A. PATHWAYS FOR THE METABOLISM OP CARBOHYDRATES
a. Introduction
The breakdown of sugar by living cells has long been recognized as one of the major sources of energy for maintaining cellular activity. It became evident that this breakdown does not occur in one giant step, i.e., carbohy-drate —» carbon dioxide + water, but rather by a series of stepwise reac-tions involving a number of intermediate compounds. The elucidation of the nature of these intermediates represents a brilliant chapter in bio-chemistry.
The early work in this field was largely carried out on two types of ma-terial, muscle tissue and yeast cells. Through the efforts of such investi-gators as Hopkins, Meyerhof, Lohmann, Parnas, Embden, Harden, Neuberg, Warburg, the Coris, and others, the details of the individual steps of sugar breakdown were worked out (83, 84)» It was discovered that free sugars were not involved in the reactions but rather that the phosphorylated sugars took part. Certain phases of sugar breakdown in muscle tissue and in yeast cells were found to be identical although the end-products were different. Later work has shown that the pathways discovered in muscle tissue and yeast cells also prevail in plants, bacteria, molds, and animal tissue other than muscle (86). More recently several additional pathways of carbohydrate breakdown have been discovered. Many of the metabolic pathways of carbohydrates are reversible and provide mechanisms for the synthesis of the wide variety of compounds that go to make up the cell.
The term glycolysis was used to denote the disappearance of carbohy-drate during metabolic activity. Warburg used the term to indicate the pro-duction of lactic acid from glucose in animal tissue. Glycolysis has also been used as a term to describe the sequence of reactions taking place in cells
82a. H. H. Horowitz and C. G. King, </. Biol. Chem. 205, 815 (1953).
83. F. Dickens, in "The Enzymes" (J. B. Sumner and K. Myrbäck, eds.), Vol. 2, Part 1, p. 624. Academic Press, New York, 1951.
84. F. F. Nord and S. Weiss, in "The Enzymes" (J. B. Sumner and K. Myrbäck, eds.), Vol. 2, Part 1, p. 684. Academic Press, New York, 1951.
85. P. K. Stumpf, in "Chemical Pathways of Metabolism" (D. M. Greenberg, ed.), Vol. 1, p. 67. Academic Press, New York, 1954.
Starch Ï D-Galactose
1 l
GlycogenJ D-Galactose 1-phosphate
1 /
D-Glucose 1-phosphate D-Mannose D-Glucose > D-Glucose 6-phosphate < D-Mannose 6-phosphateU I
u
D-Fructose > D-Fructose 6-phosphate
I Î I
D-Fructose 1-phosphate > D-Fructose 1,6-diphosphate
11
Dihydroxyacetone ; D-Glyceraldehyde 3-phosphate
1-phosphate ][
D-Glyceric acid 1,3-phosphate
11
D-Glyeerie acid 3-phosphate
u
D-Glyceric acid 2-phosphate
11
Acetaldehyde + C02 < Pyruvic — "phospho-enol-pyruvic acid"
] [ acid Ethanol J, ^
Krebs L-Lactic acid Cycle
(Citric acid cycle)
FIG. 8. Embden-Meyerhof-Parnas scheme of glycolysis.
during the anaerobic breakdown. Weinhouse (86) has pointed out that glycolysis should not be used to designate a particular pathway of sugar utilization but rather only to denote sugar disappearance.
b. The Embden-Meyerhof-Parnas Scheme of Glycolysis
The best-known glycolytic pathway is that studied especially in muscle tissue and yeast cells. This pathway, sometimes known as the Embden-Meyerhof-Parnas (EMP) scheme, is shown in Fig. 8. The reactions take place under anaerobic conditions. In yeast the end-products are ethanol and carbon dioxide, whereas in muscle tissue the end-product is L-lactic acid. The EMP scheme is operative in a great many tissues and organisms and apparently represents the major pathway of carbohydrate breakdown.
86. S. Weinhouse, Ann. Rev. Biochem. 23, 125 (1954).
768 G. R. NOGGLE
The existence of the E M P pathway is based on several lines of evidence:
the presence of the intermediates in the system, the isolation and charac-terization of one or more of the enzymes involved, and the metabolism or disappearance of one or more of the intermediates when added to a tissue extract.
