1UT:T1uJ,JIIE,1
`
`_!0EM;’-i
`Principles of
`Biochemistry
`
`David L. Nelson
`Michael M. Cox
`
`Includes CD-ROM
`Understand Biochemistry
`
`BUTAMAX 1026
`
`

`
`THIRD EDITION
`
`etiiili;ipilus Ui (cid:9)
`
`iuriefflistry
`
`Professor of Biochemistry
`University of Wisconsin(cid:151)Madison
`
`Professor of Biochemistry
`University of Wisconsin(cid:151)Madison
`
`WORTH PUBLISHERS
`
`

`
`Lehuinger Principles of Biochemistry Third Edition
`
`David L. Nelson and Michael M. Cox
`
`Copyright ' 2000, 1993, 1982 by Worth Publishers
`
`All rights reserved
`Printed in the United States of America
`
`Library of Congress Cataloging-in-Publication Data
`
`Nelson, David L.
`Lehninger principles of biochemistry / David L. Nelson, Michael M. Cox.(cid:151) 3rd ed.
`p. cm.
`Includes index.
`ISBN 1-57259-153-6
`1. Biochemistry. I. Nelson, David L. (David Lee), 1942- II. Cox, Michael M. III. Title.
`QD415 .L44 2000
`572(cid:151)dc2i
`
`99-049137
`
`Printing: 5 4 3 2 (cid:9)
`
`Year: 04 03 02 01 00
`
`Development Editor: Morgan Ryan, with Linda Strange and Valerie Neal
`Project Editor: Elizabeth Geller
`Art Director: Barbara Rusin
`Design: Paul Lacy
`Production Supervisor: Bernadine Richey
`Layout: York Graphic Services and Paul Lacy
`Photo Editor: Deborah Goodsite
`Illustrations: Susan Tilberry (with Alan Landau and Joan Waites), J.B. Woolsey & Associates,
`Laura Pardi Duprey, and York Graphic Services
`Molecular Graphics: Jean-Yves Sgro
`Composition: York Graphic Services
`Printing and Binding: R.R. Donnelley and Sons
`
`Cover (from top to bottom): Cut-away view of GroEL, a protein complex in-
`volved in protein folding; cut-away view of tobacco mosaic virus, an RNA virus;
`ribbon model of a 0-barrel structural domain from UDP N-acetylglucosamine
`acyltransferase; cut-away view of the F 1 subunit of ATP synthase, with bound
`ATP shown as a stick structure; mesh surface image of the electron-transfer
`protein cytochrome c, with its heme group shown as a stick structure.
`Cover images created by Jean-Yves Sgro.
`
`Illustration credits begin on p. IC-1 and constitute a continuation of the
`copyright page.
`
`Worth Publishers
`41 Madison Avenue
`New York, NY 10010
`
`(cid:9)
`

`
`Part lii Bioenergetics and Metabolism
`
`15. Rates of Turnover of y and 13 Phosphates of
`ATP If a small amount of ATP labeled with radioactive
`phosphorus in the terminal position, [y- 32PJATP, is
`added to a yeast extract, about half of the 32P activity
`is found in P 1 within a few minutes, but the concentra-
`tion of ATP remains unchanged. Explain. If the same
`experiment is carried out using ATP labeled with
`32P in
`the central position, [13-32P]ATP, the 32P does not ap-
`pear in P1 within such a short time. Why?
`16. Cleavage of ATP to AMP and PP 1 during (cid:9)
`Metabolism The synthesis of the activated form of (cid:9)
`acetate (acetyl-00A) is carried out in an ATP-dependent (cid:9)
`process:
`
`Acetate + CoA + ATP -* acetyl-CoA + AMP + PP
`(a) The G’(cid:176) for the hydrolysis of acetyl-CoA to
`acetate and CoA is -32.2 kJ/mol and that for hydroly-
`sis of ATP to AMP and PP, is -30.5 kJ/mol. Calculate
`G’(cid:176) for the ATP-dependent synthesis of acetyl-CoA.
`(b) Almost all cells contain the enzyme inorganic
`pyrophosphatase, which catalyzes the hydrolysis of
`PP1 to P1. What effect does the presence of this enzyme
`have on the synthesis of acetyl-CoA? Explain.
`17. Energy for H Pumping The parietal cells of the
`stomach lining contain membrane "pumps" that trans-
`port hydrogen ions from the cytosol of these cells
`(pH 7.0) into the stomach, contributing to the acidity
`of gastric juice (pH 1.0). Calculate the free energy re-
`quired to transport 1 mol of hydrogen ions through
`these pumps. (Hint: See Chapter 13.) Assume a tem-
`perature of 25 (cid:176)C.
`18. Standard Reduction Potentials The standard
`reduction potential, E’(cid:176), of any redox pair is defined
`for the half-cell reaction:
`Oxidizing agent + n electrons -p reducing agent
`The E’(cid:176) values for the NAD/NADH and pyruvate/
`lactate conjugate redox pairs are -0.32 and -0.19 V,
`respectively.
