Q Carbohydrate Metabolism Pyruvie acid has been established as the key intermediate substang, in the metabolism of earbohydrates by bacteria. Almost alll six-, five., ang four-carbon compounds are converted initially to pyruvate, from which substance further catabolic or synthetic reactions proceed. Because of its key position, it was thought best to deal first with the origin of pyruvic acid. Glucose Metabolism The main carbohydrate compound that serves as carbon source for bacteria is glucose. Glucose is converted to pyruvic acid mainly via four different pathways, which have been named after those rescarehers who discovered and established them or according to their main components: 1, Embden-Meyerhof-Parnas (EMP) pathway 2. Warburg-Dickens or hexose monophosphate (HMP) pathway 3. Entner-Doudoroff (ED) pathway 4, Phosphoketolase (PK) pathway The first two pathways also function in mammalian tissue and in yeasts. However, there are signifieant differences between the terminologies used by biochemists and by microbiologists. The EMP pathway is very often referred to as “glycolysis,” or the “glycolytic” or “anaerobie” pathway. AS used by mammalian biochemists, the term “glycolysis” refers to the path way by whieh glycogen or glucose is converted anaerobically via pyruvic acid to lactic acid. Only in the homofermentative lactobacilli is the con plete pathway, including the conversion of pyruvic acid to lactic acid found. Many other microorganisms use this pathway to pyruvic acid, but 208gp METABOLISM 209 ol ent metabolism of ic ac Pyruvic acid varies from o1 ne group of bac- is used 1 not only by annerobie bacteria, sequ 2 the au nother. The EMP pathwa: in 0") facultative an pact aerobic pathway. Som acrobie bactesin, His thereto vot mn oe ve nner ; acaae via th se annerabie bacteria, such erefore not an notin oxchsive pathway for the ete takng the EMP pativeny wi ¥ for the anaerobic. utilix tion of glucose, ser“ best be restricted to its mammalian tion is f ¢ au fermentation.” = sidered as Synonymous with fermentation nadt all chee vs as “fermentative” pathway in and all the different ancient history and with progress in biochemics Tic retodige it ha amarious meanings. As defined in this hook bal nawledge i hae js restricted to those pathw 7 ia 2 Chapter 8), “fer- ics y ch the terminal elec an organic compound. Where the terminal ‘Sesaptor Is sen urded as oxidative and the 7 > or . . © lone of res n. The latter is cither acrobic (using oxygen) or (using inorganic compounds other than oxygen) pisregarding for the moment whether the various pathways are func- igual ender acrobie or anacrobie conditions, each will be traced to the tity of the formation of pyruvie acid, which is an intermediate common to ‘As the problem of glucose transport will be dealt with sepa- them all (89)- P » rately (see p- 241), this aspeet is also disregarded for the time being. bio beet term that needs clarifies gmbden-Meyerhof-Parnas Pathway The EMP hacteria (Enterobacteriaceae, Lacto ete.) and shows the overall reaction = 2 pyruvate + 4ATP + 2(NADH + H*) pathway, outlined in Fig. 5.1, is widely distributed among pacillaceae, saccharolytic clostridia, glucose + 2ATP + 2NAD* kdown is a phosphorylation step that glucose brea (ATP: p-hexose 6-phos- The first step (i) in the 'd the enzyme hexokinase requires 1 mole of ATP an photransferase, EC 2.7.1.1) CHO. CHO ndow dou HOCH ate APP HOeH HEOH 4p" — + 5000 cal/mole HOOH CH,OH H,COPOs glucose 6-phosphate glucose5, GARBOTIYDRATE yp, AR uy Me 210 glucose aT Es 1. (eC 2714 ke glucose 6-phosphate GEC 5.3.1.9 | fructose 6-phosphate ATP. EC 2.7.111 Apr. fructose 1,6-diphosphate (iv) EC 41.213 a (v) EC 5.3.1.1 == slyceraldehyde 3-phosphate dihydroxyacetone phosphate NADY. (vi) EC 1.2,1.12 NADH + Ht Py 1,3-diphosphoglycerate ADP (vii) EC 2.7.2.3 c ATP 3-phosphoglycerate (viii) EC 2.7.5.3 | 2-phosphoglycerate (ix) EC auf, 4,0 phosphoenol pyruvate (x) EC 2.7.1.40, apr ATP. pyruvate Fig. 6.1. The Embd len-Meye leyerhof-Parnas (EMP) pathway of glucose utilizat; METABOLISM 21 a. terms “hexokinase” and « a phe tem Pentokinase” include all enzymes that catalyze te ene pipe Phosphoryl residue of ATP to any one of oe ee only the toe 0 i C5 or C-6 monosuccharide, Jn actual fact, toed in the ronction, (73 - “Ol group of the free sugar scems# to. volvee Hon (72). Enzyime-catalyzed phosphorylations of be inv A : " aroxyl groups occupying othe . hydroxy] BrOUD pying T positions on the sugar molecule have ty of certain coenzymes and cofactors. eon observed only in the ribose moi Hexokinase, the prototy pe of this group of enzymes, was first described in yeasts. ‘The distribution of these enzymex among, mieroorgantsme shown in Table 5.1. The hexokinnses of bacterial origin appear to have a far greater substrate specificity than those of mammalian or yeast origin: phe yeast hexokinase is capable of phosphorylating a number of different sugars and appears to be specific for the 3,4,5,6 region of the molecule but not for the 1,2 part, provided, of course, that no larger groups are attached to them (94). Brain and Aspergillus hexokinases are broadly similar but differences occur between the two; e.¢., brain enzyme is much less exacting with regard to substitution on C-5, and Aspergillus enzyme with regard to the C-4 atom. Because of this, galactose can serve almost as well as | TABLE 5.1 Herokinases and Pentokinases of Microbial Origin Enzyme Substrate Produet Source Hexokinase Glucose, mannose, Hexose G-phosphate Yeast, Neurospora fructose, 2-deoxy- crassa, Pseudomo- glucose, ete. nas ‘putrefaciens, Borrelia recurrentis Glucokinase Glucose Glucose 6-phosphate P. saccharophila, ~ Staph, aureus ‘Mannokinase ‘Mannose Mannose 6-phos- _—P.. saccharophila phate Galactokinase Galactose Galactose 6-phos- 8. fragilis phate Fructokinase Fructose Fructose 6-phos- _—_-P, saccharophila phate Ribokinase Ribose, 2-deoxy- Ribose 5-phosphate Bacteria, yeast ribo. Ribulokinase 1-Ribulose, arabitol Ribulose S-phos- Bacteria phate Xylulokinase p-Xylulose Xylulose S-phos- Bacteria phate N-Acetylglu- N-Acetyl glucos- _-N-Acetyl glucos- General bacterial eosamine kinase amine