The metabolism of isotopically labeled substrates has also been used to indicate the presence or absence of a particular glycolytic pathway. Kosh-land and Westheimer (87) studied the fermentation of glucose-1-C14 by yeast and found that the distribution of radioactivity in the fermented products was in accord with the EMP pathway. If the EMP path is opera-tive, the fermentation should proceed as shown in reaction (25). They
H C * = 0
D -Fructose Triose phosphate 1,6-diphosphate
D -Gly ceraldehy de 3-phosphate
isolated the ethanol and found that the specific activity of the methyl group was about 95% that of the glucose used; these results gave quantitative support to the EMP pathway. Some radioactivity was found in the C02
and indicated another pathway of glycolysis.
The details of the Embden-Meyerhof-Parnas glycolytic pathway are thoroughly discussed in several recent articles (83,84,86,88). The compara-tive biochemistry of glycolysis is discussed by Stumpf (85).
87. D. E. Koshland, Jr., and F. H. Westheimer, J. Am. Chem. Soc. 72, 3383 (1950).
88. J. B. Neilands and P. K. Stumpf, 'Outlines of Enzyme Chemistry" p. 246.
Wiley, New York, 1955.
c. The Hexose Monophosphate Shunt (HMS) Scheme of Glycolysis
During work on the detailed analysis of glycolysis in various organisms, evidence accumulated for the existence of other pathways of carbohydrate breakdown than the E M P pathway. Enzymes were found which catabolized Ό-glucose in a number of different ways. The work of Warburg, Lipmann, and Dickens showed that D-glucose 6-phosphate was oxidized to D-gluconic acid 6-phosphate by a TPN-specific dehydrogenase. In yeast extracts the D-gluconic acid 6-phosphate is further oxidized in a reaction accompanied by carbon dioxide evolution. Scott and Cohen (89) demonstrated that D-ribose 5-phosphate was formed during this reaction. It was shown (74) that D-ribose 5-phosphate formation is preceded by D-ribulose 5-phosphate, and that an isomerase exists which catalyzes an equilibrium between these two pentose phosphates. The following reaction sequence (26) was suggested.
The postulated keto-acid intermediate has not been detected. D-Gluconic acid 6-phosphate is formed from D-glucose 6-phosphate by way of the
δ-lac-COOH tone (90), a reaction analogous to the bromine oxidation of glucose (Chap-ter VI) as follows:
770 G. R. NOGGLE
Evidence from a number of sources indicated that pentose phosphates were metabolized in a series of reactions that resulted in the formation of hexose monophosphates and hexose diphosphates. Several enzyme steps are involved in these transformations. The reaction between D-ribulose 5-phosphate and D-ribose 5-phosphate to form D-sedoheptulose 7-phos-phate and D-glyceraldehyde 3-phos7-phos-phate is catalyzed by an enzyme known as transketolase (91). This enzyme is found in plant, animal, and bacterial cells. Thiamine pyrophosphate (TPP) and Mg ions are required as co-factors. The rmechanism of the reaction was suggested (92) as shown in reaction (28).
HaCOH
c=o I I
HCOH HCOH H2COP03H2
D-Ribulose 5-phosphate
H C = 0 HCOH
TPP Enzyme Transketolase
H2COH
H C = 0 H2COH
I I
^ HCOH + H C = 0
I !
H2C OP03H2 TPP · Enzyme D-Glyceraldehyde "Active
glycol-3-phosphate aldehyde' ' H2COH
C = 0
I
HOCH
(28)
HCOH + H C = 0 ^ HCOH + TPPEnzyme HCOH TPP-Enzyme HCOH
I I
H2COP03H2 HCOH
D-Ribose 5-phosphate
H2COP03H2
D-Sedoheptulose 7-phosphate
The specificity of purified transketolase is rather broad, and several com-pounds have been shown (93) to act as donors of "active glycolaldehyde."
Included in these compounds are D-ribulose 5-phosphate, D-sedoheptulose 7-phosphate, D-fructose 6-phosphate, L-erythrulose, and hydroxypyruvic acid. A number of aldehydes have been shown to act as "active
glycolalde-90. O. Cori and F. Lipmann, J. Biol. Chem. 194, 417 (1952).
91. G. de la Haba, I. G. Leder, and E. Racker, Federation Proc. 12, 194 (1953).
92. B. L. Horecker, P. Z. Smyrniotis and H. Klenow, J. Biol. Chem. 205, 661 (1953).
98. B. L. Horecker, The Brewers Digest 28, 214 (1953).
hyde" acceptors, including glycolaldehyde, D-erythrose 4-phosphate, D-ri-bose 5-phosphate, and D-glyceraldehyde 3-phosphate. The carbohydrates acted upon by transketolase have both eis and trans configurations at carbon atoms 3 and 4. However it has been reported (73b) that highly puri-fied transketolase is specific for D-xylulose 5-phosphate. Plant transketolases may have the same specificity (94). The activity of D-ribulose 5-phosphate in the transketolase reaction is due to the presence of a phosphoketopento-epimerase which converts D-ribulose 5-phosphate to D-xylulose 5-phosphate.
