JouRAL OF BACrOLOGY, June 1975, p. 1274-1282 Copyright @ 1975 American Society for Microbiology
Vol. 122, No. 3 Printed in U.S.A.
Purification and Regulatory Properties of Pyruvate Kinase from Veillonella parvula STEPHEN K. C. NG' AND IAN R. HAMILTON* Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3 Received for publication 6 March 1975
The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations of phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg2+ ions with S... values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 mM PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (K1 = 3.4 mM), and this inhibition was only slightly affected by increasing concentrations of Mg2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP > fructose-1,6-P2 > dihydroxyacetone-P > glucose-6-P > fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was signiflcantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M. plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP. Abundant information is available on pyruvate kinase (EC 2.7.1.40) in carbohydrate-fermenting microorganisms such as yeast (9, 29), Escherichia coli (17, 30), Brevibacterium flavum (23), Azotobacter vinelandii (15), Acetobacter xylinum (3), and Bacillus lichenformis (27, 28). However, there are conflicting reports as to the presence of pyruvate kinase in members of the obligate anaerobic genus Veillonella, shown to be incapable of metabolizing carbohydrates as an energy source (5, 12, 20, 25). Rogosa et al. (26) showed that V. alcalescens VH-11 and V. parvula BYR-2 were unable to ferment carbohydrates because they lacked hexokinase and, hence, were incapable of converting hexoses to the corresponding hexose' Prent addres: Department of Biochemistry, University of Westm Ontario, London, Canada.
phosphates. However, when crude extracts of V. alcalescens VH-11 were supplemented with yeast hexokinase, [IC ]pyruvate was produced from [14C ]glucose, suggesting that these extracts contained all of the remaining enzymes in the glycolytic pathway, including pyruvate kinase. This was confirmed when it was shown that lactic acid was produced after the incubation of glucose-6-phosphate (P) with extracts supplemented with commercial lactic dehydrogenase. Contrary to these findings are the more recent results by Michaud and Delwiche (19), obtained with V. alcalescens Cl isolated from sheep rumen. These authors were unable to detect hexokinase, phosphoglyceromutase, or pyruvate kinase activity in this organism. These workers proposed that the earlier observations by Rogo-
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PYRUVATE KINASE FROM V. PARVULA
sa's group (26) did not actually verify the presence of pyruvate kinase, but rather indicated a pathway to pyruvate that effectively circumvented this enzyme. In view of the controversy over the presence of pyruvate kinase in species of Veillonella and the importance of this enzyme in microbial gluconeogenesis, we undertook to examine this question in detail with the oral strain V. parvula M,. Since we had shown previously that crude, dialyzed extracts of this organism contained an active pyruvate kinase (22), purification and characterization of the enzyme were undertaken. The results indicate that, although V. parvula M4 cannot ferment carbohydrates, the pyruvate kinase isolated from this nonglycolytic organism is regulated in a manner similar to those found in glycolytic organisms. MATERIALS AND METHODS Bacteriological. Cells of V. parvula M4 were grown in Rogosa 1% lactate broth (24) and harvested in the late exponential phase by centrifugation at 13,000 x g for 15 min. The cells were washed once with phosphate buffer (50 mM, pH 6.5) containing 20 mM mercaptoethanol and finally suspended in this buffer at a cell concentration of 20 to 30 mg (dry weight) per ml. Preparation of crude extract. Crude, cell-free extracts, prepared from the above suspensions by anaerobic sonic disruption in a Branson Sonifier (Heat Systems, Plainview, N.Y.) for 20 min, were dialyzed overnight at 4 C against 2 liters of the above buffer (21). The dialyzed extracts were then concentrated to 5 ml by the Diaflo ultrafiltration method (Amicon Ultrafiltration, Lexington, Ky.) using a PM 30 filter with pure nitrogen gas (65 lb/in2). The concentrated extract was then diluted to 50 to 100 