ENERGY TRANSFORMATIONS IN LIVIN G MATTER A SURVEY BY H. A. KREBS H. L. KORNBERG AND MEDICAL RESEARCH COUNCIL UNIT FOR RESEARCH IN CELL METABOLISM, DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF OXFORD WITH AN APPENDIX BY K. BURTON WITH 21 FIGURES SPRINGER-VERLAG BERLIN· GOTTINGEN . HEIDELBERG 1957 SONDERABDRUCK AUS ERGEBNISSE DER PHYSIOLOGIE, BIOLOGISCHEN CHEMIE UND EXPERIMENTELLEN PHARMAKOLOGIE NEUNUNDVIERZIGSTER BAND - 1957 ISBN 978-3-540-02189-6 ISBN 978-3-642-86577-0 (eBaak) DOI 10.1007/978-3-642-86577-0 ALLE RECHTE, INSBESONDERE DAS DER UBERSETZUNG IN FREMDE SPRACHEN, VORBEHALTEN OHNE AUSDRUCKLICHE GENEHMIGUNG DES VERLAGES 1ST ES AUCH NICHT GESTATTET, DIESES BUCH ODER TEILE DARAUS AUF PHOTOMECHANISCHEM WEGE (PHOTOKOPIE, MIKROKOPIE) ZU VERVIELFALT IGEN © BY SPRINGER-VERLAG OHG. BERLIN -G{)TTINGEN -HEIDELBERG 1957 Preface This survey was written at the invitation of the Editors of the "Ergebnisse der Physiologie". Its aim is to present the more recent progress in the know ledge of biological energy transformations. Since it was intended for a review journal, the reader was taken to be familiar with the fundamentals of current biochemistry, as described in the standard textbooks. It was not the object to compile an extensive collection of facts. The survey is limited to aspects of wider interest, and the main emphasis has been on the general unifying principles which emerge from the great mass of detailed ob servations. The article is reprinted in the hope that it may be useful in this form to advanced students and research workers in biochemistry and related subjects. H. A. KREBS H. L. KORNBERG Table of Contents2 Page 1. The Key Position of Adenosine Triphosphate . . . 213 2. The Three Phases of Foodstuff Degradation. . . . 213 3. The Energy-Yielding Steps of Intermediary Metabolism 215 4. The Build-up of Phosphate Bond Energy ..... 221 5. Alternative Pathways of Anaerobic Fermentation in Micro-organisms. 227 6. Alternative Pathways of Glucose Oxidation . 237 7. The Path of Carbon in Photosynthesis . . . 243 8. Utilization of Energy for Chemical Syntheses 249 9. Control of Energy-Supplying Processes . . . 262 10. A Special Feature of ATP as an Energy Store. 271 11. Evolution of Energy Transforming Mechanisms 273 Appendix by K. BURTON Free Energy Data of Biological Interest 27S References . . . . . . . . . . . . . . . 285 The last 15 years have witnessed great advances in the analysis of the energy transformations in living organisms. From the vast amount of detailed inform ation which has been amassed, a picture emerges revealing some striking characteristics of the chemical organization of living matter. Although meta bolic processes are both diverse and complex, the number of basic components is relatively small. 2 The following abbreviations have been used in the present survey: AMP = adenosine sf-phosphate; ADP = adenosine diphosphate; ATP = adenosine triphosphate; DPN = diphosphopyridine nucleotide; DPNH2 = reduced diphospho pyridine nucleotide; IDP = inosine diphosphate; ITP = inosine triphosphate; GDP = guanosine diphosphate: GTP = guanosine triphosphate: P = inorganic phosphate; CoA and CoA-SH = coenzyme A; TPN = triphosphopyridine nucleotide; TPNH = 2 reduced triphosphopyridine nucleotide; TPP = thiamine pyrophosphate. The Three Phases of Foodstuff Degradation 213 It has long been established that the innumerable different proteins of living matter are all built up from the same set of about 20 amino acids. It is now evident that this economy of basic units has a counterpart in dynamic biochemistry. Most of the essential machinery for the main chemical processes of living matter is provided by a comparatively small number of enzymes and coenzymes. The same catalysts are used in the different types of fermentations, in cell respiration, and in the reactions by which the constituents of living matter are synthesized. It is true that these basic mechanisms are supplemented by many additional enzymes. This may be taken as obvious in view of the innumerable different forms in which life presents itself. But these obvious species differences which strike the eye have tended to obscure the fact that many basic features are common to many different forms of life. It is the object of this survey to discuss these common features in the sphere of energy transformations. 