The D-sedoheptulose 7-phosphate formed during the cleavage of the pentose phosphates is metabolized by the following reaction (29). The en-zyme catalyzing this reaction is called transaldolase and is supposed to act by transferring a dihydroxyacetone group. Only D-sedoheptulose 7-phos-phate and D-fructose 6-phos7-phos-phate have been shown to act as dihydroxy-acetone donors, and D-glyceraldehyde 3-phosphate and D-erythrose 4-phos-phate as dihydroxyacetone acceptors (95).
H2COH
94. J. A. Bassham, S. A. Barker, M. Calvin, and U. S. Quarck, Biochim. et Bio-phys. Ada 21, 376 (1956).
95. B. L. Horecker and P. Z. Smyrniotis, / . Biol. Chem. 212, 811 (1955).
772 G. R. NOGGLE
The tetrose phosphate formed in the preceding reaction (29) was identi-fied as D-erythrose 4-phosphate (96) on the basis of its participation in several reactions. The tetrose phosphate was found to react in an aldolase-catalyzed reaction with dihydroxyacetone phosphate to yield
D-sedohep-H2COP03H2
tulose 1,7-diphosphate (reaction (30)). The tetrose phosphate was also found to act as an acceptor of "active glycolaldehyde" derived from pen-tose phosphate. In this reaction (31), catalyzed by transketolase, D-frucpen-tose 6-phosphate is formed.
The various reactions of the hexose monophosphate shunt have been summarized in Fig. 9. By this pathway it is possible to oxidize completely glucose 6-phosphate without having to go through the E M P pathway or the tricarboxylic acid cycle. A more detailed account of this oxidative
path-96. B. L. Horecker, P. Z. Smyrniotis, H. H. Hiatt, and P. A. Marks, / . Biol. Chem.
212, 827 (1955).
D-Glucose
D-Glucose 6-phosphate
D· Fructose D-Erythrose 6-phosphate 4-phosphate
D-Fructose 1,6-diphosphate "
\ D-Glucono-5-lactone 6-phosphate
D-Gluconic acid 6-phosphate
*. D-Ribulose /
5-phosphate "~~ . D-Ribose ' 5-phosphate
D-Glyceraldehyde D-Sedoheptulose 3-phosphate 7-phosphate
Dihydroxyacetone phosphate
FIG. 9. The hexose monophosphate shunt scheme of gly-colysis. [After Gunsalus, Horecker, and Wood (97).]
way will be found in several reviews (93, 97). The relative importance of this pathway as compared to the E M P pathway has been studied with the aid of isotopically labeled substrates. As indicated by reaction (25), if glucose-1-C14 and glucose-6-C14 are respired by comparable tissue samples, the contribution of C14 to the C02 given off will be the same. If the HMS pathway is operative, the C02 from glucose-1-C14 will be initially higher in C14 than that from glucose-6-C14 by virtue of the following reaction (32) :
H C * = 0 HCOH
| HOCH
| HCOH
| HCOH
| H2COP03H2
C*02
+
H2COH
|
_>
c= o
* 1
HCOHHCOH
1
|
(32)
H2COP03H2
D-Glucose D-Ribulose 6-phosphate 5-phosphate
97. I. C. Gunsalus, B. L. Horecker, and W. A. Wood, Bacterial. Revs. 19, 79 (1955).
774 G. R. NOGGLE
A. Yield of C1402 from glucose-6-C14 . , , . ,
If the ratio ΛΓ. ..—r „.,„ , = ττ^τ l s determined, a value of Yield of Cu02 from glucose-1-C14
unity would be expected if the E M P pathway is operating. A value of less than one would indicate the existence of the HMS pathway. This technique has been used on plant and animal tissue to evaluate the rela-tive importance of the various glycolytic pathways. Corn root tips were found (98) to convert glucose to C02 by way of the EMP glycolytic route.
The tissues of other plants were found to respire glucose by both the E M P route and the HMS pathway. A study (99) of several rat tissues indicated that in diaphragm tissue the E M P pathway was followed, whereas in the kidney tissue the EMP pathway was accompanied by the HMS route.
