Metabolism. Glycolysis

Metabolism Part 1: Glycolysis or the EmЬden-Meyerhoff Pathway Metabolism (from the Greek metabolas, changeaЫe) h88 been described 88 the sum total of the chemical reactions carried out Ьу Ьiological systems. It includes processes Ьу which cells: 1) capture energy from their surroundings, 2) convert nutrients into the building Ыocks for synthesis of macromolecules such 88 polysaccharides, proteins, and nu­ cleic acids, 3) synthesize the macromolecules needed for cell growth and replication, and 4) degrade macromolecules to obtain energy or to salvage their building Ыocks for future construction. It is useful to divide metaЬolism into cataЬolic and anabo­ lic sequences of reactions or pathways. Catabolism (from the Greek katabole, throwing down) refers to processes which break down or degrade compounds into smaller, simpler molecules such 88 the lactate ion, ethanol, СО2, NH3, etc. Catabolic pathways are invariaЬly accompanied Ьу а net rele88e of free energy, and one of the goals of metabolism is to capture at least some of this energy in the form of "high energy" compounds such 88 adenosine triphosphate (АТР). Anabolism (from the Greek anabole, rising up) describes sequences of reactions in which incre88ingly more complex molecules are synthesized at the expense of АТР. The adjective "intermediary" is often used to descriЬe metabolism Ьесаuве both catabolic and anaЬolic processes occur in а series of small, discrete steps that pass through а numЬer of intermediates on their way to the end product or products. There are many re88ons why metaЬolism involves long sequences of reactions that gradually modify the structure of а compound. It is much e88ier for an enzyme to catalyze small changes in the structure of а substrate such 88 glucose than it is to 88k the enzyme to Ьind glucose and simulta­ neously oxidize а11 six carЬon atoms. Breaking metabolism into а numЬer of small steps also makes it easier to capture some of the energy given off in catabolic reactions. It allows the cell to synthesize the many intermediates which serve 88 precursors for the Ьiosynthesis of the polysaccharides, pro­ teins, and nucleic acids needed for cell growth. It provides а way of converting fats or proteins into carbohydrates, or vice versa, 88 conditions require. Finally, it provides а number of places where а reaction can Ье either stimulated or inhiblted, and therefore allows the cell to exert finely tuned control over the rate of metaЬolic reactions. Anaerobic (literally: without air) organisms flourish in the absence of oxygen. Тhеу cannot utilize 02 in their metabo­ lism and are often poisoned Ьу its presence. AeroЬic organ­ isms, on the other hand, need 02 to survive. 0rganisms that have the abllity to live under either aeroblc or anaeroblc conditions are called facultative. Тhis paper focuses on а sequence of reactions known as glycolysis or the EmЬden-Meyerhoff pathway. Glycolysis is the principal source of energy for anaeroblc organisms and the first step in the degradation of glucose in aeroblc organ­ isms. Тhis pathway breaks down glucose to either pyruvate, lactate or ethanol and captures part of the energy rele88ed to synthesize АТР from ADP and phosphate. Blochemlcal Energetlcll Adenosine triphosphate (АТР) is the primary source of chemical energy for а seemingly endless variety of Ьiological processes. It fuels processes 88 diverse 88 Ьioluminescence; the transport of ions and molecules across cell membranes; the contraction of muscles; the synthesis of carbohydrates, fats, proteins, and nucleic