JOURNAL OF BACTERIOLOGY, May 1971, p. 456-467 Vol. 106, No. 2 Copyright ( 1971 American Society for Microbiology Printed in U.S.A.

Mechanism for Regulating the Distribution of Glucose Carbon Between the Embden-Meyerhof and Hexose-Monophosphate Pathways in Streptococcus faecalis ALBERT T. BROWN1 AND CHARLES L. WITTENBERGER Microbial Physiology Section, Laboratory of Microbiology, National Institute of Dental Research, Bethesda, Maryland 20014

Received for publication 5 February 1971

Glucose-adapted Streptococcus faecalis produced little if any "4CO2 from glu- cose-1-'4C, although high levels of glucose-6-phosphate dehydrogenase (EC 1. 1. 1.49) and 6-phosphogluconate dehydrogenase (EC 1. 1. 1.44) were detected in cell-free extracts. Metabolism of glucose through the oxidative portion of the hexose-monophosphate pathway was shown to be regulated in this organism by the specific inhibitory interaction of the Embden-Meyerhof intermediate, fructose-1,6- diphosphate (FDP), with 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase activity was unaffected by FDP. The S. faecalis 6-phosphogluconate dehydrogenase was partially purified from crude extracts by standard fractionation procedures and certain kinetic parameters of the FDP-mediated inhibition were investigated. The negative effector was shown to cause a decrease in Vmax and an increase in the apparent Km for both 6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP). These effects were apparently a consequence of the ligand interacting with the enzyme at a site distinct from either the substrate or the coenzyme sites. Among the evidence supporting this was the fact that ,B-mer- captoethanol blocked completely FDP inhibition, but had no effect on catalytic activity. The possibility that the regulation of 6-phosphogluconate dehydrogenase activity by FDP might be of some general significance was suiggested by the obser- vation that this enzyme from several other sources was also sensitive to FDP.