Liver tissue was found to respire glucose almost exclusively by the HMS route.
i. Other Pathways of Glucose Utilization
Several other pathways of glucose catabolism have been discovered in different microorganisms. In Pseudomonas saccharophila it was observed (100) that D-gluconic acid 6-phosphate was degraded to pyruvic acid and tri ose phosphate. It was postulated that an intermediate 2-keto-3-deoxy-D-gluconic acid 6-phosphate was formed. Subsequently this intermediate was isolated and characterized (101). The following pathway was suggested.
0 = C O H
The use of isotopically labeled substrates has also revealed other path-ways of glucose metabolism. It was found that Leuconostoc mesenteroides fermented glucose to yield one mole each of lactate, ethanol, and C 02.
98. H. Beevers and M. Gibbs, Plant Physiol. 29, 322 (1954).
99. B. Bloom, M. R. Stetten, and D. Stetten, Jr., J. Biol. Chem. 204, 681 (1953);
B. Bloom and D. Stetten, Jr., </. Am. Chem. Soc. 75, 5446 (1953).
100. N. Entner and M. Doudoroff, J. Biol. Chem. 196, 853 (1952).
101. J. MacGee and M. Doudoroff, J. Biol. Chem. 210, 617 (1954).
Using C14-labeled glucoi
{102), , the following scheme was shown
+ C02
Glucose-1-C14 yielded labeled carbon dioxide. Glucose-3,4-C14 gave car-binol-labeled ethanol and carboxyl-labeled lactate. Several enzymes have been isolated from the organism, but the mechanism of the reaction remains obscure.
Some microorganisms are able to oxidize glucose to gluconic acid and 2-ketogluconic acid without going through phosphorylated intermediates.
The mechanism of these reactions are poorly understood. The pathways of carbohydrate metabolism in microorganisms are thoroughly discussed in a review by Gunsalus, Horecker, and Wood (97).
B. INTERCONVERSION OF THE SUGARS
The glycolysis schemes shown in Figs. 8 and 9 indicate that D-glucose, D-fructose, and the trioses are the major sugars involved. It is known, however, that other carbohydrates may be converted to D-glucose and D-fructose and, thus, enter the glycolytic pathways. Sugars can also be synthesized from D-glucose and D-fructose, indicating that either the en-symes responsible for the interconversions are reversible or that other enzymes are present which catalyze the reverse reaction. Some of the rela-tionships between the hexoses are shown in Fig. 6 (p. 760). Not all of the enzymes for these reactions have been isolated and characterized.
The transformation of D-galactose 1-phosphate to D-glucose 1-phosphate has been shown to be catalyzed by an enzyme known as galactowaldenase.
Leloir and cp-workers (108) demonstrated that the reaction required a coenzyme that was identified as uridine diphosphate glucose
(si/ra-D-gluco-102. I. C. Gunsalus and M. Gibbs, J. Biol. Chem. 194, 871 (1952).
108. R. Caputto, L. F. Leloir, R. E. Trucco, C. E. Cardini, and A. C. Paladini, J. Biol. Chem. 179, 497 (1949) ; R. Caputto, L. F. Leloir, C. E. Cardini, and A. C. Pala-dini, ibid. 184, 333 (1950). For more recent work and concepts, see H. M. Kalckar, Science 125, 105 (1957).
776 G. R. NOGGLE
L-Arabinose
D-Arabinose ^ D-Ribulose
D-Ribose 1-phosphate D-Ribose D-Ribose 5-phosphate 1
\\ > —> Deoxyribose
^ D-Ribulose 5-phosphate J 5-phosphate
\\ î
D-Xylulose 5-phosphate Deoxyribose
î 1-phosphate D-Xylulose
D-Xylose
FIG. 10. Interrelationships of pentose and phosphates (After Glock, (77)).
pyranosyluridine 5'-pyrophosphoric acid, UDPG). It was postulated that the interconversion from D-galactose 1-phosphate to D-glucose 1-phosphate took place in two steps as follows:
D-Galactose 1-phosphate + UDP glucose ^
D-glucose 1-phosphate + UDP galactose (35)
UDP galactose ;=± UDP glucose (36)
The conversion of D-glucose 1-phosphate to D-glucose 6-phosphate is catalyzed by an enzyme known as phosphoglucomutase {104). A cof actor for this reaction was identified as D-glucose 1,6-diphosphate {105). The following reaction mechanism was suggested:
Only catalytic amounts of the diphosphates are required for the reaction.
The phosphoglucomutase also acts on D-mannose phosphate and D-ribose phosphate and is thought to catalyze the interconversion of D-mannose 1-phosphate to D-mannose 6-1-phosphate and D-ribose 1-1-phosphate to D-ribose
m. G. T. Cori, S. P. Colowick, and C. F. Cori, J. Biol. Chem. 124, 543 (1938).