1. The Key Position of Adenosine Triphosphate The concept is now firmly established (LIPlVIANN 1941) that the chemical energy set free in the breakdown of foodstuffs (unless it generates heat) is transformed into a special kind of chemical energy before it is converted into other forms of energy, such as mechanical work in muscle or osmotic work in secreting glands. This special form of chemical energy is that stored in the pyrophosphate bonds of adenosinetriphosphate (ATP). Living matter shares the requirement of a special fuel with man-made ma chines. In a steam engine, the energy obtained from the combustion of a variety of fuels must be converted into steam pressure before it can do mechani cal work. In an internal combustion engine it is the high pressure which carries out work. An electric motor must be supplied with electrical energy in a specified form to do work. The first major stage, then, of the energy transformations in living matter culminates in the synthesis of pyrophosphate bonds of ATP, at the expense of the free energy of the degradation of foodstuffs. The overall thermodynamic efficiency of this process is estimated at 60~ 70 %. This is high when compared with the efficiency of man-made machines depending on the burning of a fuel as a source of energy. The greater efficiency is possible because living matter is not a heat engine, but a "chemical" engine organised in a special manner. The Three Phases of Foodstuff Degradation 2. The release of energy by the combustion of foodstuffs in living matter may be said to proceed in three major phases (Table 1). In phase I the large molecules of the food are broken down to small con stituent units. Proteins are converted to amino-acids, carbohydrates to hexoses 214 H. A. KREBS and H. L. KORNBERG: Energy Transformations in Living Matter Table 1- The three main phases of energy production from foodstuffs (KREBS 1953b) Phase Outline of chemical change carbohydrates proteins fats I t t t hexoses about 20 amino acids glycerol; fatty acids hexoses glycerol several fatty acids several several several j amino acids amino acids amino acids amino acids (alanine, (3 leucines, (glutamic (aspartic serine, tyrosine, acid, acid, tyrosine. lactic acid cysteine) phenyl- histidine, phenyl- II I ! alanine) prolines, alanine) t I arginine) j pyruvic acid I II I 1 t acetyl coenzyme A IX-ketoglu- oxaloacetic taric acid acid acetyl coenzyme A IX-ketoglutaric acid oxaloacetic acid III I I I t tricarboxylic acid cycle and fats to glycerol and fatty acids. The amounts of energy liberated in the stages of phase I are relatively small. The free energy of hydrolysis of the glucosidic bonds of starch or glycogen is about 4.3 kgcal (BURTON & KREBS 1953), of the peptide bonds of the order of 3.0 kgcal (HUFFMAN 1942) and of an ester of the order of 2.5 kgcal, per mole (see KAPLAN 1951). This means that about 0.6 % of the free energy of polysaccharides and proteins, and about 0.1 % of that of triglyceride fats, is liberated in phase I. These quantities are not utilised except for the generation of heat. The reactions of this phase merely prepare the foodstuffs for the energy-giving processes proper: they oc cur mainly in the intestinal tract and in tissues when reserve material is mo bilised for energy production. In phase II the diversity of small molecules produced in the first phase - three or more different hexoses, glycerol, about twenty amino acids and a number of fatty acids - are incompletely burned, the end-product being, apart from carbon dioxide and water, one of three substances: acetic acid in the form of acetyl coenzyme A, IX-ketoglutarate or oxaloacetate. The first of these three constitutes the greater amount: two-thirds of the carbon of carbohydrate and of glycerol, all carbon atoms of the common fatty acids and approximately half the carbon skeleton of amino acids yield acetyl coenzymeA. IX-Ketoglutaric acid arises from glutamic acid, histidine, arginine, citrulline, ornithine, proline and hydroxyproline; oxaloacetic acid from aspartic acid and, The Energy-Yielding Steps of Intermediary Metabolism 215 through malic acid, from part of the benzene ring of tyrosine and phenyl alanine. The details of the pathways cannot be discussed here in full [see KREBS 1954(a), GREENBERG 1954, LANG 1952, STETTEN 1955, KNOX 1955, VOGEL 1955, COON, ROBINSON & BACHHAWAT 1955]; the main stages are out lined in Table 1. One matter of principle, however, should be emphasized: the number of steps which living matter employs in order to reduce a great variety of different substances to three basic units is astonishingly small; it could certainly