Glucose-6-phosphate occupies a pivotal posi- sedoheptulose-7-phosphate in addition to ery- tion in carbohydrate dissimilation, forming as it throse-4-phosphate (34). The first enzyme unique does, a branch point between the Embden-Mey- to the hexose-monophosphate pathway, glucose- erhof and hexose-monophosphate pathways. The 6-phosphate dehydrogenase, is inhibited in Esch- importance of this was stressed (25, 29, 33), but erichia coli by reduced nicotinamide adenine relatively little attention has been given to a com- dinucleotide (NADH) and is activated by spermi- prehensive consideration of factors which may op- dine and certain other polycations (28, 29). Con- erate to regulate the distribution of glucose trol of the E. coli enzyme by NADH inhibition is carbon between these two major pathways. Sev- apparently not a universal regulatory mecha- eral instances of inhibition of key enzymes by nism, however, since the enzymes from mam- diverse metabolites were reported, however, malian tissue (J. Passonneau et al., Fed. Proc., which are interpretable in regulatory terms. For 25:219, 1966) and Pseudomonas aeruginosa (16) example, the Embden-Meyerhof enzyme glucose- are insensitive to NADH but are strongly inhib- 6-phosphate isomerase from mammalian sources ited by adenosine triphosphate (ATP). The glu- was found to be inhibited by both 6-phosphoglu- cose-6-phosphate dehydrogenases from Hydro- conate (13, 22, 23) and erythrose-4-phosphate genomonas facilis (30) and from yeast (1, 8, 12) (10), and the enzyme from yeast is inhibited by are also sensitive to ATP, although their re- I Present address: Department of Oral Biology, School of sponse to NADH was not reported. Another Dental Medicine, University of Connecticut Health Center, hexose-monophosphate enzyme, ribose-5-phos- Farmington, Conn. 06105. phate isomerase, was reported to be inhibited by 456 VOL. 106, 1971 EMBDEN-MEYERHOF AND HEXOSE-MONOPHOSPHATE PATHWAYS 457 6-phosphogluconate in Pediococcus pentosaceus faecalis MR cells were stored as a lyophilized powder (3). at -20 C until used, whereas S. mutans cells were re- Streptococcus faecalis seemed to be an appro- suspended in 0.05 M Tris-hydrochloride buffer (pH 7.5) priate organism for studying systematically the and used immediately for the preparation of cell-free extracts. factors that operate to regulate the flow of glu- Dried cells of Candida utilis (stock number YCU) cose carbon between the Embden-Meyerhof and and Saccharomyes cerevisiae (stock number YSC) hexose-monophosphate pathways. This homofer- were obtained from Sigma Chemical Co., St. Louis, mentative organism was shown to possess the Mo., and were washed once with 0.05 M Tris-hydro- enzymes glucose-6-phosphate dehydrogenase, 6- chloride buffer (pH 7.5) before use. phosphogluconate dehydrogenase, and transketo- Lactobacillus plantarum ATCC 11739 was obtained lase when grown with glucose as the primary from M. Rogosa and was cultivated in the medium of energy source (J. T. Sokatch, A. P. Prieto, and de Man, Rogosa, and Sharpe (19). E. coli strain 0127: I. C. Gunsalus, Bacteriol. Proc., p. 112, 1956; B8 (Difco) was provided by T. Tempel and was grown in Brain Heart Infusion Broth. Cultures of both orga- 32). The presence of transaldolase under the nisms were harvested from the stationary phase of same growth condition is uncertain (32), al- growth, and the cells were washed once with 0.05 M though its function in gluconate-adapted cells Tris-hydrochloride buffer (pH 7.5) before use. was inferred from labeling studies (31). S. fae- Preparation of cell extracts. Lyophilized cell powder calis, therefore, has the potential to utilize at (S. faecalis MR) or wet cell paste (S. mutans) was re- least the oxidative portion of the hexose-mono- suspended in 0.05 M Tris-hydrochloride buffer (pH 7.5), phosphate pathway for glucose dissimilation. and the cell suspensions were disrupted by treatment Other studies, however, established that the sole with a Branson Sonifier for 20 min at maximal voltage product of the glucose fermentation is lactic acid output. The disrupted cell suspensions were then centri- (7, 24), and that the lactate is derived from glu- fuged in the cold for 30 min at 37,000 x g, and the supernatant fluids were collected by decantation. The cose more or less exclusively through the crude, cell-free extracts usually contained between 10 Embden-Meyerhof pathway (24). and 20 mg of protein per ml and were used immedi- In this communication, we report results of a ately for enzyme assays. study designed to gain insight into the factors Chemicals. The coenzymes, glucose-6-phosphate, involved in channeling glucose preferentially disodium-6-phosphogluconate, streptomycin sulfate, through the Embden-Meyerhof pathway in S. and fructose-I , 6-diphosphate, were all products of faecalis. It will be shown that this is accom- Sigma Chemical Co., St. Louis, Mo. Ultrapure ammo- plished, at least in part, by the specific inhibition nium sulfate was used in all enzyme fractionation pro- of 6-phosphogluconate dehydrogenase by the cedures and was obtained from Mann Research Labo- ratories. Glucose-l-14C and gluconate-1-_4C were pur- Embden-Meyerhof intermediate, fructose-1,6- chased from the Amersham/Searle Corp. diphosphate. A preliminary report of these find- Analytical procedures. Protein was determined by ings has appeared (A. T. Brown and C. L. Wit- the biuret method (9), or, in the case of preparations tenberger, Fed. Proc., 29:399, 1970). containing high concentrations of ammonium sulfate, the method of Warburg and Christian was used as de- scribed by Kalckar (14). MATERIALS AND METHODS Glucose was assayed with glucose oxidase (Wor- Organisms and culture conditions. S. faecalis strain thington Glucostat reagents) after removing cells from MR (17) and S. faecium strain N-55 were obtained samples by centrifugation. The pH of all samples was from Jack London, and S. faecalis IOCI was obtained adjusted to neutrality before carrying out the glucose from T. Shiota. The organisms were grown anaerobi- determinations. cally in a complex medium under conditions previously Enzyme assays: 6-phosphogluconate dehydrogenase. described (37) except that the final concentration of 6-Phosphogluconate dehvdrogenase activity was meas- glucose in the medium was 0.5% (w/v). In cases where ured by following the increase in absorption at 340 nm compounds other than glucose were employed as pri- resulting from the 6-phosphogluconate-dependent re- mary energy sources, all were sterilized separately and duction of nicotinamide adenine dinucleotide phosphate added aseptically to the medium at a final concentra- (NADP). All measurements were made at room tem- tion of 0.5% (w/v). perature with a Beckman model DB-G spectropho- All strains of S. mutans (11) were supplied by H. V. tometer equipped with a Sargent model SRLG re- Jordan. These organisms were grown in the same com- corder. The standard assay system contained disodium plex medium used for the cultivation of S. faecalis 6-phosphogluconate, 2.0 mM; NADP, 0.25 mM; Tris- MR, except that the yeast extract concentration was hydrochloride buffer (pH 7.5), 100 mM; and water to a increased to 0.5% (w/v) and 0.05% Tween 80 was also final volume of 1.0 ml. Reactions were initiated by the added. Growth conditions for S. mutans strains were addition of crude cell extract or