`(a) Which conjugate pair has the greater tendency
`to lose electrons? Explain.
`(b) Which is the stronger oxidizing agent? Explain.
`(c) Beginning with 1 M concentrations of each re-
`actant and product at pH 7, in which direction will the
`following reaction proceed?
`
`Pyruvate + NADH + H (cid:9)
`lactate + NAD
`(d) What is the standard free-energy change
`(G’(cid:176)) at 25(cid:176)C for the conversion of pyruvate to lactate?
`(e) What is the equilibrium constant (K,) for this
`reaction?
`
`19. Energy Span of the Respiratory Chain Ele c-
`tron transfer in the mitochondrial respiratory chain
`may be represented by the net reaction equation
`NADH + H + 02 (cid:9)
`H20 + NAD
`(a) Calculate the value of E’(cid:176) for the net reaction
`of mitochondrial electron transfer.
`
`(b) Calculate G’(cid:176) for this reaction.
`(c) How many ATP molecules can theoretically
`be generated by this reaction if the free energy of ATP
`synthesis under cellular conditions is 52 kJ/mol?
`
`20. Dependence of Electromotive Force on Co n-
`centrations Calculate the electromotive force (in
`volts) registered by an electrode immersed in a solu-
`tion containing the following mixtures of NAD and
`NADH at pH 7.0 and 25 (cid:176)C, with reference to a half-cell
`of E’(cid:176) 0.00 V.
`(a) 1.0 mM NAD and 10 mm NADH
`(b) 1.0 mm NAD and 1.0 mivi NADH
`(c) 10 mm NAD and 1.0 mm NADH
`
`21. Electron Affinity of Compounds List the fol-
`lowing substances in order of increasing tendency to
`accept electrons: (a) a-ketoglutarate + CO 2 (yielding
`isocitrate); (b) oxaloacetate; (c) 02; (d) NADP.
`
`22. Direction of Oxidation-Reduction Reactions
`Which of the following reactions would you expect to
`proceed in the direction shown under standard condi-
`tions, assuming that the appropriate enzymes are pre-
`sent to catalyze them?
`(a) Malate + NAD -f
`oxaloacetate + NADH + H
`(b) Acetoacetate + NADH + H -
`fi-hydroxybutyrate + NAD
`(c) Pyruvate + NADH + H -
`
`lactate + NAD
`(d) Pyruvate + 0-hydroxybutyrate -p
`lactate + acetoacetate
`(e) Malate + pyruvate -* oxaloacetate + lactate
`(f) Acetaldehyde + succinate
`
`ethanol + fumarate
`
`fIycoIysis and the
`ata bo Ii sm of Hexoses
`
`n-Glucose is the major fuel of most organisms and occupies a central posi-
`tion in metabolism. It is relatively rich in potential energy; the complete
`oxidation of glucose to carbon dioxide and water proceeds with a standard
`free-energy change of -2,840 kJ/mol. By storing glucose as a high molecu-
`lar weight polymer such as starch or glycogen, a cell can stockpile large
`quantities of hexose units while maintaining a relatively low cytosolic os-
`molarity. When energy demands suddenly increase, glucose can be released
`quickly from these intracellular storage polymers and used to produce ATP
`either aerobically or anaerobically.
`Glucose is not only an excellent fuel, it is also a remarkably versatile
`precursor, capable of supplying a huge array of metabolic intermediates for
`biosynthetic reactions. A bacterium such as Escherichia coli can obtain
`from glucose the carbon skeletons for every amino acid, nucleotide, co-
`enzyme, fatty acid, or other metabolic intermediate needed for growth. A
`comprehensive study of the metabolic fates of glucose would encompass
`hundreds or thousands of transformations. In higher plants and animals,
`glucose has three major fates: it may be stored (as a polysaccharide or as
`sucrose), oxidized to a three-carbon compound (pyruvate) via glycolysis, or
`oxidized to pentoses via the pentose phosphate (phosphogluconate) path-
`way (Fig. 15-1).
`This chapter describes the individual reactions of glycolysis and the en-
`zymes that catalyze them; fermentation, the operation of glycolysis under
`anaerobic conditions; and the pathways that produce the starting material
`for glycolysis from rionglucose hexoses, disaccharides, and polysaccharides.
`Using glycolysis as an example of a metabolic pathway under tight regula-
`tion, we discuss the general principles of metabolic control. We conclude
`with a brief description of the catabolic pathway that leads to pentoses.