not be equalled with the tools at present available to the organic chemist. acetyl coenzyme A The three end-products of the second phase are metabolically 2H~oacelae closely interrelated. They take part in phase III of the energy L-malafe !H0 release: the tricarboxylic acid 2 cycle, the common "terminal" fumarale cis-aconifole pathway of oxidation of all food 2H~ H20~ stuffs. The cycle is shown in succinale iso-cilrate Fig. 1; it sets out how one acetic coenZymeA~H20 -fZH acid equivalent is burned to succinyl coenzyme A oKalosuccinale carbon dioxide and water, a 2H+C02~A / /f series of di- and tri-carboxylic «-ketogluforate acids appearing as intermediate C~ Fig. 1. The tricarboxylic acid cycle stages. Substances which enter the cycle (coenzyme A, HPl after the initial condensation of 1 molecule of acetyl coenzyme A As each step of intermediary and 1 molecule of oxaloacetate are written inside the cycle; substances which arise are written outside. During one turn metabolism requires a specific of the cycle, one acetic acid equivalent is completely enzyme, it is evident that a oxidized. The 4 pairs of H atoms which arise ultimately common pathway of oxidation react with 0, to form 1w9a5t0e,r1 9[f5o4r (faulr]t.h er details see KREBS results in an economy of chemical tools. Surveying the pathway of the degradation of foodstuffs as a whole, one cannot but be impressed by the relative simplicity of the arrangement, in as much as the total number of steps required to release the available energy from a multitude of different substrates is unexpectedly small. 3. The E!lergy -Yielding Steps of Intermediary Metabolism The economy of stages does not, however, end here. Energy is not released at every step of intermediary metabolism. Most, though not all, of the avail able energy contained in the foodstuffs is liberated when the hydrogen atoms removed by dehydrogenations react with molecular oxygen to form water. Such reactions occur in phase II and in phase III. Roughly one-third of the total energy of combustion is set free in phase II and two-thirds in phase III. 216 H. A. KREBS and H. L. KORNBERG: Energy Transformations in Living Matter Table 2. Free energy changes in the breakdown of foodstuffs The figures in brackets refer to the free energy change L1 G in kilocalories per mole reactants at 25°; PH 7.0; 0.2 atm 02; 0.05 atm CO •• concentrations of other reactants at unit actitivity. The pairs of H atoms removed are assumed to be oxidized to HIO by molecular oxygen. Fatty acids are oxidized as acyl coenzyme A derivatives. The value given for the oxidative deamination of amino acids is an average figure varying a little from amino acid to amino acid. For further particulars see BURTON and KREBS (1953). KAPLAN (1951). and Tables 9-13. Glycogen Glucose Fatty acid IX-Amino acid (1 glucose equivalent) 1 +H,O -2H(-36) l+H,O -2H(-43) I (-57.0) (-50.1) + fJ-hydroxyacid IX-ketonic acid NH3 -+ z lactate +- l-ZH(- 50) lactate fJ-ketonic acid I 1-2H (-45.8) pyruvate + coenzyme A CO + coenzyme A (-3.0) z+- -2H(-60) -+ acetyl coenzyme A +- 1 + oxaloacetate (-7.5) citrate 1-H,O (+ 2.04) cis-ac1oni tate + H,O (-0.45) isocitrate Jl'l- COl 2 H (-54.4) IX-ketoglutarate Jl'l CO + H,O - 2H (-70.1) 2 succinate 1-2H(-35.1) fum1arat e + H,O (-0.88) malate 1-2H (-44.3) oxaloacetat e Data on the magnitude of the free energy changes have recently become available and are given in Table 2. Even with a common terminal pathway of oxidation and the shortness of the routes in phase II. the number of stages where utilizable energy is set free is still considerable (see Table 3). There are four such stages in the tricarboxylic acid cycle. In phase II there are at least twenty-nine different oxidative reactions. though more than half of these belong to one type of reaction, the oxidative deamination of amino acids. The Energy-Yielding Steps of Intermediary Metabolism 217 Table 3. Number of different oxidative stages at which energy becomes available "Phase" refers to the classification given in Table 1 ; energy is liberated when mole cular oxygen is the oxidizing agent. Phase I ............ . o Phase II: Carbohydrate -+ acetyl coenzyme A 2 Fatty acids -+ acetyl coenzyme A 2 Glycerol1 -+ acetyl coenzyme A Amino acids: Oxidative deamination of amino acids! (about) 16 Oxidative decarboxylation of corresponding IX-ketonic acids3 0 29 Additional oxidation of phenylalanine and tyrosine leading to malic and acetoacetic acids 4 • • • • • • • • • • • • 4 Oxidation of proline and hydroxyproline to glutamic acid 2 Oxidation of arginine to glutamic acid. . . . . . . . . 