amine 6-phosphate distribution ¢ From Crane (72).212 5. cannonynrany, ~ glue osphorylation of hexoses ang i ever, is governed by more specific enzymes; nt pace te tractoe by ketohexokinase (15 lvcogg any “onaes, kinases are quite specific for a particular stigar at , es the ribulokinase (EC 271.16) of Aerobacter aerogenes phosp}yn tree p- and 1-ribulose. . . slate ‘All kinases require the participation of ATP as the phox, and also a metal, normally Mg?t. The actual number of digg Phaty Kinases continues to Brow, AS ‘ean well be expeeted from tha Rinae aeurrence of glucose throughout the living forma, exception of glucokinase, are inhibited 1, ed by Hexokinases, with the 386). A mechanism for ADP control of he, bli, d (G0) that invoked an ATP compartment sy he compartment and is also phosphoryl, lene > aust, be compartmented either with wt thy, Mth geo as a substrate. The Pb 6-phosphate ( yeast was propose diphosphate enters which means that AT! hexokinase. The rate of glucose util ation in a cell that has rapid glucose ¢, and that has little glucose 6-phosphate hydrolysis has been shown ty 4 ant he hexokinase step, which has been located in soluble and nites ° dete, fractions of mammalian cells (248) and is strongly inhibited eho 6-phosphate (71). This inhibition is competitive (132) with appt Van Thiedemann and Bor presence of excess However, arcorved that inorganie phosphate offsets the inhibitory effect of gi) Grphosphate in Ehrlich ascites cell homogenates and concluded that gy ition by glucose 6-phosphate is overcome by inorgani at phate. They also suggested that this might have an important rehie the Pasteur effect. Rose et al. (331) traced the offeot of inorganic phosph, in stimulating glucose utilization in red blood cells to this ree specific inhi inorganic phosphate. It is possible, however, that glucose 6-phosphate, as a regulatory sub. stance of hexokinase, is a consequence rather than a cause of metabol Guo H,COH HCOH bo I HOGH —_gucosephosphatefbomerse, HOH HCOH neon HCOH AF’ = Ocal/mole HOOH HAOPOS” H;,COPO;- glucose 6-phosphate fructose 6-phosphate213 samt «pou . ae pexokinase could effect a limitation on the size of the oF gnereloret. pool only. fe thet ae wh phos! (ji) in the BMP pathway is the isomerization of glucose rose Od SHEP “ge G-phosphate (3500) by the enzyme glucosephos- er’, oc , fructoss : y) Bone Sate #0 Tr aucose-G-phosphate ketol-isomerase, EC 5.3.1.9). greene’ gphorylation step (iii) requires a accond mole of ATP pate Second PP. the most important enzyme in the pathway, phos- mene coal 2e4 (ATP:p-fructose-6-phosphate 1-phosphotransferase, EC dF gokinas and tol ofr rhea) m n,con icoro,- co co 1 are apr yoOCcH HOCH neon ~Phosphotrustokinase neon HCOH AF’ = + 5000 cal/mole = HCOH H,COPO; H,COPO,- tose 6-phosphate fructose 1,6-diphosphate fruct mI enzyme is & most complex enzyme, in terms of both kinetics and al structure, for it is inhibited by ATP (226), by citrate (303, 304, and to a lesser degree by Mg?*. In contrast, phosphofructokinase ctivated or reactivated by NH,* (2), K+, inorganic phosphate, ADP, yAMP, S/-AMP, and fructose 6-phosphate 304). There is strong svidence that cach of these activators acts at a different site on the enzyme molecule. In considering possible structures that might give the kinetic properties of phosphofructokinase, it is important to keep in mind the jact that citrate and ATP are synergistic in action; each lowering the inhibition constant of the other. Phosphofructokinase is a unique enzyme of the EMP pathway in faculta- tive anaerobes in that it is not involved in any of the other types of carbo- hydrate degradations. This enzyme therefore plays an important function in glucose utilization (217). Evidence indicates that phosphofructokinase may be the rate-limiting factor of the EMP pathway. Its suppression by oxidation has led to the proposals that the enzyme is the mediator of the Pasteur effect, (see p. 609). There is also other evidence indicating that phosphofructokinase is endowed with many properties that make it well adapted for regulation 'n the cell. Three such properties are recognized: (a) kinetie properties phy 313)>5. CARDOHYDRAy, E Nyy en, 214 7); (v) reversible dissociation ay ‘allostery) of the ct yme (17); ¢ ' of Callostery) of Uh ivi enzyme (178, 259); and (6) aggro, the ty enzyme to an inact S ystem in : _polymer 8y8 ae yme ina monomer 1 Mueleotides aid on the hexose Phony heey, Lies depend on mda in the molecular configuration gf loan perch uructuiral change, the oes come Mory (258). Alor ae 1 of innetive subunits ( ti ). r iether Phe, ble to the orm (386) is (a) the transforma i o the enzy ns yeast: (38 y the ac cheorved, in is to an active form (8). Only the ‘A aye orm (4 in active Se nee A similar transforma’ jon has been Obsen*ht, ble to ATP inhibition: which the enzyme from strictly 2erobicay, Ty Escherichia coli K 12, in nsitivity and the enzyme from anaery rob > cells exhibited no ATP sensitivit; 1 ve (377). Ils was ATP sensitive ( . inet every microorganism. The enzyme from ArUhrobacter crystattonoig i aly as well as that from lactobacilli (97), hes pecn fennel to follow Sin! Michaelis-Menten kinetics with respect to 4 a ructose S-Phosppeht | Different temperatures also appear to effect the kinetics of the eazy Thermostable phosphofructokinase from an extreme thermophilie acta Me appears to change its substrate effect (408). Whereas the enzyme follone simple Michaelis-Menten kinetics with respect to fructose S-phosphee and ATP, it is phosphoenol pyruvate that turns the normal Saturati curve for fructose 6-phosphate into a sigmoidal one independent te ATP concentration. In the cold, in contrast, the enzyme undergoes in activation eaused by dissociation (216). This makes any Suggestion for, mechanism difficult, although attempts have been made (116), In summary, phosphofructokinase is influenced by no leas than ning metabolites, eight of which (viz., ATP, ADP, AMP, I6-P, FDP, Pi, citrate PEP) are all affected, or capable of being affected, by phosphofructokins activity. Many ef these in turn determine through equilibria the level of other