`
`i yc Uy S
`In glycolysis (from the Greek glykps, meaning "sweet," and lpsis, meaning
`"splitting") a molecule of glucose is degraded in a series of enzyme-
`catalyzed reactions to yield two molecules of the three-carbon compound
`pyruvate. During the sequential reactions of glycolysis, some of the free en-
`ergy released from glucose is conserved in the form of ATP and NADH. Gly-
`colysis was the first metabolic pathway to be elucidated and is probably the
`best understood. From the discovery by Eduard Btichner (in 1897) of fer-
`mentation in broken extracts of yeast cells until the clear recognition by
`Fritz Lipmann and Herman Kalckar (in 1941) of the metabolic role of high-
`energy compounds such as ATP, the reactions of glycolysis in extracts of
`Yeast and muscle were central to biochemical research. The development of
`
`Glycogen,
`starch, sucrose
`
`storage
`
`i cose
`
`oxidation via
`pentose phosphate (cid:9)
`pathway (cid:9)
`
`oxidation via
`glycolysis
`
`Ribose 5-phosphate (cid:9)
`
`Pyruvate
`
`1 -1
`Major pathways of glucose utilization in cells of higher
`plants and animals. Although not the only possible fates
`for glucose, these three pathways are the most significant
`in terms of the amount of glucose that flows through
`them in most cells.
`
`Fritz Lipmann
`1899-1986
`
`Herman Kalckar
`1908-1991
`
`527
`
`

`
`528 (cid:9)
`
`Part I II Bioenergetics and Metabolism
`
`methods of enzyme purification, the discovery and recognition of the j
`portance of coenzymes such as NAD, and the discovery of the pivotal meta
`bolic role of ATP and other phosphorylated compounds all came out
`studies of glycolysis. The glycolytic enzymes of many species have lon g
`since been purified and thoroughly studied.
`Glycolysis is an almost universal central pathway of glucose catabolism
`the pathway with the largest flux of carbon in most cells. The glycolyq
`breakdown of glucose is the sole source of metabolic energy in some mam
`malian tissues and cell types (erythrocytes, renal medulla, brain, and
`sperm, for example). Some plant tissues that are modified to store starch
`(such as potato tubers) and some aquatic plants (watercress, for example)
`derive most of their energy from glycolysis; many anaerobic microorga n
`isms are entirely dependent on glycolysis.
`Fermentation is a general term for the anaerobic degradation of glu-
`cose or other organic nutrients to obtain energy, conserved as ATP. Because
`living organisms first arose in an atmosphere without oxygen, anaerobic
`breakdown of glucose is probably the most ancient biological mechanism for
`obtaining energy from organic fuel molecules. In the course of evolution,
`the chemistry of this reaction sequence has been completely conserved; the
`glycolytic enzymes of vertebrates are closely similar, in amino acid se-
`quence and three-dimensional structure, to their homologs in yeast and
`spinach. Glycolysis differs among species only in the details of its regulation
`and in the subsequent metabolic fate of the pyruvate formed. The thermo-
`dynamic principles and the types of regulatory mechanisms in glycolysis are
`common to all pathways of cell metabolism. A study of glycolysis can there-
`fore serve as a model for many aspects of the pathways discussed later in
`this book.
`Before examining each step of the pathway in some detail, we will take
`a look at glycolysis as a whole.
`
`An Overview: Glycolysis Has Two Phases
`The breakdown of the six-carbon glucose into two molecules of the three-
`carbon pyruvate occurs in ten steps, the first five of which constitute the
`preparatory phase (Fig. 15-2a). In these reactions, glucose is first phos-
`phorylated at the hydroxyl group on C-6 (step ). The D-glucose 6-phos-
`phate thus formed is converted to n-fructose 6-phosphate (step ') which
`is again phosphorylated, this time at C-i, to yield D-fructose 1,6-bisphos-
`phate (step Q. For both phosphorylations, ATP is the phosphoryl group
`donor. As all sugar derivatives in the glycolytic pathway are the n isomers,
`we will omit the D designation except when emphasizing stereochemistry.
`Fructose 1,6-bisphosphate is next split to yield two three-carbon
`molecules, dilhydroxyacetone phosphate and glyceraldehyde 3-phosphate
`(step '); this is the "lysis" step that gives the pathway its name. The di-
`hydroxyacetone phosphate is isomerized to a second molecule of glycer-
`aldehyde 3-phosphate (step fi), ending the first phase of glycolysis. Note
`that two molecules of ATP must be invested to activate the glucose mole
`cule for its cleavage into two three-carbon pieces; later there will be a good
`return on this investment. To sum up: in the preparatory phase of glycolY
`sis the energy of ATP is invested, raising the free-energy content of the in-
`termediates, and the carbon chains of all the metabolized hexoses are con -
`verted into a common product, glyceraldehyde 3-phosphate.