105. C. E. Cardini, A. C. Paladini, R. Caputto, L. F. Leloir, and R. E. Trucco, Arch. Biochem. 22, 87 (1949).
Proteins Carbohydrates
Α,,,)η0 acids < ■ " — » « " « » . pyru'vate
Oxalacetic acid Acetyl-CoA
Lipides
Fatty acids
il Citric acid , cis-Aconitic acid : Malic acid
Fumaric acid
Succinic acid
Isocitric acid
Oxalosuccinic acid
Succinyl-CoA 7=
C0+ 2
or-Ketoglutaric acid C0+ 2
FIG. 11. The citric acid cycle.
5-phosphate. Complete discussion of these and other reactions involved in hexose interconversion are to be found in reviews by Racker (64) and Leloir (78, 106).
The problem of the interconversion of the pentoses and pentose phos-phates has been extensively studied during recent years. Many of these findings are discussed in reviews by Lampen (107) and Glock (77) and sum-marized in Fig. 10.
D-Ribose can be formed by way of the HMS glycolytic pathway as well as by way of the condensation of two-carbon and three-carbon fragments.
The deoxypentoses apparently arise only by the condensation of two-car-bon and three-cartwo-car-bon fragments. The dotted lines in Fig. 10 indicate inter-conversions whose pathways are not thoroughly understood. In certain strains of bacteria, L-arabinose is metabolized but the intermediate prod-ucts are not known.
The hexose monophosphate shunt pathway ties together hexose and pentose metabolism and provides reactions for the interconversion of these two groups of sugars. In addition this pathway also provides for the metab-olism of the three-, four-, and seven-carbon sugars (108).
C. PATHWAYS OF PYRUVATE METABOLISM
Whether carbohydrate is metabolized by the E M P pathway or the HMS pathway, pyruvic acid is the common end-product. Pyruvate may be
106. L. F. Leloir, Advances in Enzymol. 14, 193 (1953).
107. J. O. Lampen, / . Cellular Comp. Physiol. 41, Suppl. 1, 183 (1953).
108. S. S. Cohen, in "Chemical Pathways of Metabolism" (D. M. Greenberg, ed.), Vol. 1, p. 173. Academic Press, New York, 1954.
778 G. R. NOGGLE
metabolized in many different ways depending upon the type of organism and whether the conditions are aerobic or anaerobic. The details of many of these pathways are discussed in several recent reviews (97, 109).
One of the major pathways of pyruvate metabolism is through the "Krebs cycle" or citric acid cycle (110, 111). This pathway (Fig. 11) provides for the complete oxidation of pyruvate to carbon dioxide and water. During this oxidation, a great deal of the energy that is available from the carbo-hydrate is conserved as high-energy phosphate (112) by a process known as "oxidative phosphorylation." Originally there was some difficulty in explaining the mechanism for the entrance of pyruvate into the citric acid cycle. However, evidence of the participation of "active acetate" in carbo-hydrate, fat, and protein metabolism eventually led to the discovery of coenzyme A (CoA) by Lipmann (113). Lynen and Reichert (114) were able to isolate "active acetate" and show that it was acetyl-CoA. The for-mation of acetyl-CoA from pyruvate was discussed earlier (reactions (14) to (17)). The acetyl-CoA then condenses with oxalacetate and enters the citric acid cycle. The biochemistry of the formation of coenzyme A and its participation in a variety of biochemical reactions are discussed in a num-ber of reviews (115).
109. E. S. G. Barron, in "Modern Trends in Physiology and Biochemistry" (E. S.
G. Barron, ed.), p. 471. Academic Press, New York, 1952.
110. S. Ochoa, Advances in Enzymol. 15, 183 (1954).
111. H. A. Krebs, in "Chemical Pathways of Metabolism" (D.M. Greenberg, ed.), Vol. 1, p. 109. Academic Press, New York, 1954.
112. F. E. Hunter, Jr., in "Phosphorus Metabolism" (W. D. McElroy and B. Glass, eds.), Vol. 1, p. 297. Johns Hopkins Press, Baltimore, 1951.
US. F. Lipmann, J. Biol. Chem. 160, 173 (1945).
114. F. Lynen and E. Reichert, Angew. Chem. 63, 47 (1951).
116. H. A. Barker, in "Phosphorus Metabolism" (W. D. McElroy and B. Glass, eds.), Vol. 1, p. 204. Johns Hopkins Press, Baltimore, 1951; E. R. Stadtman, J. Cellular Comp. Physiol. 41, Suppl. 1, 89 (1953).