mg/ml with 0.1 M phosphate buffer (pH 7.0) containing 20 mM mercaptoethanol and stored at 4 C under nitrogen until used. Crude extracts were dark reddishbrown in color, which, on storage in the absence of a reducing agent, rapidly faded to light pink. Loss of the reddish-brown color was coincident with the loss of pyruvate kinase activity. However, if the extracts were stored at a minimum concentration of 50 mg/ml under nitrogen at 4 C in phosphate buffer (50 mM, pH 7.0) with 20 mM 2-mercaptoethanol, activity was preserved with a loss of only 5.1% of the original activity in 2 months. The samples were not frozen, since freezing and thawing always resulted in a rapid decrease in enzyme activity. Purification of the enzyme. The purification of pyruvate kinase in V. parvula M4 was initiated by removing the nucleic acids from the concentrated sonic extract by dropwise addition of protamine sulfate (0.02% final concentration). After mixing for 30 min at 4 C, the suspension was centrifuged at 35,000 x g for 20 min and the supernatant was purified further. Attempts were made to eliminate deoxyribonucleic acid by incubating the extract with crystalline deoxyribonuclease (1 Ag/ml) for 1 h at 37 C; however,
1275
pyruvate kinase activity was lost during this procedure. The supernatant obtained after protamine sulfate treatment was subjected to ammonium sulfate fractionation at 4 C. Since more than 75% of the pyruvate kinase activity was precipitated between 10 and 30% saturation with ammonium sulfate while removing 50% of the total protein, this salt concentration was used during purification and routinely resulted in almost a twofold increase in specific activity. Column chromatography. The 10 to 30% ammonium sulfate fraction (1 to 4 ml) was subjected to gel filtration on a column (5 by 100 cm) of Sephadex G-200. The protein (40 to 80 mg) was eluted from the column with tris(hydroxymethyl)aminomethanehydrochloide buffer (50 mM, pH 7.0) and monitored continuously at 280 nm. The fractions (3 ml) containing enzyme activity were pooled and concentrated by the Diaflo ultrafiltration method. All procedures were carried out at 4 C. Further purification of the enzyme was undertaken by passing the Sephadex G-200 concentrate through a column (1.5 by 90 cm) of Sephadex G-100. The protein was eluted with phosphate buffer (50 mM, pH 6.5), and the fractions containing enzyme activity were pooled and concentrated as described previously. The preparation obtained at this stage was purified 126-fold (see Table 1) and constituted the purified enzyme used in the studies to be reported here. Pyruvate kinase assays. Pyruvate kinase was assayed by the conversion of the pyruvate formed from phosphoenolpyruvate (PEP) and adenosine 5'-diphosphate (ADP) to lactate with commercial lactic dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH). The oxidation of the NADH was monitored in a Unicam SP800 dual-beam spectrophotometer at 340 mn as described previously (22). Unless otherwise specified, each cuvette contained (millimolar): PEP, 4; ADP, 5; MgSO4, 10; NADH, 5; phosphate buffer (pH 7.0), 50; with 3.6 U of commercial lactic dehydrogenase, and extract preparation in 1 ml. Kinetic experiments routinely employed 20 Ag of purified pyruvate kinase. When dialyzed crude extracts were employed, 5 mM sodium arsenite was added to inhibit the degradation of pyruvate to acetate and CO2 by pyruvate dehydrogenase present in these preparations (22). Pyruvate kinase was assayed in the direction of pyruvate formation in the above assay without lactic dehydrogenase and NADH by measuring the amount of the pyruvate hydrozone formed upon the addition of 0.2 ml of 0.1% 2,4-dinitrophenylhydrazine in 2 N HCI (6). Pyruvate kinase was also assayed in the direction of PEP formation by measuring the formation of [32P]PEP and [32PJADP after incubations with pyruvate and [y-32Pladenosine 5'-triphosphate (ATP) and [a-32P]ATP, respectively (22). Other enzymes. The above [a-32P]ATP assay was also used to test for the presence of PEP synthetase and pyruvate dikinase activity in enzyme preparations of V. parvula M4. The presence of enolase activity in purified pyruvate kinase preparations was measured in the pyruvate-orthophosphate kinase spectrophotometric assay by determining the increased oxidation of NADH with 2-phosphoglycerate (5 mM)