partially purified en- the same as those described for S. faecalis MR. zyme. Initial reaction rates were linear under the condi- Both S. faecalis MR and S. mutans cells were har- tions described, and were proportional to enzyme con- vested from the stationary phase of growth and were centration within the ranges employed. One unit is de- washed one time with 0.05 M tris(hydroxymethyl)ami- fined as the amount of enzyme required to catalyze the nomethane (Tris)-hydrochloride buffer (pH 7.5). S. reduction of I Amole of NADP per min under the 458 BROWN AND WITTENBERGER J. BACTERIOL. assay conditions described. Specific activity is ex- 37,000 x g for 20 min. The crude, cell-free extract was pressed as units per milligram of protein. In the case of dialyzed overnight against 9 liters of the KCI-KHCO, fructose-1 , 6-diphosphate inhibition studies, reactions solution, and the protein concentration was thkn ad- were initiated with 6-phosphogluconate immediately justed to about 10 mg per ml by dilution of the di- after mixing of the other reactants. If the enzyme was alyzed extract with the KCI-KHCO3 solution. Solid preincubated with FDP for 2 min before starting the ammonium sulfate was next added to the diluted, di- reaction with 6-phosphogluconate, the initial reaction alyzed fraction to 50% saturation, and the protein pre- rate was nonlinear and increased with time. cipitate was removed by centrifugation and discarded. Glucose-6-phosphate dehydrogenase. Glucose-6- To the supernatant fluid, solid ammonium sulfate was phosphate dehydrogenase activity was measured by again added to 70% saturation. The resulting protein following the increase in absorption at 340 nm result- precipitate was removed by centrifugation, redissolved ing from the glucose-6-phosphate-dependent reduc- in 9.0 ml of distilled water and dialyzed overnight tion of NADP. The standard assay and fructose-1,6- against 4 liters of distilled water. A precipitate which diphosphate inhibition studies were carried out exactly formed during dialysis was removed by centrifugation, as described for 6-phosphogluconate dehydrogenase, and the supernatant fluid served as the source of 6- except that 2.0 mM glucose-6-phosphate was substituted phosphogluconate dehydrogenase. This fraction, which for 6-phosphogluconate. Enzyme units and specific ac- contained 6.63 mg of protein per ml, had a specific ac- tivity were as described for 6-phosphogluconate dehy- tivity 3.3 times greater than that present in the crude, Preparation of cell suspensions and reaction mix- cell-free extract. tures for isotope studies. Cultures were grown with glu- Partial purification of S. faecalis 6-phospbogluconate tures for isotope studies. Cultures were grown with glu- dehydrogenase. All steps were carried out at 4 C unless cose or gluconate as the primary energy source and otherwise specified. cells from 150 ml of medium were harvested from the Step 1: crude extract. A crude, cell-free extract was stationary phase of growth and washed once with 0.01 prepared as described previously from I g (dry weight) M potassium phosphate buffer (pH 7.0). Cells were then of lyophilized S. faecalis MR cells suspended in 40 ml resuspended in the same buffer so that a 1:100 dilution of 0.05 M Tris-hydrochloride buffer (pH 7.5). of each suspension gave an optical density at 660 nm of 0.100 (Beckman model DB spectrophotometer). Step 2: streptomycin sulfate treatment. A 40% solu- Reaction vessels were 10-ml Erlenmeyer flasks con- tion of streptomycin sulfate (in water) was added drop- taining a center well. Each flask contained in the main wise with constant stirring to the crude, cell-free ex- compartment: potassium phosphate buffer (pH 7.0), 72 tract until a final concentration of 3.5% was attained. mM; 1.0 ml of the appropriate cell suspension, and dis- The resulting precipitate was removed by centrifuga- tilled water to a final volume of 2.7 ml. To each center tion and discarded. well was added 0.2 ml of 1.9 N NaOH. Vaccine stop- Step 3: ammonium sulfate fractionation. Solid am- pers were inserted into the flasks, and each flask was monium sulfate was slowly added to the supernatant gassed with purified N2 through syringe needles for 5 fluid from step 2 with constant stirring until a final min. Flasks were then placed at 37 C for 15 min to concentration of 50% saturation was obtained. The allow for temperature equilibration. Reactions were protein precipitate was removed by centrifugation and initiated by injecting through the vaccine stoppers ei- was discarded. ther 0.1 ml of glucose-1-'4C (50 umo!es; 0.5 gCi) or 0.1 Step 4: ammonium sulfate precipitation. To the step ml of gluconate-1-14C (50 smoles; 0.5 qCi). Flasks 3 supernatant fluid was added additional ammonium were incubated at 37 C for 3 hr, after which time the sulfate until a final concentration of 75% saturation reactions were terminated by injecting 0.2 ml of 6 N was reached. The resulting protein precipitate was col- H2SO4 into the main compartment. Incubation was lected by centrifugation and was redissolved in a min- continued for an additional 30 min to insure complete imal volume of 0.05 M Tris-hydrochloride buffer (pH trapping of any evolved 14CO2 in the center well. 7.5). Center well contents were then removed quantitatively Step 5: gel filtration. The step 4 fraction was next and placed in counting vials, which contained 6 ml of placed on a Sephadex G-200 column (2.5 by 45 cm) by ethanol and 10 ml of 0.4% 2, 5-diphenyloxazole (PPO) using the upward flow technique. The column was pre- in toluene. Vials were counted in a Packard Tri-Carb viously equilibrated with 0.05 M Tris-hydrochloride liquid scintillation spectrometer. Deviations from this buffer containing 0.5 mM iB-mercaptoethanol pH 7.5) basic procedure are noted in individual experiments. and was eluted with the same buffer. Effluent fractions Partial purification of guinea pig liver 6-phosphoglu- of 5 ml each were collected, and those showing the conate dehydrogenase. Repeated attempts to measure highest specific activity were pooled and stored at 4 C. 6-phosphogluconate dehydrogenase activity in crude Although A-mercaptoethanol was found to stabilize extracts of guinea pig liver yielded erratic results. The the enzyme to storage, this compound interfered with following procedure, however, produced a preparation some of the assays as will be discussed later. Therefore, which gave fully reproducible results. All steps were the ,3-mercaptoethanol was removed routinely from the carried out at 4 C. Two guinea pig livers were placed in enzyme at the beginning of each day's work by passing a cold mortar with about 50 ml of 0.15 M KCI con- a small volume of the step 5 fraction over a Sephadex taining 0.16 mM KHCO,. The livers were mascerated G-25 column. with a pestle and made up to 120 ml with the KCl- A summary of the results from the complete purifli- KHCO, solution. The suspension was treated for I min cation procedure is given in Table I, where it may be with a Branson Sonifier at maximum voltage output, seen that about a 20-fold increase in specific activity and cellular debris was removed by centrifugation at was achieved with a 63% recovery of the enzyme. VOL. 106, 1971 EMBDEN-MEYERHOF AND HEXOSE-MONOPHOSPHATE PATHWAYS 459 TABLE 1. Summary ofpurification procedure for 6-phosphogluconate dehydrogenase from S. faecalis MR