acids; and Ьoth N2 and СО2 fixa­ tion. Although we са11 АТР а "triphosphate", it is actually an acid anhydride like acetic anhydride. о о 11 11 11 АТР is а mixed anhydride made Ьу condensing adenosine monophosphate (АМР) with а phosphate ion (НРО42- or Н2РО4-, depending on рН) to form adenosine diphosphate (ADP). lf we let the symЬol "Ad" stand for adenosine, this "phosphorylation" reaction can Ье written as follows, о ь о ь Ad-O-i-02- + H-O-t-02- + н+ АМР Ad-O-t-O-f-0з - + Н20 AnaeroЬlc v81'8U8 AeroЬlc Procesиa When you conвider the numЬer of reactions each cell car­ ries out, the number of different cells within even the sim­ plest plants and animals, and the V88t differences between species of plants and animals, the amount of information needed to describe metaЬolism would seem virtually limit­ less. Fortunately, metabolism is simplified Ьу two factors. First, metabolism exhiblts only minor variations within the cells of а species, or even among the myriad cells of different вресiев. Second, metaЬolic processes are coupled through а backЬone of essential reactions that can Ье organized into pathways such 88 "glycolysis", and an understanding of а limited number of these pathways can yield а great deal of information aЬout the entire process of metaЬolism. It iв often useful to distinguish Ьetween anaeroblc, aero-, Ьiс, and facultative organisms when discussing metaЬolism. о 2 СНз-С-ОН - СНз-С-О-t-СНз + Н� acetic acid acetic anhydride ADP Phosphorylation of the diphosphate then gives the triphos­ phate (АТР), ? Ь ? Ь Аd-0-Р-О-Р-ОЗ- ADP + НРО.2- + н+ о о о Ad-O-�-O-i-0-�-04- + Н2О Ь ЬАТР Ь сап Ье written for the productв of these reactionв than for the reactantв, and hydrolysis therefore increases the reso­ nance вtabllization (4). The only way for anaeroblc organiвms to synthesize АТР is to make "super high-energy" phosphates which are even less staЫe to hydrolysis than АТР or ADP. Most of the steps in glycolyвis are therefore designed to build super high­ energy phosphate intermediates such as phosphoenolpyr­ uvate and 1,3-diphosphoglycerate which contain enough en­ ergy to convert ADP to АТР. 4- О О 0 1 1 1 О-Р-О-Р-О-Р-О-СН2 1 о 1 о 1 о о phoвphoenolpyruvate Flgure 1. Slnl<:tw'e of АТР. да 0 ' = -31 kJ/mol но он Enzyme Nomenclature ·whose complete вtructure iв вhown in Figure 1. The key to understanding the role of АТР in metaЬoliвm is appreciating what is meant when АТР is referred to as а "high energy" phosphate. Phosphate esters such as glucose 6-phosphate and anhydrides such as АТР are unstaЫe to hydrolysis. Вefore we сап compare their free energies of hydrolysis, however, it is important to recognize that а strict adherence to the def"шition of standard state causes prob­ lems when dealing with Ьiological systems. Тhе standard state for reactions involving the н + ion would have а н + ion concentration of exactly 1.000 М. Тhis is clearly unrealistic for Ьiological reactions which are much more likely to occur near рН 7. Тhе "standard state" for Ьiochemical reactions is therefore defined as рН 7, and this convention is indicated Ьу using the symЬol да 0 ' instead of да 0 • Тhе difference between да 0 and да 0 ' for а reaction which liЬerates or consumes а single н + ion is 40.0 kJ/mol. The standard free energy of hydrolysis of а typical phos­ phate ester such as glucose 6-phosphate or fructose 6-phos­ phate is rougbly 15 kJ/mol. Тhе free energy of hydrolyвis of АТР to ADP {or ADP to АМР) is twice as large. АТIМ- + Н!