1276
J. BACTERIOL.
NG AND HAMILTON
as the substrate instead of PEP. PEP carboxykinase and PEP carboxylase were assayed by converting to malate the oxaloacetate formed from PEP with commercial malate dehydrogenase, and NADH. The reaction mixture for PEP carboxykinase contained (millimolar): PEP, 5; ADP, 5; NADH, 0.5; NaHCOa, 5; MgSO4, 5; 3.6 U of malate dehydrogenase; and 20 pg of V. parvula M4 enzyme preparation. For the PEP carboxylase assay, ADP was omitted from the reaction mixture. Pyruvate carboxylase was assayed by converting to malate the oxaloacetate formed from radioactive pyruvate and isolating the labeled malate by paper chromatography. The assay contained (millimolar): sodium [3-"4Cjpyruvate (5.4 x 104 dpm/pmol), 5; ATP and NADH, 0.5; NaHCO3, 5; 3.6 U of commercial malate dehydrogenase; and enzyme preparation in 1 ml of phosphate buffer (50 mM, pH 7.0). The reaction was stopped by adding 0.5 ml of 0.1% 2,4-dinitrophenylhydrazine in 2 N HCl. The a-keto acid hydrozone precipitate was sedimented by centrifugation at 35,000 x g for 15 min at 4 C, and the ["4C]malate was isolated from the supematant by paper chromatography as described previously (21). Pyruvate dehydrogenase was determined by measuring the evolution of 14CJC02 from [1-4C lpyruvate. The reaction mixture contained (millimolar): sodium [1-14Cjpyruvate (5.8 x 104 dpm/pmol), 5; MgSO4, 5; and 20 pg of isolated enzyme in 2 ml of phosphate buffer (50 mM, pH 7.0). Assays were carried out in tubes containing tight-fitting serum stoppers through which polyethylene cups were suspended. Reaction was allowed to proceed at 37 C for 30 min and then were stopped by injecting 0.2 ml of 4 N HCI by syringe. After an additional 30-min incubation period, 0.2 ml of 1 N hyamine hydroxide was added by syringe to the polyethene cup to absorb ["4C]CO, produced during the reaction. The tubes were again incubated for 30 min, the contents of the cup was washed into a counting vial with 3 ml of methanol and 10 ml of scintillation liquid, and the radioactivity was determined. In all cases, one unit of enzyme is defined as the formation of 1 Amol of product per min. Enzyme specific activity is defined as enzyme units per milligram of protein. Electrophoresis. Acrylamide gel electrophoresis was carried by the procedures outlined by Davis (4). The gels were stained for 1 h in a solution of 1% amido shwartz stain in 7% acetic acid. Analyses. Protein was assayed by the methods of
et al. (16) and Layne (14). The latter method employed with samples containing 2-mercaptoethanol because this reducing agent interfered with the Lowry method. Materials. All radioactive materials were purchased either from NEN Ltd. (Montreal, Canada) or from the Radiochemical Centre (Amersham, England). Commerical enzymes and metabolites were obtained from Boehringer-Mannheim Corp. (New York). Column materials were purchased from Pharmacia (Montreal).
Lowry
was
RESULTS
Enzyme purification. Data on the purification of pyruvate kinase are given in Table 1. The single most useful step in the procedure was the chromatography of the 10 to 30% saturated ammonium sulfate fraction on Sephadex G-200, which resulted in a 25-fold increase in specific activity with no loss in total activity (Fig. 1). Subsequent column chromatography on Sephadex G-100 resulted in a doubling of the specific activity and produced a preparation containing only one major protein band after polyacrylamide disc-gel electrophoresis (Fig. 2). The minor contaminating bands were devoid of activity for enolase, PEP carboxylase, PEP carboxylkinase, pyruvate carboxylase, PEP synthetase, pyruvate-orthophosphate dikinase, and pyruvate dehydrogenase. From the Sephadex elution profiles, it was estimated that the molecular weight of the enzyme was approximately 150,000. Optimal conditions. Maximum activity of the purified enzyme in the forward direction was observed at pH 7.0 when assayed with 50 mM phosphate buffer (Fig. 3A). The optimum for the reverse reaction, when assayed in phosphate and glycine-NaOH buffers with pyruvate and [.y-_2PJATP, was pH 8.0. Furthermore, enzyme activity increased with increasing temperature up to 45 C (Fig. 3 B). All routine enzymes assays were carried out at 33 C and, at this temperature and at pH 7.0, PEP utilization was linear for at least 50 min with enzyme concentrations up to 200 ;tg.