Total Speciric Fraction Total units protnpoen activity RecoveryR (m) (units/mg)

1. Crude extract ...... 405 840 0.0771 100 2. Streptomycin-S04 treatment ...... 390 710 0.0884 96.2 3. 0-50% (NH4)2S04 supernatant fluid ...... 365 280 0.2090 90.1 4. 50-75% (NH4)2SO4 precipitate ...... 327 168 0.3135 80.7 5. SephadexG-200eluate ...... 255 25.1 1.6334 63.0

RESULTS TABLE 2. Levels ofglucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in cell extracts Evidence for control of Embden-Meyerhof and ofS. faecalis MR and various strains of S. mutans hexose-monophosphate pathway activity in vivo. Preliminary evidence for some form of in vivo Enzyme Activities control glucose two of carbon flow between these Organisma (units/mg of protein)' pathways was obtained from studies in which the ability of various streptococci to produce labeled G6PD 6PGD CO2 from glucose-1-"4C was compared with the S. faecalis MR 0.0659 0.0740 relative levels of glucose-6-phosphate dehydro- genase and 6-phosphogluconate dehydrogenase in S. mutans strains corresponding cell extracts. During the course of FA-1 0.0016 <0.0003 this survey, it was found that a number of strains SL- I 0.0016 <0.0003 of S. mutans were unable to grow on gluconate 21-Typical 0.0024 < 0.0003 (A. T. Brown, unpublished data) and, as shown 6715-9 0.0016 <0.0003 in Table 2, this particular group of organisms 3720 0.0016 <0.0003 possessed extremely low levels of glucose-6- NCTC 10449 0.0016 < 0.0003 phosphate dehydrogenase and even lower levels a Organisms were grown anaerobically in complex of 6-phosphogluconate dehydrogenase compared medium with glucose as the primary energy source. with those found in S. faecalis. In spite of the Cells were harvested and washed, and cell extracts were fact that S. faecalis contained levels of the two prepared. enzymes ranging between 30 and 100 times h Glucose-6-phosphate dehydrogenase (G6PD) and 6- higher than those observed in S. mutans strains, phosphogluconate dehydrogenase (6PGD) were assayed it was no more active in metabolizing glucose in crude extracts of the indicated organism. through the oxidative portion of the hexose- monophosphate pathway than was S. mutans. This is shown in Table 3 where it may be seen calis MR also produced substantial amounts of that cell suspensions of S. faecalis produced little labeled CO2 from gluconate-I-14C, and this was more labeled CO2 from glucose-J-"4C than did almost completely inhibited when unlabeled glu- the S. mutans strains. cose was included in the reaction mixture (Fig. 1). These results strongly suggested that one or The observed glucose effect could be explained more factors were operating in S. faecalis to re- if glucose or a product derived from it inhibited strict the flow of glucose carbon through the the activity of gluconate permease, the gluconate hexose-monophosphate pathway. It seemed kinase or 6-phosphogluconate dehydrogenase. likely, moreover, that the control site was either The first two possibilities were considered un- glucose-6-phosphate dehydrogenase or 6-phos- likely since glucose-adapted cells, which do not phogluconate dehydrogenase or both, since these depend on gluconate permease or gluconate ki- are the enzymes involved in producing labeled nase for glucose utilization, also appeared to CO2 from glucose-1-14C. have a severe restriction imposed on the amount Additional information on the in vivo regula- of glucose passing through the hexose-mono- tion of the hexose-monophosphate pathway was phosphate pathway (Table 3). An effect at the sought by examining the effect of certain com- level of 6-phosphogluconate dehydrogenase syn- pounds on the ability of gluconate-adapted cells thesis was also excluded by the observation that to produce labeled CO2 from gluconate-1-14C. the specific activity of this enzyme, as well as Sokatch and Gunsalus (3 1) demonstrated pre- that of glucose-6-phosphate dehydrogenase, was viously that gluconate-adapted S. faecalis IOCI virtually the same in extracts of cells grown on a produced large amounts of "4CO2 from glu- variety of carbon sources (Table 4). This con- conate-l-_4C. Resting cell suspensions of S. fae- firms and extends a prior observation of Sokatch 460 BROWN AND WITTENBERGER J. BACTERIOL. TABLE 3. "4CO2 production from glucose-1i-4C by glucose-adapted cell suspensions ofS. faecalis MR and various strains ofS. mutans ,uMoles of "4CO2 Glucose 14CO, evolved/ Organisma used" evolved 100 (gmoles) (umoles) gmoles of w(.2 1C-I-Gluconote glucose 0 used