metabolites.. Consequently, cell composition may be determined to & considerable extent by the kinetic parameter of the particular brand of phosphofructokinase present in the cell. (see also Pasteur effect, p. 609 and regulation on p. 256). The second key enzyme of the EMP pathway catalyzes reaetion (iv), |_| which is a cleavage of fructose 1,6-diphosphate to 2 moles of triose phe | 4 phates. The enzyme is fructose-diphosphate aldolase (fructose-1,0-diphe & phate:p-glyceraldehyde-3-phosphiate-lyase, EC 4.1.2.13) or in the olf ‘ 4, ‘ literature also “zymohexase.” This enzyme is even more widely distributed than phosphofructokinase, as it is lon a key enzyme in gluconeogenes Meyerhof and Lohmann discovered this enzyme in 1934 (275) and propo the above reaction; they thus confirmed, experimentally, a view expres* x_ yeTATOLIOM sate nM 215 ow 1,coPe; Lo 11,COPO, 1,011 f t=o 4inydroxyacetone noe fructono-diphonphate bhoaphatn ugomt Aldolane ugon ‘HO - nto; 1,COPO, AF » +4000 ent/mote boro: fructose * 1, 6-aiphosphate elyceraldehyde : 3-phoaphate |. Embden in. 1913 (98, 133). The term “zymohexase”’ has been used to by jate the combined activity of two independent eatalyaes frectwme Giphosphate aldolase and triosephosphate isomerase (377), although the Me has been used to refer to aldolase free of isomerase (174), Marburg and Christian (390), who first reported the Purification and stallization of the aldolase from rat muscle, brought the fires ecidone tat there exist differences between the m exist ‘ammalian and the yeast aldolases. Investigations into the nature of bacterial aldolases indicated that these enaymes were inhibited by chelating agents (eg., EDTA) and that this jnbibition could be overcome with metal ions (23, 26, 139, 249, 368, 396). TABLE 5.2 Some Differences between Class I and Class II Aldolases ee. FON 3142 Metal ion Chelation reversal of Optimal Source inhibition inhibition pH range Class I Muscle No - 6.7-8.5 Liver No _ bovine 7.29.2 rabbit 7.5-8.0 Green pea No _ 5.9-8.8 Glass IT Anacystis nidulans Yes Fett 8.0-8.5 Clostridium perfringens Yes Fett; Cort 7.57.9 Brucella suis Yes Fett; Mn 7.0-7.5 Aspergillus niger Yes Fett; Cott Znt*; Mnt* Bacillus stearothermophilus No —ee5, CARBOHYDRATE apy, 216 res led Richards difference and . hese and other ¢ metal ion-independg. 4 Bu ccumulation of t rototype leat cqtte, ider aldolase the P sidered the prototy,,” (C, ponsider ee jane was con .2). Differences beta hy Idolase es, whereas yeast #! Table 5. Sent, (Class I) aldolases Cimsidered to be owing ithe, = in molecule, with the phe vere C0) d Class IT aldolase we 11 protei , milarities in the ove, or to the existence of qigctin 7 [dolase had only half the jy" : vase remaining equiv site in enc eaves also found that yeast # eeu, active sites, It was al . lar weight of the muscle aldolase. { aldolase has been possible ag q A concise idea of the ane inert dihydroxyacetone-Phosphate Aldoteat of the discovery tne formed by treating rabbit Ti Candida vais compound could be form: S ce of dihydroxyacetone Phosphat, aldolases with borohydride in the presen The a (324) to I) ensy™ ion-depe' Class I and existence of dissi Ri I ond: CH,0OH c~o CHO | a | C-N-CH,),—CH —oO + HiN—(CH2)<—CH — I l CH2—O—{P) NH NH CH:—O—(P) i 2 dihydroxyacetone phosphate | NaBH reduction cH,-oH ond I HC—NH—~{(CH)>)4—CH I I CH,—O-{P) NH DHAP aldolase (152, 153). It was shown that the linkage of the substrate carbonyl group to the enzyme involved the formation of a Schiff base intermediate with the eamino group of lysine residue of the enzyme protein. Grazi et al. (152, 153) concluded that the lysine was located at the active site of the protein and that the Schiff base formation occurs during the enzyme- catalyzed aldol condensation and the transfer reactions. Both yeast and muscle aldolase have been shown to proceed almost exclusively by an ordered reaction sequence enzyme + FDP = GAP + enzyme — DHAP (lacking a proton on a-carbon) enzyme + DHAP(332)METABOLISM wo | 217 a of this reaction sequence haye indi is cated the followi re wing steps —— 5—DH oo = E_p ‘ Et == ep = --d” ~G DY = E_pG eG “EPs indicates dihydroxy, pH indica‘ 5 ) ‘yacetone phos; hs 7 re ane . Tse 'e Phate; FE, ; wise base intermediate; and E-D-, intermediate gree (eave: B-DH, soon ED, product following omerization step: Gea or raen « Geaphate; and DG, condensation product, ie, mye Blyceraldehyde Sitter ef al. (382) considered that the tutelirine ee. « ¢-H bond formation, i.c., the protonexchange remsth ee” involves the “he enayme is generally intracellular and is fornd nn oo im ean (320) although the lave ‘ound in the soluble fraction , the enzyme has been observed in the diphosphate, a sixfold augmentation of aldol (223), which may result from a specific interaction of ionized FDP and the enzyme molecule rather than from a nonspecific anion stimulation. The greatest difference between the mammalian and bacterial aldolase, however, is the existence of large numbers of isozymes of the former (13, 320). Attempts are being made to use this enzyme as a model of biochemical evolution (245a, 336). This attempt may be particularly useful also for the evolution of the bacteria, as the Class IT aldolases turn out to be much more heterogenous in regard to stability, inhibition, and reactivation compared to the Class I aldolases. As no isozymes have been reported for Cus II aldolases as yet, to the author's knowledge, this heterogeneity may indieate a similar diversity. Fructose-diphosphate aldolase was found tobe different from spores and vegetative cells of Bacillus cereus (337), and aldolase from Escherichia coli reacts differently from the one isolated from Lactobacillus casei var. rhamnosus (96). Whereas the enzyme from Escher- ‘chia coli was extremely unstable in a buffer containing mereaptoethanol, the “dlobacillus enzyme depended on this sulfhydryl compound for its ac- tity. The fruetose-diphosphate aldolases from Thermus aquaticus and ae stearothermaphitus are extremely heat stable (130). Despite all althomy tons) all bacterial aldolases belong to the Class IT al ugh there are indications of the existence of multiple forms in Taste Taldon, 8? (195a, 195b) and Escherichia coli K-12 (98a) and a an lolase in Escherichia coli (Crookes? strain) (375a). However, it wor lase activity was observed218 5. CARRONYD Rag, va AY be more helpful if the Class II aldolases could be subgroupeg «, “ey a ; m, parisons easier. . The cleavage of fructose 1,6-diphosphate to 2 moles of ridge would lead to accumulation of one of the triose Phosphate,. 