О- ADP- + НРО42- + н+ да 01 = -31 kJ/mol АТР and ADP are therefore often referred to as "high­ energy" phosphates Ьecause the energy stored in one of these "high-energy" phosphates is large enough to drive the phosphorylation of а вugar such as glucose or fructose. glucose + АТР - glucose 6-phosphate + ADP да 0 ' + ADP + н + - pyruvate + АТР • -16.7 kJ/mol There are several reasons why the standard free energy of hydrolyвis of either АТР or ADP is unusually large. At а typical phyвiological рН, the polyphosphate backЬone car­ ries а number of negatively charged oxygen atoms, which ·repel each other strongly. Hydrolysis of АТР to ADP and phosphate ion, or ADP to АМР and phosphate, alleviates some of that repulвion (1). Hydrolyвis also increases the total ionic charge, Before discussing the individual steps in glycolyвis it might Ье useful to look at the nomenclature of enzymes. Most enzymes are named Ьу liвting the suЬstrate and then the type of reaction they catalyze. An enzyme that oxidizes alcohols to aldehydes Ьу removing hydrogen, for example, would Ье called an alcohol dehydrogenase. An important class of enzymes for discussions of interme­ diary metaЬoliвm are the kinase enzymes, which catalyze the transfer of а phosphate from АТР to an alcohol to form а phosphate ester or phosphomonoester. АТР о 1 R-O-r-02- о + н+ Тhе phosphorylation of one of the -ОН groups оп glucose to form glucose 6-phosphate, for example, is catalyzed Ьу en­ zymes known as hexokinase and glucokinase. Mutase en­ zymes, on the other hand, catalyze the intemal transfer of а phosphate within а substrate, such as phosphoglyceromu­ tase which convertв 3-phosphoglycerate to 2-phosphoglycer­ ate. An isomerase catalyzes the conversion of one isomer to another. Isomerase enzymes may catalyze reactions that in­ volve oxidation-reduction, such as the conversion of an alde­ hyde {glucose 6-phosphate) into а ketone {fructose 6-phos­ phate), but there is no net change in the oxidation state of the substrate. Dehydrogenase enzymes catalyze reactions in which there is а net change in the oxidation вtate of the subвtrate. Although the reactionв catalyzed Ьу а dehydro­ genase are usually reversiЫe, they are always named in the direction of oxidation. Reduction of acetaldehyde to etha­ nol, for example, is catalyzed Ьу the enzyme alcohol dehy­ drogenase. CIIJcolJ8l8 Glycolysis {literally: glucose dissolution) is а sequence of 10 or 11 enzyme-catalyzed reactions shown in Figures 2 and 3 that degrade а molecule of glucose into two molecules of either pyruvate, lactate or ethanol, with the net synthesis of two moleculeв of АТР. Stsp 1: Phosphorylatlon of Glucosв The first step is an enzyme-catalyzed phosphorylation of glucose at С 6 to form а phosphate ester. glucoвe and therefore the contribution of solvation to the free energy of the productв (2). Тhе free energy associated with the reaction Ьetween the н + ion given off during hydrolysis and the surrounding buffering medium also driveв these reac­ tions to completion (3). Finally, more resonance вtructures ADP R-o-н'J. + АТР � glucose 6-phoвphate + ADP да 0 ' = -17 kJ/mol Although this step consumes а molecule of АТР, it has the advantage of laЬeling the glucose molecule with а higbly charged phosphate group that helps suЬsequent enzymes recognize and bind the appropriate suЬstrates (5). Step 2: lsomвrlzatlon of Glucose 6-Phosphatв Stвp 4: C/eavage of Fructose 1,6-D/phosphatв Glucose 6-phosphate is then converted from an aldehyde (aldose) into а ketone (ketose). phoophopucoioome,ue) fructose 6-phosphate glucose 6-phosphate In the presence of base, aldehydes or ketones with an a­ hydrogen can undergo а reversiЫe reaction known as an aldol condensation. да 0 ' = +2 kJ/mol Тhе equilibrium constant for this reaction slightly favors the starting material (К, = 0.4), but the reaction readily pro­ ceeds to the right due to the relatively large quantities of glucose 6-phosphate which build up in the cell. о о- 11 1 н• СНз-ё-СНз - СНз-С-СНз \,сН.