0a.) S. faecalis MR 48.1 0.38 0.79 CL) w -J S. mutans strains 0 FA-l 39.3 0.14 0.36 SL- I 21.8 0.08 0.37 21 -Typical 37.8 0.15 0.40 6715-9 41.9 0.21 0.50 3720 44.4 0.30 0.68 NCTC 10449 48.6 0.37 0.76 a All organisms were grown in glucose-containing, 50 100 150 complex medium, and cell suspensions were prepared TIME IN MINUTES from freshly harvested cells. FIG. 1. Effect of unlabeled glucose on "4CO, produc- bReaction mixtures were as described in Materials tion from gluconate-1_-4C by cell suspensions ofS. fae- and Methods except that the main compartment of calis MR. Cell suspensions and reaction mixtures were each flask contained 1.0 ml of the designated cell sus- prepared as described in the text. Separate reaction pension and 1.7 ml of complex growth medium minus flasks were used for each of the indicated time inter- glucose. Complex growth medium was added because vals. The cell suspensions incubated with labeled glu- cell suspensions of S. mutans strains were unable to conate plus unlabeled glucose (0) metabolized 20.0 metabolize glucose effectively in 0.067 M potassium Amoles ofglucose after iS0 min. phosphate buffer (pH 7.0) alone (C. L. Wittenberger and M. P. Palumbo, unpublished data). Each reaction TABLE 4. Effect of various growth substrates on the was initiated after gassing with N2 by the addition of levels of glucose-6-phosphate dehydrogenase and 6- glucose-1-_4C (50 gmoles; 0.5 MCi). phosphogluconate dehydrogenase in cell extracts - ofS. faecalis MR and Gunsalus (31), who reported that both of these enzymes were present in glucose- as well as Enzyme activity gluconate-grown S. faecalis lOCI. We tentatively Growth substratea (units/mg of protein)" concluded, therefore, that the likely site for regu- lation was 6-phosphogluconate dehydrogenase G6PD 6PGD and that the likely effector for regulating the ac- Sodium D-gluconate ...... 0.0691 0.0740 tivity of this enzyme was one or more interme- D-Glucose ...... 0.0788 0.0675 diate compounds from the Embden-Meyerhof D-Fructose ...... 0.0482 0.0643 pathway. D-Ribose ...... 0.0772 0.0723

Effect of various Embden-Meyerhof intermedi- D-Mannitol ...... 0.0675 0.0691 ates on 6-phosphogluconate dehydrogenase ac- D-Sorbitol ...... 0.0675 0.0659 tivity. A number of Embden-Meyerhof interme- DL-Serine ...... 0.0370 0.0498 diates were tested for their effect on 6-phos- a 5. faecalis was cultivated in complex medium with phogluconate dehydrogenase activity. Of all the the compounds indicated serving as the primary energy compounds tested singly and in various combina- source. tions, only fructose-I , 6-diphosphate (FDP) was ' Cell extracts were prepared from freshly harvested, found to be an effective inhibitor. None of the washed, cell suspensions, and assays for glucose-6- compounds or combinations thereof shown in phosphate dehydrogenase (G6PD) and 6-phosphoglu- Table 5 acted as inhibitors of glucose-6-phos- conate dehydrogenase (6PGD) were carried out imme- phate dehydrogenase from the same organism. It diately. appeared, therefore, that FDP was the com- pound responsible for directing the flow of glu- Some general information concerning the na- cose carbon primarily through the Embden- ture of the FDP-mediated inhibition of 6-phos- Meyerhof pathway in S. faecalis and that it did phogluconate dehydrogenase was sought next so by inhibiting specifically 6-phosphogluconate through a study of several kinetic properties of dehydrogenase. the enzyme. VOL. 106, 1971 EMBDEN-MEYERHOF AND HEXOSE-MONOPHOSPHATE PATHWAYS 461 Coenzyme specificity. 6-Phosphogluconate TABLE 5. Effect of various Embden-Meyerhof dehydrogenases from different sources were intermediates on 6-phosphogluconate dehydrogenase shown to possess different coenzyme specificities. from S. faecalis MR The enzyme from C. utilis, for example, is highly specific for NADP (26) whereas the enzyme Per cent inhibi- tion at a final from Leuconostoc mesenteroides reacts with ei- Compounda concn ofb ther nicotinamide adenine dinucleotide (NAD) or NADP, although the activity with NAD is about 1.0 mM 5.0 mM 25 times greater than it is with NADP (2). The Glucose-6-phosphate (G-6-P) 0...... 0 partially purified enzyme from S. faecalis (frac- Glucose-I-phosphate (G-1-P) ...... <5 0 tion 5, Table 1) appears to be specific for Fructose-6-phosphate (F-6-P) ...... <5 12 NADP. Significant levels of activity could not be Fructose-I-phosphate (F-I-P) ...... <5 <5 detected with NAD as the coenzyme under the Fructose-i ,6-diphosphate (FDP) ... 63c 78c standard assay conditions described above. If, Glyceraldehyde-3-phosphate 0...... O however, the NAD concentration was increased 3-Phosphoglycerate ...... 0...... <5 from 0.25 to 4.0 mM, a low but detectable rate of 2-Phosphoglycerate ...... <5 <5 NADH formation was observed which was de- Phosphoenol pyruvate ...... 0..... <5 pendent on 6-phosphogluconate. The reaction Pyruvate ...... 0 0 Lactate ...... 0 0 rate these conditions was still about under only ATP ...... <5 0 6% of that observed with NADP. As will be G-i-P + G-6-P ...... 0...... <5 documented in another publication, we found F-l-P + F-6-P ...... <5 <5 that S. faecalis adaptively forms a separate and G-i-P + F-i-P ...... 0...... 0 distinct NAD-specific 6-phosphogluconate dehy- G-i-P + F-6-P ...... 0...... 0 drogenase during growth on gluconate. The low F-i-P + G-6-P ...... 0...... 0 level of 6-phosphogluconate dehydrogenase ac- G-6-P + F-6-P ...... 0...... 0 tivity observed with NAD in the partially puri- a Au compounds were tested at the concentrations fied preparation from glucose-adapted cells, shown in the standard 6-phosphogluconate dehydro- therefore, may have been due to contamination genase assay. Combinations of compounds were each of this preparation with a basal level of the present at the final concentrations indicated. Each NAD-linked enzyme. assay contained 0.07 units of partially purified S. fae- Effect of FDP on the Km for NADP and 6- calis 6-phosphogluconate dehydrogenase (fraction V, phosphogluconate. The reaction rate was found to Table 1) and was initiated by the addition of 6-phos- be a hyperbolic function of the coenzyme concen- phogluconate. tration in the presence of a saturating amount of b Results are expressed as the percent decrease in the initial reaction rate (AA3. nm/min) due to the com- substrate and was also a hyperbolic function of pound or combination shown. the substrate concentration in the presence of c It is important to note that in some preliminary excess coenzyme. The NADP and 6-phosphoglu- enzyme fractionation procedures, 5 mM ethylenedi- conate saturation curves remained hyperbolic aminetetraacetate (EDTA) was included in all of the when FDP was included in the reaction. The in- buffers. The resulting 6-phosphogluconate dehydro- hibitor, however, raised significantly the ap- genase preparations were either completely refractory parent Km for both substrate and coenzyme and to the FDP effect shown here or, on occasion, showed also lowered Vmax (Fig. 2A and 2B). The inhibi- a greatly reduced sensitivity to the effector. Removal of EDTA tion, therefore, appears to be of the mixed type free from these preparations by dialysis or by gel filtration failed to restore FDP sensitivity to the (36). enzyme. EDTA was not included in any of the prepara- Incomplete inhibition of 6-pbosphogluconate tions used in this study. dehydrogenase activity by FDP. One of the char- acteristic features of the FDP-mediated inhibi- hibitor interacted with the enzyme at a site dis- tion was the inability of the effector to inhibit the tinct from either the coenzyme or the substrate reaction completely when both NADP and 6- binding sites. This will be considered in more phosphogluconate were present at concentrations detail later. Another possibility was that there which were saturating in the absence of FDP. were actually two distinct forms of the enzyme: The maximum inhibition observed with FDP was one which was completely sensitive to FDP and only about 80% under these conditions (Fig. 3). another which was completely insensitive to the It is also apparent in Fig. 3 that the glucose-6- inhibitor. Clearly, the two explanations are not phosphate dehydrogenase from S. faecalis is mutually exclusive. The possible existence of quite refractory to the FDP effect. FDP-sensitive and FDP-insensitive forms of the One explanation for the inability of FDP to enzyme in S. faecalis is an unresolved question at inhibit the reaction completely was that the in- present. It is interesting to note, however, that 462 BROWN AND WITTENBERGER J. BACTERIOL.