7 triosephosphate isomerase (D-glyceraldehyde-3-phosphate og, s Towel, EC 5.3.1.1) (53, 276) ensures that neither of the two p,,, duet Cty 4 cumulate. H,COPO;- H.copo,- triosephosphate isomerase DEE OHCOH duon CHO glyceraldehyde 3. Phosphate dihydroxyacetone phosphate The equilibrium lies toward the right (glyceraldehyde 3-Phosph ite) long as the EMP pathway functions in the proper way. : The following step (vi) is a combined oxidation and Phosphyo, step, which is catalyzed by the enzyme slyeeraldehyde-phospjin cn _ NAD* NADH + H* . H,COPO; H,COPO; HCOH HCOH cuo ¢=o + HS-enzyme S-enzyme glyceraldehyde 3-phosphate H,COPO, H,COPO, HCOH HEOH + HyPO, 1 ¢=0 co—opo, S-enzyme AF =+2000cal/mole + HS-enzyme 1, 3-diphospho- D-glycerate hydrogenase [p-glyceraldehyde-3-phosphate: NAD oxidoreductase (phos- phorylating), EC 1.2.1.12]. This enzyme is unique insofar as it also tran fers acyl groups (137). It is composed of four chemically similar subunits, o which each is able to bind 1 mole of NAD*, and it also contains four SH groups, one of which is highly reactive and participates in the enaymat reaction (393). The reactivity of the thiol group is brought about by the binding of NAD+, as this would lower the pK of the particular SH eon (379). The importance of the coenzyme NAD* therefore lies in the at® tion of the thiol group and not in the normal redox step. Adenine nucle”prABOLisM, a Ts 219 has ATP (127, 128), ana gide8) SUF activity strongly” When Substrates as r-serine (365) inhibit er Bas Ie in they Lot this inhibition could result tbe OP rticipation of the enzyme ‘asteur effect or in general meta- iq the P' 7 4 in trol will be discussed be}, je con! ‘ elow. Thi : polic with the ATP ions Lhe complexit; r 7 even, 108 thet ae contrel AED inhibition, certainly of the renation, how. ticipation pte in Bacillus stearothermay, ae’ ehayme waa leg fount be ghermos hily is sti ermophilus (364) also found to be heme thermophily 8 still unknown, » although the mechanism ol igh-energy compound . the hie! 1,3-dipho step vi) one Phosphnnte group to form't mole al Ree dar elas or the catalytic H,COPO,, ADP ATP HCOH —\/ H,CoPo, —oPo; CoH coo” ( form 1 AF = -27,000 cat/mote 1, 3-diphospho- beglycerate 3-phospho- elycerate tion of phosphoglycerate kinase (ATP. ae Noase, EC 2.7.2.3), ‘Phospho-p-glycerate 1-phospho- The conversion of 3-phosphoglycerate to 24 3 2 phospho, i ii) results from the interaction of 2,3-diphosphoglveerste eral hen coro; ¥,cOPO; - Moot + Heopo; —Prewphe TDS aie FOPO; HCOPO; + : con Togs elveeromutase 5 HGOPO; s-phospho- 2, 8-diphospho- 2, 8-dtphospho- 1 giyeeric acid p-glyceric acid bvglyceric acid dyer mesa phosphoglyceromutase _ (2,3-diphospho-p-glycerate:2-phospho-p-glycerate phosphotransferase EC 2.7.5.3). The phosphate group is transferred to the 2 position in the molecule. Because 2,3-diphospho-p-glycerate is re- generated during this reaction, it is used in a cycling process by this par- ticular reaction. ‘The formation of phosphoenolpyruvate (reaction ix) is obtained by the action of enolase (2-phospho-p-glycerate hydro-lyase, EC 4.2.1.11) (370). H,coH 1,0 oH, ueopoy ————> Goro; coo" AF =0cal/mole COO 7 7 phospho- 2-phospho enolpyruvic acid glyceric acid5. cARnONYDR,, 7m ry lecular el ‘ ion it ected with an intramol leetron a ee reactiad Is conn an “intramolecular oxidation rag bi, sont tt Mare concentrated around C-2 of phonphrelttig, tion.” The electron d formation result 1, "Oly" This concentration and the double-bon rae ane tin fel, savy between the negative loaded phosphate and carboxyl groups, tlt ht the inner energy tension of the molecule. ae rearrangement jt 1nerendt : iu jore energy-rich compound ¢h,, "the phosphoenolpyruvate ® ® Wate is one of the key inter ®” 2p'™at ree! Phosphoenolpyruvate is 0! the key intermed; ~Phav™ Foe eases aystem, as will be seen in Chapter 7. "ata, "0h. anaplerotic sequence 8) 1 ts : The phosphate group of phosphoenolpyruvate can now, ;, reaction (reaction x), be transferred to ADP with the enya” the ° ban » cn, ADP ATP a dopo; 4 b=o ~' ar = -27,000 cal/mote boo" pyruvic acid 220 phospho- enolpyruvic acid kinase (ATP:pyruvate phosphotransferase, EC 271.40). Ty, | completes the pathway, with the formation of pyruvie acid ang an tin Ad. tional mole of ATP. Because of the central position of phosphoens) as far as anaplerotic and catabolic metabolism is concerned, thera pyruvate kinase underlies certain controls. The kineties of thi, Mt from Escherichia coli exhibit sigmoidal velocity curves, with hoop ® pyruvate as the variable gubstrate (253), that can be converted to ¢ 2° hyperbola on addition of AMP. The specific substrate required ig Naw ADP~ (271), with ATP being a strong inhibitor (41) and fructose Loa. phosphate a good activator (253). Pyruvate kinase, in other words, ik second allosteric enzyme in the EMP pathway. It is not likely to play role in gluconeogenesis, as the reverse reaction from pyruvate to phomi! enolpyruvate is almost impossible because of its unfavorable equilibria, under physiological conditions (9). The organism overcomes this bani by inducing a number of enzymes, such as phosphoenolpyruvate synthetase (30-32), pyruvatephosphate dikinase (9, 323), and a system that ss oxalacetate as intermediate. Although these enzymes are used mainly in gluconeogenesis (glucose synthesis) of acetate- or dicarboxylic acii grown cells (28), they are mentioned here to show that the pyruvate kinase reaction may well be a regulatory enzyme. Phosphoenolpyruvate synthetase in Hscherichia coli (31) catalyzes the reaction ATP + pyruvate = AMP + PEP + PiABOLISM 5 ue ot been purified. [: = has P - [@P}-Labe! me P } led ATP experiments indicated 1 eee ae not 8 equally probable (sore aes im involvil 7 wat