`The energy gain comes in the payoff phase of glycolysis (Fig. 15-2b)
`Each molecule of glyceraldehyde 3-phosphate is oxidized and ph osphOry
`lated by inorganic phosphate (not by ATP) to form 1 ,3bisphosphoglycerate
`(step (D). Energy is then released as the two molecules of 1,3-bisphosph 0
`glycerate are converted to two molecules of pyruvate (steps ' through
`
`

`
`Chapter 15 Glycolysis and the Catabolism of Hexoses
`
`529
`
`ure 15-2
`The two phases of glycolysis. For each
`molecule of glucose that passes through
`the preparatory phase (a), two molecules
`of glyceraldehy 3-phosphate are formed;
`both pass through the payoff phase (b).
`pyruvate is the end product of the second
`phase of glycolysis. For each glucose mol-
`ecule, two AIR are consumed in the
`preparatory phase and four ATP are pro-
`duced in the payoff phase, giving a net
`yield of two AIR per molecule of glucose
`converted to pyruvate. The number beside
`each reaction step corresponds to its num-
`bered heading in the text discussion. Keep
`in mind that each phosphoryl group, rep-
`resented here as fi, has two negative
`c harges ( (cid:151) POr).
`
`(a) (cid:9)
`
`Glucose
`
`first (cid:9)
`priming
`reaction
`
`j) (cid:9)
`
`ATP
`
`APP
`
`Glucose 6-phosphate
`
`Fructose 6-phosphate
`
`second (cid:9)
`priming (cid:9)
`reaction (cid:9)
`
`(,3
`
`ATP
`
`ADP
`
`HO(cid:151)CH 2
`5,O
`
`\II
`OH H
`HO H
`
`O
`
`Preparatory phase
`
`Phosphorylation of glucose
`and its conversion to
`glyceraldehyde 3-phosphate
`
`OH
`
`0
`
`H
`
`HO (cid:9)
`
`OH H
`>10
`H
`
`H OH
`fi (cid:151) O (cid:151) CHO CH,(cid:151)OH
`
`H HO
`
`H (cid:9)
`
`OH
`
`OH H
`
`Fructose 1,6-bisphosphate
`
`fi - O (cid:151) CH>O C.H2 (cid:151) O ---fi
`
`cleavage
`of 6-carbon
`sugar
`phosphate to
`two 3-carbon
`sugar
`phosphates
`
`Glyceraldehyde 3-phosphate
`+
`Dihydroxyacetone phosphate
`
`I U15
`
`(b)
`
`Glyceraldehyde 3-phosphate (2)
`2P1
`2NAD
`
`oxidation and (cid:9)
`phosphorylation (cid:9)
`
`2 NADH + H
`
`1,3-Bisphosphoglycerate (2)
`
`first ATP-
`
`forming reaction D (cid:9)
`
`(substrate-level
`phosphorylation
`
`2ADP
`
`2 ATP
`
`3-Phosphoglycerate (2)
`
`2-Phosphoglycerate (2)
`
`fi1L
`fi I H 21120
`
`Phosphoenolpyruvate (2)
`2APP
`
`second ATP-
`forming reaction
`(substrate-level
`phosphorylation)
`
`2 ATP
`
`Pyruvate (2)
`
`KH HO)
`H (cid:9)
`OH
`
`OH H
`
`OH H
`
`fi - 0(cid:151) CH2 ---(cid:151) CH20H
`0
`
`Payoff phase
`
`fi(cid:151)O(cid:151)CHC(cid:151)CH(cid:151)C
`
`0 (cid:9)
`
`OH H (cid:9)
`
`Oxidative conversion of
`glyceraldehyde 3-phosphate to
`pyruvate and the coupled
`formation of ATP and NADH
`
`0
`fi (cid:151) O (cid:151) CHC (cid:151) CH-- C\
`H Ofi
`
`0
`fi_ O_CHC_?H_C\
`OH
`
`0
`
`-
`
`OH 0
`
`CH2= ?_ C \
`0 0
`
`0
`jj_C \
`
`CH3
`
`0 0
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`
`530 (cid:9)
`
`Part I II Bioenergetics and Metabolism
`
`Much of this energy is conserved by the coupled phosphorylation of
`molecules of ADP to ATP. The net yield is two molecules of ATP per 1le
`
`cule of glucose used, because two molecules of ATP were invested in t
`-
`preparatory phase. Energy is also conserved in the payoff phase in the f or
`
`mation of two molecules of NADH per molecule of glucose.
`In the sequential reactions of glycolysis, three types of chemical trans
`formations are particularly noteworthy: (1) degradation of the carb0
`skeleton of glucose to yield pyruvate, (2) phosphorylation of ADP to ATp
`by high-energy phosphate compounds formed during glycolysis, and (3)
`transfer of a hydride ion with its electrons to NAD, forming NADH. The fate
`of the pyruvate depends on the cell type and the metabolic circumstance s.