I I 1 140 l lF 1-- edly within the same pH range (Fig. 4). A. Additional support for FDP acting at an inde- FDP Km6PGA 30 pendent site came from studies on the effect of (mM) (mM) ,B-mercaptoethanol on the enzyme. This com- 0 .026 pound was found to have no effect on catalytic .125 .048 .250 .057 I/V 20 activity, but inhibition by FDP was always ob- served to be significantly less when the sulfhydryl compound was included in the standard assay 10 system. When catalytic activity and sensitivity to FDP inhibition were compared in the absence and in the presence of increasing concentrations -60 -40 -20 0 20 40 60 of ,B-mercaptoethanol, a fB-mercaptoethanol con- l/E6PGA m Molar] centration was reached (10 mm in this case) which completely prevented inhibition by FDP, XF X but which had no effect on the uninhibited reac- tion rate (Fig. 5). Although the mechanism by which ,B-mercaptoethanol prevents FDP inhibi- tion is not known at present, the fact that it can block the inhibition completely without affecting catalytic activity indicates that FDP is bound at a site separate and distinct from either the coen- oIo 80

-60 -40 -20 0 20 40 60 I/(NADP m Molar] FIG. 2. Ejfect offructose-1,6-diphosphate (FDP) on the apparent Km of6-phosphogluconate dehydrogenase 60 for (A) 6-phosphogluconate (6PGA) and (B) NA DP. Assays were carried out as described in the text except that the 6PGA and NADP concentrations were varied z as shown. Fraction 5 (Table I) served as the source of 0 enzyme. Symbols: (A) 0, no FDP; 0, plus 0.125 mM I- FDP; 0, plus 0.250 mM FDP; (B) 0, no FDP; 0, plus z 0.250 mM FDP; 0, plus 0.500 mm FDP. Each reaction 40 z mixture in A contained 0.078 units of enzyme, and w each reaction mixture in B contained 0.069 units of w enzyme. a. two forms of 6-phosphogluconate dehydrogenase have been found in C. utilis (26), which differ 20 from one another in molecular weight, electro- phoretic mobility, and several other properties. No information is available, however, on their relative or lack of it, to inhibition by sensitivity, Glucosee-6-Phosphate Dehydrogenase -----IA- FDP. ,\%UCi " , . e A separate binding site for the negative O 2 3 4 5 10 effector. As just indicated, one possible explana- FRUCTOSE-1,6-DIPHOSPHATE (m Molar) tion for the inability of FDP to inhibit 6-phos- FIG. 3. Effect of increasing concentrations offruc- phogluconate dehydrogenase activity completely tose-I,6-diphosphate (FDP) on the activity of 6-phos- was that FDP interacted with the enzyme at a phogluconate dehydrogenase and glucose-6-phosphate site distinct from the catalytic site. This interpre- dehydrogenase. The assay procedure used for botI_Cn- tation of the results found support in the obser- zymes was as described in the text. Where indicated, FDP was included in the reaction mixtures at the final vation that catalytic activity and the degree of concentrations shown. Fraction 5 (Table 1) was the inhibition by FDP responded differentially to source of 6-phosphogluconate dehydrogenase and changes in pH. Catalytic activity increased some- crude, cell-free extract of S. faecalis MR served as a what between pH 7.0 and 8.5, whereas the ability source of glucose-6-phosphate dehydrogenase. Each of FDP to inhibit the reaction decreased mark- assay contained 0.064 units of the enzyme indicated. VOL. 106, 1971 EMBDEN-MEYERHOF AND HEXOSE-MONOPHOSPHATE PATHWAYS 463