mechanis' ‘6 ® Pyrophosphory! tranafer osphorylenolpyruvate + 1.0 — pap wo + Pi aA mechanism involving an. adenosine Pyrophosphoryl transf : fer ATP + pyruvate — ADP-enolpyruvate + Pi ADP-enolpyruvate + HAO — AMP 4 pup idence seems to f ' grnout Sy ehucidated. “Vor the first reaction, the mechanism has got beste, orthophosphate dikinase (ATP: Petpotransferase EC 2.7.9.1) catalyzes the, reaction 7 “"*boPhosphate p Pyruvate + ATP + Pi = PEP + AMP + PPi q has only been purified as yet from Bacter: "Spe third way PEP can be synthesized fro whereby pyruvate is first carboxylated to form oxalacetate, which in turn j decarboxylated to phosphoenolpyruvate (PEP), The two enzymes involved in this system are pyruvate carboxylase and PEP-carboxykinase (g). Both reactions require the presence of ATP (254) and/or free fatty jrids and CoA (187) and are therefore not favored. The EMP pathway thus produces two molecules of ATP and 2 moles oj NADH + H*, which are used for biosynthesis and as hydrogen donor, respectively, in the further breakdown of pyruvate. The 2 moles of NADH + H* are formed during the production of 2 moles of pyruvate from 1 mole of glucosé at the 1,3-diphosphoglyceric acid level. This meta- bolic breakdown of glucose to pyruvate can be used by most of the anaerobic microorganisms, such as yeast and fermentative bacteria, because no oxygen is necessary. We know, however, that it is not restricted to these particular groups (23, 309, 340). ‘otdes symbiosus (323). m pyruvate is via oxalacetate, Warburg-Dickens or Hexose Monophosphate (HMP) Pathway The hexose monophosphate pathway of glucose metabolism carries a number of names, e.g., ‘‘shunt” and “pentose cycle.” It is made up of a ‘ather complicated series of reactions, which can be carried out by many5. CARBOHYDRATE, Men, 222 i i polize glucose via the EMP or microorganisms oD. cecherichia coli, for example, uses the a, er tucose Utilization but also My, (54, 64, 142, 266, 309), OA oO way mainly during - ee regulation on P- 255) of the glucose mae pathway to 20-9070 mefaciene uses the combination ED ang ‘ i :44 (15a). ; Mp : The ores 7 Srnes conversion to einooee S-phosphate is identical to reaction (i) of the EMP pet ind Po here, howe tian HMP pathway departs from the EMP pati wey (Fig. 5.2). Glue ever, wt phate is not isomerized to fructose 6-phosphate but is oxidize > Oh glucose ATP () BC 2.7.11 ADP glucose 6-phosphate NADP” (i) ED 1.11.49) NADPH + HY peglucono-8-lactone 6-phosphate (ui) EC 3,1.1.17 6-phosphogluconate ‘NADP* (iv) EC 1,1,1.44 © NADPH +H” p-xylulose S-phosphate —___)__rtbutose 8-phosphate —__) _ EC 5.1.3.1 +O, EC 5.3.1.6 Srethowy S-phosphate (WWEC 2.2.44 (wit) glyceraldehyde 3-phosphate | t ‘eset 1p dinydroxyacetone phosphate EC 5.3.14 wo canays (wo | Be 2.24.2 fructose 1, 6- >. —. diphoophate “fructose 6-phosphate erythrose 4-phosphate ayluloee 5 psp | Ga) } Ec 3.1.3.1 Gai) | EC 5.3.1.9 xii) | EC 22d fructose 6-phosphate phosp! glucose 6-phosphate fructose 6-fhosphate slyceraldeyt ‘S-phosphale tye slucose 6-phosphate Ly glucose 6-phosphate mm Fig. 6.2. Hexose monophosphate (HMP) pathway of glucose utilization.LisM 223 180) pum wo #. On g wou | NADP* NADPH + it* —-) Gort \ / HCOOH worn 9 HOCH eo HCOH ue ng — H,COPOs H,Copo; «thoaphate Preteen 0-lactone pP-linked glucose-6-phosphate deh: it? the NADP oxidoreductase, EC 1.1.1.49) virogensien (>-glucose- T nosphate? Nono-dlactone G-phosphate. Thecnarre vais Tenetion (ii) elco t a nzyme glucose-6-phosphate roatuces ‘ase is becoming known as an important nara Kaosgmen not only by NADI te canta gtC® a it Ce 355), but also by ATP, as in the case of the (sce P. rote was exposed in studies of the metabolism of #7, jenomonas be + mixotrophic bacteria. Here, glucose-6-phosphate denegemracs and jates the use of the autotrophic carbon dioxide fixation or heterotrophic regulate ym, Although this enzyme is mainly N. i i meal. ey linbed somactaas ly NADP* linked, it can also or elue ose-6-phospbate dehydrogenase is localized exclusively in the jouble fraction of the cell and has been detected in a wide variety of cells and tissues in the plant and animal kingdoms (yeast; Escherichia coli; Sarillus sublilis; Bacillus megaterium; Pseudomonas fluorescens; Zymomenaa mobilis; Leuconostoc .mesenteroides; red, blue-green, and green algae; and higher plants). The enzyme of yeast, Escherichia coli, and mammalian tissues origin is specific for NADP*. However, it has been reported (28a, 69, 405) that the enzyme lacks coenzyme specificity and is also active with NAD*. This lack of specificity could also be owing to a transhydrogenase (69), which catalyzes the reaction pseudomonads (377). NADPH + H* + NAD+ — NADP* + NADH + H+ as is the case in Pseudomonas fluorescens (405) or two different de- hydrogenases with distinctly different functional roles as in Acetobacter zylinum (28a). Although the physiological function of transhydrogenases are not fully elucidated as yet (316), they appear to exist wherever NADP*- linked dehydrogenases are functioning. The enzyme in Aspergillus flavus- oryzae is specific for both NADP* and NAD*. In Bacterium anttratum (165) and Bacillus megaterium (44), glucose dehydrogenase can metabolize slueose without the intervention of pyridine or flavin nucleotides. It is therefore assumed that a novel prosthetic group is involved.7h 7 aty 224 ct of the glucose-6-phosphate deh The produ Pt) Teh drogen > . zed (reaction to 64 almost immediately hydroly: PhOMD hog “et, “Onag ‘ey ” n ator no don work 9 H ndon Hdon Ht fon 4,CoPo, ¥,Copo,- p-glucono-0-lactone 8-phospho. 