`
`Fates of Pyruvate Barring some interesting variations in the bacterial
`realm, the pyruvate formed by glycolysis is further metabolized via one of
`three catabolic routes. In aerobic organisms or tissues, under aerobic con-
`ditions, glycolysis is only the first stage in the complete degradation of glu-
`cose (Fig. 15-3). Pyruvate is oxidized, with loss of its carboxyl group as
`CO2 , to yield the acetyl group of acetyl-coenzyme A; the acetyl group is then
`oxidized completely to CO 2 by the citric acid cycle (Chapter 16). The elec-
`trons from these oxidations are passed to 02 through a chain of carriers in
`the mitochondrion, forming 1120. The energy from the electron transfer re-
`actions drives the synthesis of ATP in the niitochondrion (Chapter 19).
`The second route for pyruvate is its reduction to lactate via lactic acid
`fermentation. When vigorously contracting skeletal muscle must function
`under low-oxygen conditions (hypoxia), NADH cannot be reoxidized to
`NAD, and NAD is required as an electron acceptor for the further oxida-
`tion of pyruvate. Under these conditions pyruvate is reduced to lactate, ac-
`cepting electrons from NADH and thereby regenerating the NAD neces-
`sary for glycolysis to continue. Certain tissues and cell types (retina, brain,
`erythrocytes) convert glucose to lactate even under aerobic conditions, and
`lactate is also the product of glycolysis under anaerobic conditions in some
`microorganisms (Fig. 15-3).
`The third major route of pyruvate catabolism leads to ethanol. In some
`plant tissues and in certain invertebrates, protists, and microorganisms
`such as brewer’s yeast, pyruvate is converted under hypoxic or anaerobic
`conditions into ethanol and CO 2, a process called alcohol (or ethanol) fer-
`mentation (Fig. 15-3).
`The focus of this chapter is catabolism, but pyruvate has anabolic fates
`as well. It can, for example, provide the carbon skeleton for the synthesis of
`the amino acid alanine. We return to these anabolic reactions of pyruvate in
`later chapters.
`
`Glucose
`
`glycolysis
`(10 successive
`reactions)
`
`anaerobic (cid:9)
`conditions (cid:9)
`
`t
`2_e (cid:9)
`
`Ethanol–CO 2
`
`Fermentation to alcohol
`in yeast
`
`aerobic
`conditions
`2co2
`
`yl-Co
`
`citric
`acid
`cycle
`
`anaerobic
`conditions
`
`ii
`
`2 Lactate
`Fermentation to
`lactate in vigorously
`contracting muscle,
`erythrocytes, some
`other cells, and in
`some microorganisms
`
`4CO 2 + 4H20
`
`Animal, plant, and
`many microbial cells
`under aerobic conditions
`
`figure 15-3
`Three possible catabolic fates of the pyruvate formed in
`glycolysis. Pyruvate also serves as a precursor in many
`anabolic reactions, not shown here.
`
`

`
`Chapter 15 Glycolysis and the Catabolism of Hexoses (cid:9)
`
`531
`
`ATP Formation Coupled to Glycolysis During glycolysis some of the energy
`of the glucose molecule is conserved in ATP, while much remains in the
`product pyruvate. The overall equation for glycolysis is
`
`
`Glucose + 2NAD + 2ADP + 2P j
`2 pyruvate + 2NADH + 2H + 2ATP + 2H 2 0 (15-1)
`
`For each molecule of glucose degraded to pyruvate, two molecules of ATP
`are generated from ADP and P 1. We can now resolve the equation of glycol-
`ysis
` two processes: (1) the conversion of glucose to pyruvate, which is
`xergornc
`
`Glucose + 2NAD - 2 pyruvate + 2NADH + 2H (cid:9)
`
`(15-2)
`= (cid:151)146 kJ/mol
`
`and (2) the formation of ATP from ADP and P, which is endergonic:
`
`(15-3)
`2 ADP + 2P, - 2ATP + 2H20 (cid:9)
`zG’2(cid:176) = 2(30.5 kJ/mol) = 61 kJ/mol
`
`The sum of Equations 15-2 and 15-3 gives the overall standard free-energy
`change of glycolysis, G(cid:176):
`
`AGIO = G(cid:176) + Gl (cid:176) (cid:151)146 kJ/mol + 61 kJ/mol
`
`(cid:151)85 kJ/mol
`
`Under standard conditions and in the cell, glycolysis is an essentially irre-
`versible process, driven to completion by a large net decrease in free en-
`ergy. At the actual intracellular concentrations of ATP, ADP, and P (see
`Box 14-2) and of glucose and pyruvate, the energy released in glycolysis
`(with pyruvate as the end product) is recovered as ATP with an efficiency
`of over 60%.
`
`Energy Remaining in Pyruvate Glycolysis releases only a small fraction
`of the total available energy of the glucose molecule. When glucose is oxi-
`dized completely to CO2 and H2 0, the total standard free-energy change is
`(cid:151)2,840 kJ/mol. Glycolytic degradation of glucose to two molecules of pyru-
`vate (AG" = (cid:151)146 kJ/mol) therefore yields only (146/2,840)100 = 5.2% of
`the total energy that can be released from glucose by complete oxidation.