-С-СНз tн.-С-СНз 11 11 о о Step 3: Phosphorylatlon of Fructosв 6-Phosphatв А second molecule of АТР is now consumed to phosphory­ late fructose 6-phosphate at С1 to form fructose 1,6-diphos­ phate. он 1 6н. fructose 6-phosphate + АТР �Ьofrucloldaue) Once again, the free energy stored in а molecule of АТР is used to drive what would otherwise Ье an unfavoraЫe reac­ tion. GLYCOLYSIS FIRST STAGE АТР он GLUCOSE GLYCOLYSIS SECOND STAGE �NАDн,н• О • 11 � у-н -l о1 I,;1�® н с-он cw-o-P-� • "2 1 0 но�н Fructose 1,6-diphosphate can Ье thought of ав the product of an aldol condensation of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. О 11 ОР \,,, .L А 6 о GLUCOSE 6-РНОSРНАТЕ //4) о ®o-t•,:-- GLYCERALDEHYDE 3-РНОSРНАТЕ О 1 F'RUCТOSE 6-РНОSРНАТЕ i 1,3-DIPНOSPНOGLYCERAТE 3-PНOSPНOGLYCERO\'L РНОSРНАТЕ АТР_�О 6�� 1 -� 3-PНOSPНOGLYCERAТE ?-cf=>o н-с-о-Р.-rlЭ 1 \ СJ,\ОН О 2-PНOSPHOGLYCERATE Р �ADP о о ®снLо-СНz С?Н,Р-Р-fРЭ Ь О1 1..:\ y-o-ir--� 0 с1 о н- - 2-н - - � с о 1 о о н �-� � н-с-он о CJ,\-0- ,;-У'АТ �он о11 �� С'1 c-OQ ADP с-о-rчfЭ 11 1 СНz О АТР � � � Ь F'RUCТOSE 1�-DIРНОSРНАТЕ � = � О 9 Р-0� Снр- о 11 да 0 ' = -14.2 kJ/mol fructose 1,6-diphosphate + ADP с;нрн о СНз-С-СН.-ё-СНз ACETALDEHYDE 11 с-н 1 н-с-он о 1 т � снр-�-0"-' GLYCERALDEНYDE DIHYDROXYACEТONE 3-РНОSРНАТЕ Н Е РНО$Р АТ Flgure 2. Re8ction sequence for lhe llrst stage о! glycolysts. LACTATE Flgure З. Rll&Cllon sequence for the seconcl stage о! glyco/ysls. phoephog!ycerate kiDue 1,3-diphosphoglycerate + ADP 3-phosphoglycerate + АТР да 0 ' = -18.8 kJ/mol dihydroxyacetone phosphate о �н 1 -ОН ----- ноЕ�� н6-он н6-он Н ? CH 20POз•­ 6н,ОРОз2- glyceraldehyde 3-phosphate fructose 1,6-diphosphate The enzyme which cleaves fructose 1,6-diphosphate to form а mixture of dihydroxyacetone phosphate and glyceralde­ hyde 3-phosphate is therefore commonly known as "aldol­ ase". fructose 1,6-diphosphate aldolue ) dihydroxyacetone phosphate + glyceraldehyde 3-phosphate да 0 ' = +24.О kJ/mol The equilibrium for this reaction strongly favors the starting material (Кс 6.25 Х 10-6). Тhе reaction is constantly pulled to the right, however, as the products of tbls reaction are consumed in subsequent steps. = Stвp 5: lnterconvвrs/on of Trlosв Phosphatвs Dihydroxyacetone phoвphate and glyceraldehyde 3-phos­ phate are both triose (С3 sugar) phosphateв. Although DHAP cannot enter the second half of the glycolytic path­ way, it can Ье converted into а вecond molecule of glyceral­ dehyde 3-phosphate Ьу an isomerase enzyme. dihydroxyacetone phosphate triooe plюophate glyceraldehyde 3-phosphate .....,""""> да 0 ' = +7.66 kJ/mol The net effect of the first five вtерв of glycolysis is to split а molecule of glucoвe into two moleculeв of glyceraldehyde 3phosphate with no net change in the empirical formula, (СН2О)п, and therefore no net oxidation or reduction. Stвp 6: Oxldatlon of G/ycвraldвhydв 3-Phosphatв Although the primary goal of glycolysis is the production of АТР, the first five steps conвume two moleculeв of АТР. The sixth step sets the stage for а reaction that produces two molecules of АТР, and therefore brings us to the break-even point. In this step, glyceraldehyde is oxidized and then phos­ phorylated to give an acyl phosphate known as