6-phosphate gluconolactonase (p-glucono-é-lactone by is known about this enzyme because of ii A second NADP+-linked oxidation oc to the formation of p-ribulose S-phosphate. ‘The reaction» hy wd aed COOH HCOH NADP* NADPH + Ht HCOOH soln bao HOOH HOOK HCOH co, HCon #H,COPO, H,COPO> 8-phospho- Fibulose gluconic acid phosphogluconate dehydrogenase [6-phosp! reductase (decarboxylating) yeast by Warburg and Christi ian in 1933 (889). ‘They obtained ey that the product was a Cs compound, moce likely a pentose phayint Which we now know to be ribulose 5-phosphate = ° I - CO, ¢=o ¢-o + wee NADP* HCOH ‘Mart H,CoH c=o0 =— c=0 I uCoH = NADPH + Ht HCon HCOH HCOH HCO Gon H,COPO, H,COPo> H,COPO; 6-phospho- luconie acia sephorie vdrolase, BC 3.1.4 15 ts quick catalytie 1): curs in reaction ho--gluconate: NaDp oxi » EC 1.1.1.44], wag 5. CARDONY Dany, Bluconic acig La. a vi Ae react on lng, 5-phosphoric acid first demonstrated a 5-phosphoric acidsont 225 w t that 3-keto-6; $ jons sugeest © ‘Phos iO _igation” “the reaction, but this ¢ Phogluconate (239) may este te in * compound hag y med erected. Although the equilibrium of 64 not yet been inte over 1egative decarboxylation, the reset, Phosphogluconate 8 avon the enzymatic fixation of UCO, ings yes Teversible, as jot tod DY SHY a by the reductive carbo; the C-1 atom of ra te xylatioy y ot pogeeme nce of NADPH ++ H+ and eatbon diocy i Moe S-phos- ajority of workers reported. pr exclusively in the soluble fracth ee oPhORlUconate de nayme is associated witha particulate fracti cell, there is ot rescens (403). &-Phosphogluconate dehydrogenase : in Peoudo- f pution in the animal and plant kingdoms to glucos, re is similar jst Mee. THE mammalian and Escherichia coli (367 ‘phosphate rose ps, but the enzyme obtained from Leyepy, ) enzymes are nats t onostoc ‘oides en 26 times the rate of with gl NADP’ NADP*. As seen with gluccse as D A ill iuces dehydrogenase, Aspergillus flavus-oryzae ¢, ‘i i aie dehydrogenases that are specific for either Nabe PHO weommgence of NADP*~ and NAD*linked 6-phompbeg eons estion of an enzymatic at the ¢ te cette in Rhizobium (264) led to the suge gaye fjon.of thizobia. serene gluconate dehydrogenase, like glucose-6- Phase js an important regulatory eng; Phosphate dehy- < :yme, for it is inhibited i ate, ATP, and possibly NADPH + H+, depending on ate i #,cou é=0 noe Hoon #,COPO; xylulose 5- phowphoric act H,COH ¢=0 - HCOH HCOH H,COPOS No ribulose 5- i phosphoric acid HCOH HOH HCOH H,COPOs ribose 5- phosphoric acidar Jd therefore play an important (90a). Tt comulation (see p. 255). rot ig hosphate ia attacked partly by two dig, w-Ribulowe TP mulosephosphate J-epimernse (Deridutga Mt “ ‘The erase, WO 5.1.8.1) converts ribulose 5-phosphate | >Dh, ym. Tel cametion ¥), whereas the ensyie FIbO%-S-phogpy Mt 3 (eribose-5-phosphate ketol-isomerase, Eo 5.8.1.0) is remponstt formation of ribose 5-phosphate (react - my: This type one fe inakes it possible for the organism to replenish ribose F-Dhoap ch, q saenemadinte forms the basic precursor for the purine, yy'hate, wen aromatic amino acid biosynthesis. ; Midin rw Poth intermediates, xylulose 5-phosphate and ribose 5. required for the cleavage reaction (vii) of the enzyme tram, Ptolagt hy (m “tf nlon transketolase quo h ied wot ggg Smaeeeet upon gh H,Copo; Hogg H¢on i ube dopo, COPO, glyceraldehy i x " 3 phosphoric sia Heo, xylulose 5- ribose 5- Ry phosphoric acid phosphoric acid oe *-Bhomtag doheptulose-7-phosphate:p-glyceraldehyde-3-phosphate gly, “ transferase, EC 2.2.1.1), which yields glyceraldehyde Bepheedtldetya, sedoheptulose 7-phosphate. SPhate ta The second cleavage reaction of this pathway (ix) cleayes b mediates from the transketolase reaction and produces p-fry, tL inte ru phate and p-erythrose 4-phosphate. tose 6hog megan ngon qe Hota ti aldol: nola. a0 HCOH + HCOOH —Zansaldolase no + Hoow H,COPO, HCOH non HCOH glyceraldehyde HOH H,COPO, = . ceral : 3"phosphorie acid H,COPO; * tian sedoheptulose hore aa fructose 6- 7-phosphoric acid phosphoric acid ahs reaction is catalyzed by transaldolase (sedoheptulose-7-phosphate: elyceraldehyde-3-phosphate dihydroxyacetonetransferase, EC 2.212)g® 227 \COH 10% 0 “— to, niton !Fansketotase | FHO 04: yoo! HCOH ron + " ‘ie Con 4,CoPo, uGOH POs fly coraldenyde nfo Ws 3 phosphorie weld H,CoPo, 1s}0re acid fructone 6- sityoric aC phosphoric acid @ maa fihon HoH —— i CH,OPOs” soe genyde 3phosphate 4 ‘Ha—O—POs giyoeral ihydroxyacetone phosphate GH.0Por cH,—0_po; ° ° cH,OH fractoreionomnate HOCH + I HCOH CHO | t HgOH HCOH - b--OPOS oe CPOs CHr 3 fructose 1,6-diphosphate uel qin0n co co ba sie Hoe HOCH 4. H,0 phatase cH HOOH HCOOH HgoH CoH CH,;—OPO,” CH,—OPO;_ fructose 1,6-diphosphate fruccose 6-phosphate HL CHO H HL qo T 2 HCOH | glucosephosphate HOCH isomerase HOCH HGOH HOH HCOH or HOCH 9: HCOH HCOH HOH CH.—oPO du,—oro He CH,—OPOs- fructose 6-phosphate glucose 6-phosphate5. CARBORYDRAn | y Bp, i rr te, like ribose 5-phosphate, is an impo, . 4 jmidine and in aromatic amino acid biogy,, for purine and pyri tion (xiii), which in carried out by qn theni 5 es ih firat cleavage reaction (vii), cleaves erythrog ain ketolase ne the |, phosphate, yielding glyceraldehyde me Ae 228 Brythrose a-phospha' with xylulose "ha hate. te p-fractose 6-phosp! actions the HMP pathway links y, ty : vage reac’ a eat creme tose G-phosphate and glyceraldehy jc" thy pathway © app Bly level. The eye! » n be complete, because fructose 6-phog,) Pho se aeane @-phosphate with a glicosephosphate ate hy converted 0 ee also in reaction (ii) of the EMP Pathan Mera product of the cleavage reaction, p-glyceraldehyde 3-phosphat, i he 8 The reverse EMP pathway and also form p-glucose S-phospha for), formation of dihydroxyacetone phosphate by triosephosphayg is". ny is identical to reaction (v). The one of both triose Phosp yet fructose 1,6-diphosphate by fructose-diphosphate aldolase ig alae a with reaction (iv) of the EMP pathway. In order to form Fructose tty phate, the organism must produce a separate enzyme, as reetig of the EMP pathway catalyzed by phosphofructokinase is an jp," (iy reaction. Fructose 1,6-diphosphate can be converted to fructose ote phate by the enzyme hexosediphosphatase (o-fructose-1,6-dipjo, J-phosphohydrolase, EC 3.1.3.11) (121, 122), and this is also an ine ble reaction. This enzyme has been reported to exist in two fo ee in rabbit liver. A recent purification from Acinetobacter (28) jy 2) an allostery to ATP and citrate. The possible importance of this a" for regulation is not as yet certain. The “end product” glucose 6p TAY is obtained by the action of glucosephosphate isomerase (EC 5.3.1.9) noted above. ne This complete HMP pathway represents what is called the « eycle” or “shunt” and has the