`The two molecules of pyruvate formed by glycolysis still contain most of the
`chemical potential energy of the glucose molecule, energy that can be ex-
`tracted by oxidative reactions in the citric acid cycle (Chapter 16) and ox-
`’dative phosphorylation (Chapter 19).
`
`impdie of Phosphorylated Intermediates Each of the nine glycolytic
`intermediates between glucose and pyruvate is phosphorylated (Fig. 15-2).
`The Phosphoryl groups appear to have three functions.
`
`1, They are ionized at pH 7, giving each glycolytic intermediate a net
`negative charge. Because the plasma membrane is impermeable to
`charged molecules, the phosphorylated intermediates cannot diffuse
`Out of the cell. Alter the initial phosphorylation, no further energy
`is necessary to retain phosphorylated intermediates in the cell,
`despite the large difference in their intracellular and extracellular
`concentrations.
`2. PhosphoT1 groups are essential components in the enz ymatic conser-
`vatio n of metabolic energy. Energy released in the breakage of phos
`Piloanhydride bonds (such as those in ATP) is partially conserved in
`the formation of phosphate esters such as glucose 6-phosphate. High-
`energy phosphate compounds formed in glycolysis (1,3 bisphospho
`8Iycte and phosphoenolpyruvate) donate phosphoryl groups to
`ADpo form ATP
`
`

`
`532 (cid:9)
`
`Part I II Bioenergetics and Metabolism
`
`3. Binding energy resulting from the binding of phosphate groups to the
`active sites of enzymes lowers the activation energy and increases the
`specificity of the enzymatic reactions (see p. 251). The phosphate
`groups of ADP, ATP, and the glycolytic intermediates form complexe s
`, and the substrate binding sites of many glycolytic enzym e5
`with Mg(cid:176)
`are specific for these Mg 2 complexes. Most glycolytic enzymes require
`
`Mg 2+ for activity.
`
`The Preparatory Phase of Glycolysis Requires ATP
`In the preparatory phase of glycolysis, two molecules of ATP are invested
`and the hexose chain is cleaved into two triose phosphates. The realization
`that phosphorylated hexoses were intermediates in glycolysis came slowly
`and serendipitously. In 1906, Arthur Harden and William Young tested their
`hypothesis that inhibitors of proteolytic enzymes would stabilize the glucose
`fermenting enzymes in yeast extract. They added blood serum (known to
`contain inhibitors of proteolytic enzymes) to yeast extracts and observed
`the predicted stimulation of glucose metabolism. However, in a control ex-
`periment intended to show that boiling the serum destroyed the stimulatory
`activity, they discovered that boiled serum was just as effective at stimulat-
`ing glycolysis. Careful examination and testing of the contents of the boiled
`serum revealed that inorganic phosphate was responsible for the stimula-
`tion. Harden and Young soon discovered that glucose added to their yeast
`extract was converted into a hexose bisphosphate (the "Harden-Young es-
`ter," eventually identified as fructose 1,6-bisphosphate). This was the be-
`ginning of a long series of investigations on the role of organic esters of
`phosphate in biochemistry, which has led to our current understanding of
`the central role of phosphoryl group transfer in biology.
`
`(13 Phosphorylation of Glucose
`In the first step of glycolysis, glucose is
`activated for subsequent reactions by its phosphorylation at C-6 to yield
`glucose 6-phosphate, with ATP as the phosphoryl donor:
`
`Arthur Harden
`1865-1940
`
`I
`
`William Young
`1878-1942
`
`HO(cid:151)CH 2
`
`0 (cid:9)
`
`HH
`OH H (cid:9)
`>10H
`
`HO
`
`H OH
`Glucose
`
`
`
`ATP ADP
`
`hexoknase
`
`0
`II (cid:9)
`6
`O(cid:151)P-0---CH 2
`
`II/’ \H
`OH H 2’
`OH
`
`HO (cid:9)
`
`H OH
`Glucose 6-phosphate
`
`= (cid:150)16.7 kJ/mol
`
`This reaction, which is irreversible under intracellular conditions, is cat-
`alyzed by hexokinase. Recall that kinases are enzymes that catalyze the
`transfer of the terminal phosphoryl group from ATP to some acceptor
`flU
`cleophile (see Fig. 14-10). Kinases are a subclass of transferases (see Table
`8-3). The acceptor in the case of hexokinase is a hexose, normally
`0-
`glucose, although hexokinase also catalyzes the phosphorylation of other
`common hexoses, such as D-fructose and D-mannose.