either 1,3diphosphoglycerate or 3-phosphoglyceroyl phosphate. glyceraldehyde 3-phosphate + NAD + + HP04"- tnoвe phoophate cleЬyd,opnaи ) 1,3-diphosphoglycerate + NADH + н+ да о , - +6.3 kJ/mol Enzymes do not oxidize or reduce а substrate directly, they only catalyze oxidation or reduction reactions. The oxidizing agent in this reaction is а coenzyme known as nicotinamide adenine dinucleotide (NAD+ ) which Ьinds to the triose phosphate dehydrogenase enzyme and picks up а hydride (Н:-) ion to form the reduced coenzyme NADH. The second hydrogen atom removed from the substrate in this oxidation is released into solution as а н + ion. Stвp 7: Synthвsis of А ТР 1,3-Diphosphoglycerate is а "super high-energy" phos­ phate which transfers а phosphate to ADP to form АТР. It should not Ье surprising that 1,3-diphosphoglycerate has an unusually large free energy of hydrolysis. The repulsion between electrons in the acyl phosphate group at С1 is very similar to the repulsion between electrons in the polyphos­ phate backЬone of АТР that enhances the free energy of hydrolysis of that compound. Furthermore, the 1,3-diphoв­ phoglycerate ion contains two higbly charged phosphate groups in close proximity. The net effect of steps 6 and 7 is to capture in the form of АТР some of the energy released when an aldehyde is oxi­ dized. Because two 1,3-diphosphoglycerate ions are formed for each molecule of glucose consumed, this step generates two molecules of АТР which compensate for the АТР con­ sumed in steps 1 and 3. Stвp В: lsomвrlzatlon of З-Phosphoglycвratв Ву transfeпing а phosphate group from С3 to С2, this step вets the stage for the вyntheвiв of another super high-energy phosphate. 3-phosphoglycerate р),оорЬок)усеnш,u...... 2-phosphoglycerate да 0 ' = +4.44 kJ/mol Stвp 9: Dвhydratlon of 2-Phosphoglycвratв Dehydration of 2-phosphoglycerate yieldв phosphoenol­ pyruvate (РЕР). 2-phosphoglycerate eoolaoe) phosphoenolpyruvate + Н2О да 0 ' = +1.8 kJ/mol Тhis iв one of the most beautiful metaЬolic reactionв. Тhе overall free energy of reaction is negligiЬly вmall, and yet the -18 reaction converts а low-energy phosphate (дG 0 'hyd kJ/mol) into а вuper high-energy phosphate (дG 0 'hyd -61.9 kJ/mol) сараЫе of transforming ADP to АТР. = Stвp 10: Synthвsls of А ТР Keto-enol equilibria lie heavily on the вide of the ketone. Rearrangement of phosphoenolpyruvate to the keto iвomer therefore provides enough driving force to transfer the phoв­ phate group from РЕР to ADP to form АТР and the pyru­ vate ion. phosphoenolpyruvate + ADP pyruvate � pyruvate + АТР да о , = -31 kJ/mol Because two phosphoenolpyruvate ionв are produced for every molecule of glucose that enterв the glycolytic pathway, the net effect of thiв вtер is the вynthesiв of two moleculeв of АТР. One molecule of АТР is consumed in each of steps 1 and 3 of glycolyвiв, and two moleculeв are produced in each of вtерв 7 and 10. The overall energy balance for glycolyвiв is therefore 2 АТР per glucose. Stвp 11: Rвductlon of Pyruvatв One more вtер iв needed to complete the glycolytic path­ way. Oxidation of glyceraldehyde 3-phosphate in step 6 was accompanied Ьу the reduction of NAD + to NADH. NAD+ , like other enzyme cofactors, is present at only low concentra­ tions in the cell. Glycolysis must therefore contain а step in which NADH is oxidized back to NAD + . Aeroblc organiвmв can recycle NADH to NAD + Ьу reduc­ ing oxygen to water. Anaeroblc organisms achieve the ваше goal Ьу reducing the pyruvate ion to either the lactate ion or ethanol. Reduction of pyruvate to lactate occurs in anaero-