'Oar Susceptibility of 6-phosphogluconate dehydro- genase from other sources to inhibition by FDP. To determine whether the regulation of 6-phos- phogluconate dehydrogenase activity by FDP was a mechanism unique to S. faecalis MR or 80h whether this might be a phenomenon of more general significance, the enzyme was tested for z its susceptibility to FDP inhibition in crude ex- 0 tracts derived from several different sources. The sources selected are known to have a complete I Embden-Meyerhof pathway for glucose dissimi- lation and also to possess both hexose-mono- z phosphate dehydrogenases. The 6-phosphoglu- u0 Percent Inhibition a. conate dehydrogenase from five bacteria, two by lOmM FDP yeasts, and guinea pig liver all showed a definite sensitivity to FDP (Table 6). It should be empha- sized that the level of FDP used in these experi- ments was relatively high (10 mM). This was 20F done in an effort to compensate for the expected presence of other enzymes in crude extracts (al- dolase, phosphatases, etc.), which would decrease the effective concentration of FDP. The argu- 7.0 8.0 9.0 ment that the concentration of FDP used here pH was unphysiological and that the effect observed FIG. 4. Effect of pH on 6-phosphogluconate dehy- is, therefore, of questionable in vivo significance drogenase activity and on the sensitivity of the enzyme cannot be dismissed in all cases. However, intra- to inhibition by fructose-1, 6-diphosphate (FDP). Frac- cellular FDP concentrations as high as 17 mM tion 5 (Table I) was the source of the enzyme, and all were reported for another homofermentative assays were carried out as described in the text, except lactic acid bacterium, L. plantarum (20). The that the pH of the Tris-hydrochloride buffer employed real physiological significance of these in vitro was as indicated. At each pH shown, the enzyme was results clearly must await a more detailed exami- assayed without (0) and with (0) 10 mm FDP. Each reaction mixture contained 0.064 units of enzyme. -FDP zyme or substrate sites. Moreover, the results further suggest that the kinetic consequences of 040 an interaction of the enzyme with its negative E a effector are manifestation of an FDP-mediated 0 change in enzyme conformation. Effect of NADH on the hexose-monophosphate dehydrogenases. The NADP-linked glucose-6- phosphate dehydrogenase from E. coli is inhib- 0.2 ited by NADH (28, 29). It was of interest to de- termine whether this inhibition might be a regu- _U I I latory mechanism superimposed upon the FDP effect on 6-phosphogluconate dehydrogenase in z S. faecalis MR. Accordingly, we tested NADH 0 20 4.0 60 8.0 10 20 30 40 as a possible inhibitor of both of the hexose- monophosphate dehydrogenases. However, ,8- MERCAPTOETHANOL(mM) NADH was found to be without effect on either FIG. 5. Desensitization of6-phosphogluconate dehy- glucose-6-phosphate dehydrogenase or 6-phos- drogenase to fructose-1,6-diphosphate (FDP) inhibition phogluconate dehydrogenase when tested at con- by 13-mercaptoethanol. The source of enzyme was frac- centrations up to 0.5 mM in the standard assays tion 5 (Table 1), and the assay procedure was as de- cell-free extracts of scribed in the text. Where indicated, various concentra- previously described. Crude, tions of 13-mercaptoethanol were included in the reac- S. faecalis MR served as a source of glucose-6- tion mixture. Assays were carried out in the absence phosphate dehydrogenase, and no NADH oxida- (0) and in the presence (0) of an amount of FDP (0.5 tion was observed during the course of the assay. mM) that gave 60% inhibition of catalytic activity in Fraction 5 (Table 1) was the source of the 6- the absence of 13-mercaptoethanol. Each reaction mix- phosphogluconate dehydrogenase. ture contained 0.071 units ofenzyme. 464 BROWN AND WITTENBERGER J. BACTERIOL. TABLE 6. Sensitivity of6-phosphogluconate conate resulted in an initial nonlinear increase in dehydrogenases from vqrious sources to inhibition by absorption at 340 nm with time (Materials and fructose-1,6-diphosphate (FDP) Methods). Frieden (6) pointed out that regulatory Enzyme activity enzymes may exist in different conformational (units/mg states which possess different kinetic properties, Source of enzymea p lnstiob)i- and that ligand-mediated changes in conforma- Minus 10 mmM(% tion may be slow relative to enzyme activity. In FDP FDP ___ such cases, the activity observed after the addi- tion of an effector would be expected to be time Streptococcus faecalis MR ...... 0771 0.0176 77.2 S. faecalis IOCI . . 0.0836 0.0160 80.9 dependent. It is possible that the time-dependent S. faecium N-55 . . 0.0675 0.0128 81.0 increase in S. faecalis 6-phosphogluconate dehy- Lactobacillus plantarum ATCC drogenase activity that occurs after preincubation 11739 .0.0627 0.0144 77.0 of the enzyme with FDP might be explicable in Escherichia coli 0127:B8 . .0996 0.0337 66.2 Saccharomyces cerevisiae . 0. 1012 0.0369 63.5 terms of Frieden's "hysteretic enzyme concept." Candida utilis . 0.1093 0.0434 60.3 Alternative explanations of the phenomenon, Guinea pig liver . 0.0765 0.0297 61 .2 however, can also be invoked and, until more in- aCell-free extracts were prepared from all of the organisms formation is available, any proposals relating to indicated by the procedure described for S. faecalis MR, and mechanism must be regarded as entirely specula- the crude extracts served as a source of the enzyme. tive. I All assay conditions were as described for S. faecalis MR The negative control of in Materials and Methods, except for the enzyme from guinea 6-phosphogluconate pig liver, where 100 mM glycyl-glycine buffer (pH 7.5) was dehydrogenase by FDP is of particular interest in used in place of 100 mM Tris-hydrochloride buffer (pH 7.5). the regulation of glucose catabolism in S. fae- cThe enzyme from guinea pig liver was partially purified. calis. This organism, like a number of other nation of the individual enzymes. The data do streptococci (38), possesses a lactate dehydro- suggest, however, that inhibition of 6-phospho- genase that has an absolute and specific require- gluconate dehydrogenase by FDP may be a fairly ment for FDP for catalytic activity (37). The fact general phenomenon. that S. faecalis converts virtually all of its glu- cose carbon to lactate under anaerobic growth DISCUSSION conditions (7, 24) indicates that an intracellular The specific inhibition of S. faecalis 6-phos- pool of FDP must be maintained to activate the phogluconate dehydrogenase by the Embden- lactate dehydrogenase. Although this organism Meyerhof intermediate, FDP, appears to repre- produced lactate from glucose more or less ex- sent a hitherto unrecognized mechanism for par- clusively via the Embden-Meyerhof pathway (24), titioning glucose carbon between the Embden- it also possesses the potential to utilize at least Meyerhof and hexose-monophosphate pathways. the oxidative portion of the hexose-monophos- The precise mechanism of the inhibition remains phate pathway for glucose dissimilation (refer- to be resolved, although certain of its features ence 32; Fig. 6). Uncontrolled use of the latter were characterized. The Km for both NADP and pathway, however, could lead to the formation of 6-phosphogluconate was raised markedly by intermediate compounds such as pentose phos- FDP and Vmax was decreased. These kinetic al- phate, possibly erythrose phosphate and even terations were apparently a consequence of the reduced nicotinamide adenine dinucleotide phos- negative effector interacting with the enzyme at a phate (NADPH), which would exceed the avail- site distinct from either the substrate or the coen- able energy (ATP) required for driving the en- zyme sites. This conclusion derives from the ob- dergonic biosynthetic reactions in which these servation that ,-mercaptoethanol completely compounds are involvd. It might be noted par- desensitized the enzyme to FDP inhibition enthetically that under anaerobic growth condi- without at all affecting catalytic activity. An tions NADPH reoxidation probably occurs ex- important unresolved question at present is why clusively by means of reductive biosynthetic reac- the enzyme cannot be inhibited completely by tions. The lactate dehydrogenase is an NAD- high concentrations of FDP. The possibility that specific enzyme (37), and, under the assay condi- the enzyme actually exists in two forms, one tions described for "reaction 1" by Keister and completely sensitive to FDP, and one completely San Pietro (15), we were unable to detect any refractory to the inhibitor is currently being in- NADPH:NAD oxidoreductase (EC 1.6.1.1) vestigated. activity in cell-free extracts of S. faecalis MR Another unresolved point of interest is the (A. T. Brown and C. L. Wittenberger, unpub- observation that incubation of the S. faecalis 6- lished data). The net result then of uncontrolled phosphogluconate dehydrogenase with FDP be- hexose-monophosphate pathway activity would fore initiating the reaction with 6-phosphoglu- be an accumulation of biosynthetic intermediates VOL. 106, 1971 EMBDEN-MEYERHOF AND HEXOSE-MONOPHOSPHATE PATHWAYS 465 Mexose-Monophosphate Embden-Meyerhof Pathway Pathway Glucose