`Hexokinase, like many other kinases, requires Mg 2 for its activity, be-
`cause the true substrate of the enzyme is not ATP 4 but the MgATP2 coim
`plex (see Fig. 14-2). Detailed studies of yeast hexokinase have shown that
`the enzyme undergoes a profound change in shape, an induced fit, when it
`binds the hexose molecule (see Fig. 8-21). Hexokinase is present in all cells
`
`

`
`Chapter 15 Glycolysis and the Catabolism of Hexoses
`
`533
`
`of all organisms. Hepatocytes also contain a form of hexokinase called hexo-
`yjnase D or glucokinase, which is more specific for glucose and differs from
`other forms of hexokinase in kinetic and regulatory properties (p. 555).
`Like the other nine enzymes of glycolysis, hexokinase is a soluble, cy-
`tosolic protein, although, as we note later, there may be organized com-
`plexes of several glycolytic enzymes (see Fig. 15-8).
`
`Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate The en-
`zyrne phosphohexose isomerase (phosphoglucose isomerase) cat-
`alyzes the reversible isomerization of glucose 6-phosphate, an aldose, to
`fructose 6-phosphate, a ketose:
`
`0
`6
`- 0-P - 0 - 2
`
`5O
`
`/1 \ 1 (cid:9)
`OH H (cid:9)
`
`OH (cid:9)
`
`HO (cid:9)
`
`2
`H OH
`Glucose 6-phosphate
`
`Mg
`
`osphohexose
`isornerase
`
`0
`II (cid:9)
`6
`O-P-O--CH2
`I (cid:9)
`I (cid:9)
`0 (cid:9)
`
`1
`CH2OH
`
`HO
`
`H (cid:9)
`
`OH
`
`OH H
`
`Fructose 6-phosphate
`
`= 1.7 kJ/mol
`
`This reaction proceeds readily in either direction, as predicted from the rela-
`tively small change in standard free energy. Phosphohexose isomerase re-
`quires Mg2 and is specific for glucose 6-phosphate and fructose 6-phosphate.
`
`fi Phosphorylation of Fructose 6-Phosphate to Fructose 1 ,6-Bisphosphate
`In the second of the two priming reactions of glycolysis, phosphofructo-
`kinase-1 catalyzes the transfer of a phosphoryl group from ATP to fructose
`6-phosphate to yield fructose 1,6-bisphosphate:
`o (cid:9)
`II (cid:9)
`6 (cid:9)
`0-P-O-CH 2 (cid:9)
`
`1
`0 CH2(cid:151)OH ATP ADP
`
`0
`0 (cid:9)
`II
`II (cid:9)
`1 (cid:9)
`6 (cid:9)
`(cid:9) (cid:151) 0-P-0- H2 (cid:9)
`CH 2 -0-P-O
`0
`0- (cid:9) (cid:176)11H02 (cid:9)
`\_:______ (cid:9)
`phosphofructokinase-1 (cid:9)
`OH
`H (cid:9)
`
`0
`
`OH
`
`Fructose 1,6-bisphosphate
`
`0 (cid:9)
`
`5HH02 (cid:9)
`OH (cid:9)
`H (cid:9)
`
`OH
`Fructose 6-phosphate
`
`= (cid:151)14.2 kJ/mol
`The reaction is essentially irreversible under cellular conditions. (This en-
`Zrrne is called phosphofructokinase-1 (PFK-1) to distinguish it from a sec-
`ond enzyme (PFK-2) that catalyzes the formation of fructose 2,6-bisphos-
`Phate from fructose 6-phosphate; see Fig. 20-8.)
`Some bacteria and protists and perhaps all plants have a phosphofruc-
`tokinase that uses pyrophosphate (PP 1), not ATP, as the phosphoryl group
`donor in the synthesis of fructose 1 ,6-bisphosphate:
`
`Fructose 6-phosphate + PP 1 J_ fructose 1,6-bisphosphate + P 1
`= (cid:151)l4kJ/mol
`Phosphofructokinasel is a regulatory enzyme (Chapter 8), one of the
`complex known. It is the major point of regulation in glycolysis. The
`aet’VitY of PFK-1 is increased whenever the cell’s ATP supply is depleted or
`en the ATP breakdown products(cid:151)ADP and AMP, particularly the
`
`(cid:9)
`(cid:9)
`(cid:9)
`

`
`534
`
`Part III Bloenergetics and Metabolism
`
`latter(cid:151)are in excess. The enzyme is inhibited whenever the cell has arnp
`ATP and is well supplied by other fuels such as fatty acids. In some orga n
`isms, fructose 2,6-bisphosphate (not to be confused with the PFK-1 reac-
`tion product, fructose 1,6-bisphosphate) is a potent allosteric activator of
`phosphofructokinase-1. The regulation of this step in glycolysis is discussed
`in greater detail later in the chapter.
`
`fi Cleavage of Fructose 1 ,6-Bisphosphate The enzyme fructose 1,6.
