glucose 就能進行反應，快速使用 glucose
B. Mg2+-required Km 值表示需要該濃度才能達到跨速作用的能力
C. Is regulated inhibited by product G6P (葡萄糖進入細胞→活化 Hexokinase→將葡萄糖磷酸
化成 G6P→極性改變，G6P 無法出去而堆積在細胞中→抑制 Hexokinase)
  glucokinase (isoform IV of hexokinase) → hormonal regulation
A. 指出現在肝臟的 hepatocyte(具有 GLUT2 能夠快速使血液中的 glucose 與 cytosol 的含量達
平衡，也提供其調控血糖的功能 inhibited by glucagon ; activated by insulin )
B. Km 值較大(Km glucose=10mM)，因此需要較高的濃度才能快速作用
C. 肝細胞中的[葡萄糖]增加，Glucokinase 就會被活化，將過多的葡萄糖轉變成 G6P，便進行
肝醣合成，即使高 G6P 仍能有效的進行作用，G6P 不會抑制 Glucokinase 的活性
D. 血糖升高會刺激胰島素的活性，而 glucokinase 是由胰島素所誘發的
E. Diabetes mellitus pt. → low insulin, low glucokinase → cannot tolerate high blood [Glc]
→ little glycogen
8. Phosphofructokinase(PFK)：
將 F6P kinase 的酵素，分成 PFK1 與 PFK2，其產物分別是 F1,6-BP 與 F2,6-BP，各有不同的影響。
總觀來說 ATP (-) citrate (-) E.T.C. (-) F-16BP (-)；ADP (+) F-26BP (+)
細胞中存在 PFK-2，其產物 F26BP 會促進 PFK-1 作用； ATP 可抑制 PFK-1
一般來說，當一連串的反應出現例如：A→B→C，則以 A→B 這一個步驟(第一步）最重要，因為
只要這個步驟出現問題，後續的反應都不會發生，但糖解作用最重要的酵素(PFK）不是第一個步
驟，理由有兩個且都與產物相關：
A. hexokinase 的產物是 G6P，G6P 除了參與糖解作用之外還參與 PPP 和肝糖之合成，而 PFK
的產物 F1,6BP 就只存在於糖解作用的路徑中，沒有參與其他反應路徑
B. hexokinase 的產物 G6P 會抑制 hexokinase 的活性，因此 G6P 越多反應的酵素活性越弱，
不利於糖解的進行；而 F1,6BP 則會活化 PFK 的活性，因此產物越多會使糖解反應加快
9. Pyruvate kinase：AMP(+) F1,6-BP(+)；ATP(-) acetyl-CoA (-) alanine (-)

18-7 Are substrates other than glucose used in glycolysis?

1. Fructose, Galactose and mannose can enter the glycolytic pathway
2. Fructose Catabolism in Liver is Unregulated and Potentially Harmful
 in liver, fructose is phosphorylated at C1 by fructokinase
 Subsequent action by fructose-1-phosphate aldolase cleaves fructose-1-P to produce DHAP (
dihydroxyacetone phosphate) and D-glyceraldehyde
 DHAP is of course an intermediate in glycolysis.
 D-glyceraldehyde can be phosphorylated by triose kinase in the presence of ATP to form G3P
 Most of the DHAP and G3P produced from fructose is converted directly to pyruvate and acetyl-
 CoA, and then to fatty acids and triglycerides in an unregulated process that can result in
accumulation of fat in the liver
 The small amounts of dietary fructose that reach the kidneys and muscle can be phosphorylated
by hexokinase to form F6P

3. Mannose Enters Glycolysis in Two Steps

 enters glycolysis at the same point as fructose is mannose
 Mannose is also phosphorylated from ATP by hexokinase, and the mannose-6-phosphate (M6P)
thus produced is converted to fructose-6-phosphate by phosphomannoisomerase

4. Galactose Enters Glycolysis via the Leloir Pathway

 galactose phosphorylated from ATP at the C-1 position by galactokinase
 Galactose-1-phosphate is then converted into UDP-galactose by galactose-1-phosphate
uridylyltransferase, with concurrent production of glucose-1-phosphate and consumption of a
molecule of UDP-glucose
 The uridylyltransferase reaction proceeds via a “ping-pong” mechanism
 G1P produced by the transferase reaction is a substrate for the phosphoglucomutase reaction
which produces glucose-6-phosphate
 UDP-galactose, is converted to UDP-glucose by UDP-glucose-4-epimerase
 galactosemia involves defects in galactose-1-P uridylyltransferase
 A. Toxic levels of galactose accumulate in individuals, causing cataracts ( 白 內 障 )and
neurological disorders
B. prevented by removing galactose and lactose from the diet
 UDP-glucose pyrophosphorylase uses Gal-1-P, reducing galactose toxicity in adults

5. An Enzyme Deficiency Causes Lactose Intolerance

 Lactose intolerance is an inability to digest lactose because of the absence of the enzyme lactase
in the intestines of adults
 can be relieved by eliminating milk from the diet
 Lactose breakdown by lactase in the small intestine provides newborn mammals with essential
galactose for many purposes, including the synthesis of gangliosides in the developing brain
 Lactase is a 𝛽-galactosidase that cleaves lactose to yield galactose and glucose → Low levels of
lactase make many adults lactose intolerant
6. Glycerol Can Also Enter Glycolysis
 Glycerol is the last important simple substance whose ability to enter the glycolytic pathway must
be considered
 This metabolite can be converted to glycerol-3-phosphate by the action of glycerol kinase
 then oxidized to dihydroxyacetone phosphate by the action of glycerol phosphate dehydrogenase
 The dihydroxyacetone phosphate thereby produced enters the glycolytic pathway