GlucI...-P

Glucono-6-Lactone-P ,,' Fructose-6-P

| , ~~~~~I Phsponwt&n

6P-Gluconate - ,_-,- FructoW1.6-diP ,#"'; I ,' / NAM 6-P-G1consto ( ,I Dehydeope _ , Di-OH-Aceton.-P Glycerakdehyde-3-P // NADPH

Iibu10#s5P + C°2 l Ribuloe.5-P + Co2 I

A

Lactate FIG. 6. Schematic representation of the relationship between the hexose-monophosphate and Embden-Mey- erhofpathways in S. faecalis. Regulatory sites are indicated as follows: 1, inhibition; A, activation. at the expense of glucose carbon that is actually strated in S. faecalis, but the enzyme from a va- required for the generation of ATP through the riety of sources, including animals, higher plants, Embden-Meyerhof pathway. This problem is other bacteria (18, 27) and yeast (35), was found apparently circumvented in S. faecalis through to be sensitive to ATP. If the S. faecalis enzyme the expediency of utilizing the same compound is similarly affected, this could result in the tem- that activates the lactate dehydrogenase, FDP, to porary diminution of Embden-Meyerhof activity inhibit the activity of the hexose-monophosphate through restriction of FDP production and the enzyme, 6-phosphogluconate dehydrogenase (Fig. consequent limitation of lactate dehydrogenase 6). Thus, under conditions where there is a cel- activity, which is absolutely required for the lular demand for ATP, glucose carbon could be reoxidation of NADH formed at the glyceralde- directed primarily through the energy-liberating hyde-3-dehydrogenase step. A decrease in FDP Embden-Meyerhof pathway by maintenance of concentration would then be expected to result in an intracellular pool of FDP. The means by a concomitant release of the negative control of which this pool is maintained is unknown, but 6-phosphogluconate dehydrogenase. This would the absolute dependence of the lactate dehydro- allow more glucose carbon to proceed through genase on FDP for activity may in itself be a suf- the hexose-monophosphate pathway with the ficient explanation. It might be speculated then formation of NADPH, pentose, and possibly that, when ATP is high and biosynthesis is fa- erythrose-4-phosphate, all of which would be vored, the intracellular concentration of FDP used for diverse biosynthetic processes. Increased would be reduced by the inhibitory interaction of activity of the hexose-monophosphate pathway ATP with phosphofructokinase. Inhibition of might also lead to an increase in the intracellular phosphofructokinase by ATP was not demon- concentration of 6-phosphogluconate, which we 466 BROWN AND WITTENBERGER J. BACTERIOL. recently found to be a potent inhibitor of the S. fermentative and heterofermentative lactic acid bacteria. faecalis glucose-6-phosphate isomerase (C. L. J. Bacteriol. 70:572-576. 8. Glasser, L., and D. H. Brown. 1955. Purification and prop- Wittenberger, unpublished data). This interaction erties of D-glucose-6-phosphate dehydrogenase. J. Biol. would prevent the accumulation of fructose-6- Chem. 216:67-79. phosphate (Fig. 6), which by analogy with other 9. Gornall, A. G., C. J. Bardawill, and M. M. David. 1949. systems (27, 35) could reverse the ATP inhibition Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751-766. of phosphofructokinase. As biosynthetic reactions 10. Grazi, E., A. De Flora, and S. Pontremoli. 1960. The inhi- consumed the available ATP, inhibition of phos- bition of phosphoglucose isomerase by D-erythrose4- phofructokinase would be relieved and FDP could phosphate. Biochem. Biophys. Res. Commun. 2:121- again accumulate. This would be expected to re- 129. 11. Guggenheim, B. 1968. Streptococci of dental plaques. direct glucose carbon through the energy-liberat- Caries Res. 2:147-163. ing Embden-Meyerhof pathway by activation 12. Horne, R. N., W. B. Anderson, and R. C. Nordlie. 1970. of the lactate dehydrogenase and inhibition of Glucose dehydrogenase activity of yeast glucose-6-phos- 6-phosphogluconate dehydrogenase. phate dehydrogenase. Inhibition by adenosine-5'-tri- phosphate and other nucleoside-5'-triphosphates and A number of different organisms are known diphosphates. Biochemistry 9:610-616. which metabolize glucose mainly through the 13. Kahana, S. E., 0. H. Lowry, D. W. Schulz, J. V. Passon- Embden-Meyerhof pathway although they also neau, and E. J. Crawford. 1960. The kinetics of phos- possess enzymes of the hexose-monophosphate phoglucoisomerase. J. Biol. Chem. 235:2178-2184. in E. Salmo- 14. Kalckar, H. M. 1947. Differential spectrophotometry of pathway. For example, coli (21) and purine compounds by means of specific enzymes. III. nella typhimurium (5), 75 to 80% of the glucose Studies of the enzymes of purine 'metabolism. J. Biol. metabolized passes through the Embden-Meyer- Chem. 167:461-475. hof pathway, whereas, in Pseudomonas natrie- 15. Keister, D. L., and A. San Pietro. 1963. Pyridine nucleo- 92% of carbon down tide transhydrogenase from spinach, p. 434. In S. P. gens, the glucose proceeds Colowick and N. 0. Kaplan (ed.), Methods in enzymol- this pathway (4). To what extent regulation of the ogy, vol. VI. Academic Press Inc., New York. two pathways in such organisms is mediated by 16. Lessie, T., and F. C. Neidhardt. 1967. Adenosine triphos- FDP-inhibition of 6-phosphogluconate dehydro- phate-linked control of Pseudomonas aeruginosa glu- genase remains to be established. The demonstra- cose-6-phosphate dehydrogenase. J. Bacteriol. 93:1337- 1345. tion here that 6-phosphogluconate dehydrogenase 17. London, J., and E. Y. Meyer. 1970. Malate utilization by a activity in crude extracts of several different or- group D Streptococcus: regulation of malic enzyme syn- ganisms is inhibited by FDP suggests that this thesis by an inducible malate permease. J. Bacteriol. deserves further consideration as a regulatory 102:130-137. 18. Lowry, 0. H., and J. V. Passonneau. 1964. A comparison mechanism of some general significance. of the kinetic properties of phosphofructokinase from bacterial, plant, and animal sources. Arch. Exp. Pathol. ACKNOWLEDGMENTS Pharmakol. 248:185-194. We thank Patricia Palumbo for very capable technical as- 19. Man, J. C. de, M. Rogosa, and M. E. Sharpe. 1960. A sistance in part of these studies. medium for the cultivation of lactobacilli. J. Appl. Bac- teriol. 23:130-135. 20. Mizushima, S., and K. Kitahara. 1964. Quantitative LITERATURE CITED studies on glycolytic enzymes in Lactobacillus plan- tarum. II. Intracellular concentrations of glycolytic in- 1. Avigad, G. 1966. Inhibition of glucose-6-phosphate dehy- termediates in glucose-metabolizing washed cells. J. Bac- drogenase by adenosine-5'-triphosphate. Proc. Nat. teriol. 87:1429-1435. Acad. Sci. U.S.A. 56:1543-1547. 21. Model, P., and D. Rittenberg. 1967. Measurement of the 2. DeMoss, R. D. 1955. Glucose-6-phosphate and 6-phos- activity of the hexose monophosphate pathway of glu- phogluconate dehydrogenases from Leuconostoc mesen- cose metabolism with the use of (1S0) glucose. Varia- teroides, p. 334. In S. P. Colowick and N. 0. Kaplan tions in its activity in Escherichia coli with growth con- (ed.), Methods in enzymology, vol. 1. Academic Press ditions. Biochemistry 6:69-80. Inc., New York. 22. Parr, C. W. 1956. Inhibition of phosphoglucose isomerase. 3. Dobrogosz, W. J., and R. D. DeMoss. 1963. Studies on Nature (London) 178:1401. the regulation of ribose phosphate isomerase activity in 23. Parr, C. W. 1957. Competitive inhibition of phosphoglu- Pediococcus pentosaceus. Biochim. Biophys. Acta 77: cose isomerase. Biochem. J. 65:34p. 629-638. 24. Platt, T. B., and E. M. Foster. 1957. Products of glucose 4. Eagon, R. G., and C. H. Wang. 1962. Dissimilation of metabolism by homofermentative streptococci under glucose and gluconic acid by Pseudomonas natriegens. J. anaerobic conditions. J. Bacteriol. 75:453-459. Bacteriol. 83:879-886. 25. Racker, E. 1965. Mechanisms in Bioenergetics, p. 207. 5. Fraenkel, D. G., and B. L. Horecker. 1964. Pathways of D- Academic Press Inc., New York. glucose metabolism in Salmonella typhimurium. A study 26. Rippa, M., M. Signorini, and S. Pontremoli. 1967. Purifi- of a mutant lacking phosphoglucose isomerase. J. Biol. cation and properties of two forms of 6-phosphoglu- Chem. 239:2765-2771. conate dehydrogenase from Candida utilis. Eur. J. 6. Frieden, C. 1970. Kinetic aspects of regulation of meta- Biochem. 1:170-178. bolic processes. The hysteretic enzyme concept. J. Biol. 27. Rose, 1. A., and Z. B. Rose. 1969. Glycolysis: Regulation Chem. 245:5788-5799. and mechanisms of the enzymes, p. 111. In M. Florkin 7. Gibbs, M., J. T. Sokatch, and 1. C. Gunsalus. 1955. and E. H. Stotz (ed.), Comprehensive Biochemistry, vol. Product labeling of glucose-1_'4C fermentation by homo- 17. Elsevier Publishing Co. VOL. 106, 1971 EMBDEN-MEYERHOF AND HEXOSE-MONOPHOSPHATE PATHWAYS 467

28. Sanwal, B. D. 1970. Regulatory mechanisms involving nic- 34. Venkataraman, R., and E. Racker. 1961. Mechanism of otinamide adenine nucleotides as allosteric effectors. Ill. action of transaldolase. 1. Crystallization and properties Control of glucose-6-phosphate dehydrogenase. J. Biol. of yeast enzyme. J. Biol. Chem. 236:1876-1882. Chem. 245:1626-163 1. 35. Viniuela, E., M. L. Salas, and A. Sols. 1963. End-product 29. Sanwal, B. D. 1970. Allosteric control of amphibolic path- inhibition of yeast phosphofructokinase by ATP. ways in bacteria. Bacteriol. Rev. 34:20-39. Biochem. Biophys. Res. Commun. 12:140-145. 30. Schindler, J., and H. G. Schlegel. 1969. Regulation der 36. Webb, J. L. 1963. Enzyme and metabolic inhibitors, vol. 1. glucose-6-phosphat-dehydrogenase aus verschiedenen General principles of inhibition, p. 160. Academic Press bakterienarten durch ATP. Mikrobiologiya 66:69-78. Inc., New York. 31. Sokatch, J. T., and 1. C. Gunsalus. 1957. Aldonic acid 37. Wittenberger, C. L., and N. Angelo. 1970. Purification and metabolism. 1. Pathway of carbon in an inducible glu- properties of a fructose-I ,6-diphosphate activated lac- conate fermentation by Streptococcus faecalis. J. Bac- tate dehydrogenase from Streptococcus faecalis. J. Bac- teriol. 73:452-460. teriol 101:717-724. 32. Sokatch, J. T. 1960. Ribose biosynthesis by Streptococcus 38. Wolin, M. J. 1964. Fructose-1,6-diphosphate requirement faecalis. Arch. Biochem. Biophys. 91:240-246. of streptococcal lactic dehydrogenases. Science 146:775- 33. Stadtman, E. R. 1966. Allosteric regulation of enzyme ac- 777. tivity. Adv. Enzymol. 28:41-154.