`bisphosphate aldolase, often called simply aldolase, catalyzes a re-
`versible aldol condensation. Fructose 1 ,6-bisphosphate is cleaved to yield
`two different triose phosphates, glyceraldehyde 3-phosphate, an aldose
`and dihydroxyacetone phosphate, a ketose:
`
`O (cid:9)
`0(cid:151)P-0(cid:151)CH2
`
`6 (cid:9)
`
`1
`
`0 (cid:9)
`H2(cid:151)O--O
`
`o
`0 (cid:9) 5HO2 (cid:9)
`OH
`H (cid:9)
`
`o (cid:9)
`H (cid:9)
`
`
`"jidolase
`
`CO O (cid:9)
`
`+
`
`CH2 OH
`
`0 H
`c
`HCOH 0
`
`C112 (cid:151) O -- P--0
`
`0
`
`Dihydroxyacetone (cid:9)
`phosphate (cid:9)
`
`Glyceraldehyde
`3-phosphate
`
`= 23.8 kJ/rnol
`
`OH H (cid:9)
`
`Fructose 1,6-bisphosphate (cid:9)
`
`The aldolase of vertebrate animal tissues does not require a divalent
`cation, but in many microorganisms aldolase is a Zn 2 -containing enzyme.
`Although the aldolase reaction has a strongly positive standard free-energy
`change in the direction of fructose 1,6-bisphosphate cleavage, in cells it can
`proceed readily in either direction. During glycolysis the reaction products
`(two triose phosphates) are removed quickly by the next two steps, pulling
`the reaction in the direction of cleavage.
`
`fi Interconversion of the Triose Phosphates Only one of the two triose
`phosphates formed by aldolase(cid:151)glyceraldehyde 3-phosphate--can be di-
`rectly degraded in the subsequent steps of glycolysis. The other product,
`dihydroxyacetone phosphate, is rapidly and reversibly converted to glycer -
`aldehyde 3-phosphate by the fifth enzyme of the glycolytic sequence,
`triose phosphate isomerase:
`
`CH2 OH
`
`- (cid:9)
`
`0
`
`CH2(cid:151)O(cid:151)P(cid:151)O
`
`Dihydroxyacetone (cid:9)
`phosphate (cid:9)
`
`O H
`
`S (cid:9)
`oose pho’e
`
`HCOH (cid:9)
`
`0
`
`CH2(cid:151)O--O’
`
`0
`Glyceraldehyde
`3-phosphate
`
`7.5 kJ/m(cid:176)
`
`1
`
`By this reaction C-i, C-2, and C-3 of the starting glucose now become chem -
`ically indistinguishable from C-6, C-5, and C-4, respectively (Fig. 15-4).
`This reaction completes the preparatory phase of glycolysis. The he
`ose molecule has been phosphorylated at C-i and C-6 and then cleaved
`form two molecules of glyceraldehyde 3-phosphate. Other hexoses, such as
`D-fructose, D-mannose, and D-galactose, can also be converted into glycer’
`aldehyde 3-phosphate, as we shall see later.
`
`tO
`
`(cid:9)
`(cid:9)
`

`
`Chapter 15 Glycolysis and the Catabolism of Hexoses
`
`535
`
`Fructose 1,6-bisphosphate
`
`Derived from
`glucose carbon (cid:9)
`
`5
`
`6
`
`Derived from
`glucose carbons
`4 or 3 (cid:9)
`
`5 or 2 (cid:9)
`
`6 or 1 (cid:9)
`
`H--!C=O (cid:9)
`I
`H(cid:150)C--OH (cid:9)
`
`3 CH2 -0-H
`
`D-Glyceraldehyde
`3-phosphate
`
`2 C=o
`
`HO(cid:150)C(cid:151)H
`4 I (cid:9)
`H(cid:151)C----OH
`
`H(cid:150)C---OH
`
`aldolase
`
`Derived from
`glucose carbon
`1
`
`2
`
`3
`
`CH2-0(cid:151)fi H(cid:151)C=O
`H--C----OH
`c==o (cid:9)
`
`CH2 OH (cid:9)
`
`CH 2 (cid:151)O
`
`Dihydroxyacetone Glyceraldehyde
`3-phosphate
`phosphate (cid:9)
`
`noose phosphate isCn erase
`
`(a)
`
`Subsequent reactions
`of glycolysis
`
`(b)
`
`flguhe 15-4
`Fate of the carbon atoms of glucose in the formation of
`glyceraldehyde 3-phosphate. (a) The origin of the
`carbons in the two three-carbon products of the aldolase
`and triose phosphate isomerase reactions. The end
`product of the two reactions is two molecules of glycer-
`aldehyde 3-phosphate. Each carbon of glyceraldehyde
`3-phosphate is derived from either of two specific
`carbons of glucose (b). Note that the numbering of the
`carbon atoms of glyceraldehyde 3-phosphate differs from
`that of the glucose from which it is derived. In glyceralde-
`hyde 3-phosphate, the most complex functional group
`(the carbonyl) is specified as C-i. This numbering
`change is important for interpreting experiments with
`glu