following sum of reactions: Potton glucose + 12 NADP* +7H,0 + ATP. 6 CO, + 12(NADPH + H') +H,P0, + app Oxidative microorganisms, however, can also use the partly comple cycle to produce pyruvate from glyceraldehyde 3-phosphate, utilizing the same enzymes of the EMP pathway that catalyze reactions (vi-x) (se p. 219). The sum of reactions for the incomplete cycle would thus be: 3 glucose + 6 NADP* + ATP 2 fructose 6-P + glyceraldehyde 3-P + 3CO, + 6(NADPH + H+) + ADP*Hj?0: If the usefulness of both the complete and the partly complete HM? pathway are compared, it could be envisaged that oxidative micBOLISM META) 220 sont most cert y Mectaal tiling the partly complete pathway to niet encry Via pyruva the TCA cycle, However, those micro- obta¥t ms which ai lize wiusone mainly through the EMP pathway robie conditions would utilize the compl ’ complete HMP pathway ror the purpose of Lepr the Precursors for their purine, pyrimidine, oO matic amino ie iosynthesis. Such microorganisms as the lacto- nd yi, which utilize solely the EMP pathway for their glucose degradation, pacil complex moda 2 compensate for the loss of biosynthetic ability. aire incomplete HMP cycle requires 1 mole of ATP and produces 2 of ATP in the formation of pyruvate. Reduced pyridine nucleotides les * oe iso formed and could function as hydrogen donors for the reduction HCHO ribose 5-P HCHO [ ribose 5-P HCHO, ribose 5-P allulose 6-P ——_allulose 6-P allulose 6-P [| | fructose 6-P fructose 6-P _ fructose 6-P xylulose 5-P _ erythrose 4-P glyceraldehyde 3-P xylulose 5-P glyceraldehyde 3-P_— dihydroxy- acetone P Scheme 5.1. Allulose 6-phosphate pathway of Pseudomonas methanica.5 CASBORYDR Ary a i, thway therefore does no of an organic compound. a order to function. As the peta to be dependent on mavme, it can function both aerobied stay A no OXON tioning of the HMP pathway is also of gi 8nd «thy » The functionnd chemoaynthetic autotrophs because ins tosynthetic and che totrophe because aim to photosyne ved by condensing carbon dioxide with ribuy carbon is dertver rived from ribulose 5-phosphate, ose 15 ah Pete nt variation of the HMP pathway has been founa Pseudomonas methanica. AS will be mentioned when rerot, ® tae, is considered (see Chapter 6), F th mathanica Onichiggs Ming stepwise via methanol, formaldehyde, and formate to ca was thought that methane and methanol were rapidly inn growing cell suspensions into sugar phosphates, whereas ¢.. was incorporated mainly into Cedicarboxylic acids (189). ‘7, work, however, revealed that it is the formaldehyde that, ig s°°" Ha into phosphorylated compounds, whereas formate is assimilated ; in and malate (207). ; : | It appears that high fixation occurs between the Oxidation k methane and formaldehyde and low fixation between formate and ely dioxide. This means that formaldehyde must be the oxidatien 1 which most of the carbon is assimilated into cell constituents, wits | observation that all the pentose phosphate cycle intermediates ith | Pseudomonas methanica and with the identification of a new hexose a phate, allulose 6-phosphate (206), it has been possible to Suggest te nism for formaldehyde incorporation. The first reaction involves ahy leths, methylation of ribose 5-phosphate and formaldehyde to allulose hoon (Scheme 5.1). With this mechanism functioning, Pseudomonas ; is able to produce most of the required intermediates for its cell matey biosynthesis. Thon «; Entner-Doudoroff Pathway This pathway is one of the most recently described pathways, discovered by Entner and Doudoroff (114) during metabolic studies of Pseudomony saccharophila. It has since been found in a number of other bacteria (26, 64, 84, 145, 218, 219, 373, 391) (see Fig. 5.3). Reactions (i), (ii), and (iii) are identical to the ones of the HMP path way, although it is not as yet certain whether or not the enzymes themselve differ in their kinetic characteristics. The marked differences of this path way from the other two is the dehydration of 6-phosphogluconate (reaetie® iv) (218) to form 2-keto-3-deoxy-6-phosphogluconate (KDPG) by § Phosphogluconate dehydratase (6-phosphogluconate hydro-lyase, a 4.2.1.12). Tt has been proposed that 6-phosphogluconate is debydr” cost METABOLISM ow 231 Blucose GEC 27.44 [CAT App lucose 6-phosphate NA Gi) EC 1.1.1,49 me NADPHY He glucono-8-lactone 6-phosphate Gil) EC 3.1.1.17 | 6-phosphogluconate (ivi EC sana | 2-keto-3-deoxy-6-phosphogluconate (W) EC 4.1.2.14 ctnoaphate 4 glyceraldehyde 3-phosphate pyruvate (wi) EC 2.2.1.1 fructose xylulose 5- iid ribulose 5- éphosphate erythrose 4-phosphate Phosphate, S57 ph oaphaie EC 2.2.1.2 (iii) sedoheptulose 7- phosphate glyceraldehyde 3-phosphate EC 2.2.1.1 xylulose 5-phosphate ribose S-phosphate Fig. 5.3. Entner-Doudoroff pathway of glucose utilization.6. CABBORSD Ray 4 ny, 232 coon ~ a Le HOCH . Bn ndon ate Oo - H2COpPo,~ : heton3 6) H,COl 2-keto-3-deoxy- Bet Phan 6-phosphogluconic acid _>-keto-3-deoxy-6-phosphogluconate, which izes frat Seoto-3-deoxy-6-phosphogluconate, POntan, ketoni bsequent cleavage reaction by phospho-2-keto-3- deoxy atlas (e-phospho-2-keto-S-deoxy:Preluconate P-glyceraldeh de 1, 1214) cleaves the 2-keto-B-deoxy-6-rhosphiog “Dlg ‘On, te phate-lyase, EC 4. first to form © ly [90K COOH eS do r nen wma 7 ndéox ce HCOH wee Hz coro ae one scoic Soren acid : into glyceraldehyde 3-phosphate and pyruvate (reaction v). This cleayag reaction is very similar to the fructose-1,6-diphosphate aldolase reaction in the EMP pathway. Intensive investigations (159, 273, 325, 359) revealai that the reaction follows the same pattern via a Schiff base intermediate and that the enzyme is a trimer, whereas the fructose-1,6-diphosphute aldolase is claimed to be a tetramer. In addition to the cleavage reaction, KDPG aldolase also catalyas the enolization of pyruvate (273). It is therefore assumed that KDP0 aldolase binds pyruvate by forming a Schifi’s base (azomethine) (15!) between the «amino group of lysine and the carbonyl group of the st strate (Fig. 5.4). The attack of the substrates is therefore initiated by electron pair of an amino nitrogen, forming a KDPG-lysine azomettise5p METABOLISM = ° 233 do g 4 T " ,-°- g + C-0 ER c~o- aot R-N—c_ow I el I B-R—NA~G Hy -c-H = i | H _—— a my moog SS ER -—OH I—C_OH “oak ago an H—C_OPO.H- Hd ope 4- (OH H i H—C_opoyH- . H H azomethine oc? H—C—o [>o- E—R_N. ! ae HC—oH + HC H—C—OPO3H- H H? Ny I Ht Pyruvyl anion, GAD-P ° 4 Zo c H cm oO [>O- _+m0 1 [So- BOK ERNE “so PORN —C—on FRN+C—0 H CH; H CH; CH; pyruvate Fig. 5.4. Proposed mechanism of a-keto-3-deoxy-6-phosphogluconic acid aldolase (Meloche and Wood (273); reprinted with the permission of Journal of Biological Chemistry]. which is rearranged with the elimination of glyceraldehyde 3-phosphate and yields a pyruvyl anion. The pyruvyl anion can then be attacked either bya proton or by a tautomeric form of glyceraldehyde 3-phosphate, yielding pyruvate in the former and KDPG in the latter case. The overall system is completely reversible. Acetobacter melanogenum and Azotobacter vinelandii also show KDPG aldolase activity as well as that of phosphogluconate dehydratase. KDPG aldolase was not detected in Leuconostoc mesenteroides 8081, Corynebacterium creatinovorans 7562, Microbacterium lacticum 8081, illus arabinosus 8014, or brewer's yeast. The triose phosphate formed during the cleavage of glyceraldehyde ‘phosphate is now free for use in the EMP pathway to pyruvate. Where5. CARBOHYDRany, ~ followed it yields 2 Cred inal Naame Subst thin pathway ie imiach of reduced NA thway, howeve 7 cose), and | mole vant feature of this pathway, ai that ; Spe more important feat precursors for pyridine 9, Mt oy, terme $0 produce | ‘amino acid biosynthesis by a Teversed jy P¥tinibly, hway utilising the same enzYMeS Aas discusgey "Xn, Tg ng ye % b Oy ‘fas well as for a phosphate rethwtuchyde 3-phosphate may condense (ree the pathway. Glyoern deh ing erythrose 4 phosphate and Sine "Se Fructose eens n ja catalyzed by transketolase (KC aan 5 phate. ae or pimerase (EC 5.1.3.1) converts xylulose Bey ib phosphate ‘hophate (reaction vii), whereas a transaldolan. Cheats ribulose oe Miehyde 3-phosphate and sedoheptulose T-phomp 22 pace hate and fructose 6-phosphate (reaction 7 te erythrose 4-phosp! 5. hate are the products from ). Rh phosphate and xylulose 5-phosphate are t! wets from ae ies 5-phosp! EC 2.2.1.1) reaction (reaction ix). Depending on the tran xetetet conditions, microorganisins are able to use the revert’ ancle to meet their pyridine and pyrimidine requirement OF, alterna Pen % len, ‘ i 0 form pyruvate from glyceralde, nt Oo eae sae Phen be vnetabolized in ways to be conn ae three different pathways could serve certain aap metabolism. The EMP pathway provides the greatest amount of i a | 2 moles, but does not produce the most important: precursors gq AT? and pyrimidine biosynthesis, ribose 5-phosphate and erythrose Sphoeph Therefore, it could be assumed that microorganisms using this Patna, must have specific growth requirements for pentoses, purines, or Pyrimig: in order to build, for example, their nucleic acids, In Seneral, onpuniens that grow only on complex media, e.g., media containing meat extra, and yeast extract, use this particular pathway (257, 326). The HMP pathway, in contrast, produces all the Precursors neg, for purine and pyrimidine biosynthesis but produces only half the amount of ATP. This pathway does not produce pyruvate directly, so the Organism must have at least part of the EMP pathway enzyme complex, from glycer. aldehyde 3-phosphate onward, to form pyruvate. It is therefore not sux prising that both pathways may be present in those organisms that dy possess the HMP pathway (see Table 5.3) and that are therefore inde pendent in regard to their purine and pyrimidine requirements. The ratio of usage of the EMP and HMP pathways can vary greatly, depending 0 environmental conditions. The Entner-Doudoroff pathway is at the other side of the HMP, where the reverse HMP pathway may be present but the direct formation vate makes it possible for it to be independent of the other two be ways. The production of 1 mole of ATP and all necessary pentoses malt9g MPTABOLISM al , 5 TABLE 5. way Estimations Microorganisms EMP HMP ED romyces cerevisiae Candida wilis oon 12 = ‘Streptomyces griseus 7 1 19-34 = penicillium chrysogenum 7 a Escherichia coli ae 23 ‘sarcina lutea to 28 Bacillus subtilis a 30 Pseudomonas aeruginosa & 26 ‘Acetomonas oxydans ~ 29 n Zymomonas mobilis 100 -_ 100 «From (62). (Reprinted wi ins taint he wien igen of John Wiley and Sons.) iPr phere are Some groups , par icularly as a number of enzymes are the sam i" ° groups of bacteria, particularly gram-negative pacteria with a deoxynucleic acid (DNA) base composition of between 72 and 70% guanine plus cytosine (G + C) (211), that are able to use this pathway alone. Table 5.3 indicates the percentage distribution of these pathways in some microorganisms. The reasons for the individual thoices are not yet quite clear. Tt was thought that oxygen may play an important role in the selection of pathway usage, since the majority of anaerobic bacteria were found to contain the EMP pathway, e.g., clostridia (12, 14, 157, 234, 263, 307, 360, coey enteric bacteria (46), spirochetes (135, 196, 175), and sarcinae (56). Those bacteria which are facultative aerobes were found to contain s combination of EMP and HMP pathway, e.g., Escherichia coli, and strict aerobes were found to contain almost exclusively the ED pathway, eg, pseudomonads and Rhizobium (203). However, the findings that the strict anaerobes Zymomonas mobilis and Zymomonas anaerobia (250) tan use only the ED pathway, that Clostridium aceticum can use a modified ED pathway (12) with gluconate as carbon source, and that homofermen- tative lactobacilli (154a, 278) can use the EMP pathway even under aerobic conditions, as Ferrobacillus ferrooxidans does, cast doubt on the early assumptions. ‘A few examples of the participation of mixed pathways and the problems they involve for metabolic studies and evaluation in general will now be given. The pathway of p-glucose metabolism in Salmonella typhimurium (120) can follow either a combination of the EMP and HMP pathways or the ED pathway. Glucose strongly inhibited catalase synthesis (222) and