2 Conversion of histidine to glutamic acid . . . . . . . . 0 Conversion of carbon chain of leu cines to acetyl coenzyme A 0 Phase III 4 1 Two more stages shared with carbohydrate. 2 A figure lower than the total number of amino acids is given because some amino acids may share one step. e.g. (glutamic acid and histidine. serine. cystine and glycine: see BACH 1952). 3 Assuming the mechanism to be the same as the analogous reaction of pyruvate. 4 See WEINHOUSE and MILLINGTON (1948. 1949). SCHEPARTZ and GURIN (1949). LERNER (1949). KNOX (1955). Their number cannot be precisely stated because the pathway of oxidation of some amino acids (e.g. tryptophan, methionine, lysine) is not yet known in every detail. There are two oxidative stages at which carbohydrate is prepared for entry into the tricarboxylic acid cycle, namely triosephosphate or lactate -+ pyruvate pyruvate -+ acetyl CoA. There are also two oxidative steps in the reactions which convert fatty acids to acetyl coenzyme A, namely fatty acid -+ a-p-unsaturated fatty acid p-hydroxy fatty acid -+ p-keto fatty acid. Glycerol derived from neutral fats, in addition to phosphorylation, requires the further special oxidative stage a-glycerol phosphate -+ triosephosphate before it joins the pathway of carbohydrate degradation. Amino acids, with a few exceptions, require at least one oxidative step each - oxidative deamina tion. In the cases of glutamic acid, aspartic acid, and alanine this one step is sufficient to produce an intermediate also encountered in either carbohydrate 218 H. A. KREBS and H. L. KORNBERG: Energy Transformations in Living Matter breakdown or in the tricarboxylic acid cycle. The exceptional amino acids referred to above, which do not require special oxidative steps, include histi dine, which yields glutamic acid anaerobically, and cysteine and glycine, which are both convertible into serine. In many other cases additional steps are required. Details about the number of energy-releasing steps in phase II of amino acid metabolism are given in Table 3. There are thus some thirty stages at which major parcels of energy are liberated. This is already a relatively small number, considering the variety of starting materials, but by some simple devices the number receives a further drastic reduction. Most of the stages listed in Table 3 are complex reactions involving several distinct steps. In many cases the first of these steps is the interaction of the substrates with pyridine nucleotides, as W ARBURG, CHRISTIAN and GRIESE (1935) first showed: substrate + pyridine nucleotide ~ (3 1) + dehydrogenated substrate reduced pyridine nucleotide ' It follows from the values of the oxido-reduction potentials of the reactants (BALL 1944, BURTON and WILSON 1952, 1953) that no appreciable amounts of energy are set free in this type of reaction, except when at-ketonic acids are oxidized, a case discussed below in more detail. The free energy of the oxi dation of the substrates does not become available when the substrate is oxi dized according to reaction (3, 1), but is set free when the reduced forms of the pyridine nucleotides, through the intermediation of flavoproteins and iron porphyrin enzymes, are reoxidized by molecular oxygen. The main stages of this reaction are the following (BALL 1944): reduced pyridine nucleotide + flavoprotein -+ reduced flavoprotein + (3,2) pyridine nucleotide (standard free energy change - 11 kgcal), + + reduced flavoprotein 2 ferricytochrome -+ flavoprotein 2 ferrocytochrome (3,3) (standard free energy change - 16 kgcal), 2 ferrocytochrome + 1/2 02 -+ 2 ferricytochrome + Hp (3,4) (standard free energy change - 25 kgcal). The over-all effect of the three reactions is the oxidation of reduced pyridine nucleotide: ° + + reduced pyridine nucleotide 1/2 2 -+ pyridine nucleotide H20 (3, 5) (standard free energy change - 52 kgcal). This scheme of three reactions, it should be emphasised, is a provisional and simplified version of the actual events (see HERBERT 1951) because more than one iron porphyrin and more than one flavoprotein can take part in the transfer of hydrogen or electrons, these intermediate carriers probably being arranged in series. The chain of the catalysts may also include substances other than the three main types, such as the factor discovered by SLATER (1949), the chemical identity of which is unknown, as well as vitamins K and E