TABLE 1. Purification of pyruvate kinase from V. parvula M. Step
Protein
1. Crude extract (40,000 x g) 2. Protamine sulfate 3. Ammonium sulfate (10-30%) 4. Sephadex G-200 5. Sephadex G-100
12,807 7,758 3,495
(mg)
139 70
a Micromoles of pyruvate formed/milligram of protein per minute.
|
Sp acta
Fold purification
activity Total(U)
0.50 0.70 1.20 30.0 63.0
1 1.4 2.4 60 126
6,400 5,400 4,200 4,200 4,400
I~~T 1.6.-1-6 -0.8 o
0805 -
C'
0
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PYRUVATE KINASE FROM V. PARVULA
VOL. 122, 1975
shows the effect of increasing concentrations of
MgSO, on the activity of pyruvate kinase in a
reaction mixture containing 4 mM PEP and 1.3 mM ATP. In the absence of ATP but with 6 mM MgSO,, the rate (63 U) was 1.86 times that of
Cs
10
20
30 40 50 6070
80
FRACTION FIG. 1. Profile of the gel filtration of the partially purified pyruvate kinase from V. parvula M4 on Sephadex G-200. Void volume was eluted prior to the collection of fractions. Enzyme activity was assayed
a
spectrophotometrically.
Enzyme kinetics. When the activity of the purified V. parvula pyruvate kinase was assayed at varying PEP concentrations (as high > 19 mM), 5 mM ADP, and 10 mM MgSO4, sigmoidal rate curves were observed (Fig. 4), indicating that they enzyme interacts with more than one molecule of PEP. Maximum activity of the enzyme was observed over a narrow range from 2 to 4 mM, with progressive substrate inhibition being observed at higher concentrations. Hill plots of the data (Fig. 4, inset) give a slope value (n) of 4.4 for PEP, suggesting that at least four molecules of PEP participate in the rate-determining step (2). With 4 mM PEP, similar sigmoidal plots (nat shown) were also observed for varying concentrations of ADP and Mg2+ ions, with slope values of 2.8 and 2.0, respectively (Table 2). Table 2 also shows that the saturation (S0.5) values (13) ranged from 1.4 mM for PEP to 2.0 and 2.4 mM for ADP and Mg2+, respectively. Effect of inhibitors. During the conversion of PEP to pyruvate in the presence of ADP, ATP is a product of the pyruvate kinase reaction and has been shown to be an inhibitor of various microbial pyruvate kinases (3, 23, 28). Thus, it was of interest to determine whether ATP had any effect on the activity of the purified V. parvula enzyme. As shown in Fig. 5A, progressive inhibition by ATP was observed at various concentrations of PEP in the presence of 5 mM Mg2+. Hill plot slope values were close to 1.0, which is indicative of an inhibitor that follows Michaelis-Menten kinetics (Fig. 5C). Inhibition by ATP was noncompetitive, with a K1 for ATP of 3.4 mM. The presence of ATP did not affect So.5 for PEP (Table 3). Holmsen and Storm (8) have observed that Mg2+ salts at concentrations above 4 mM abolished the ATP inhibition of rabbit skeletal muscle pyruvate kinase. Therefore, similar experiments were undertaken with the purified pyruvate kinase from V. parvula M,. Table 4
7 :.
Is
II
I
FIG. 2. Disc-gel electrophoresis patterns of: I, crude extract (275 Mg); II, 10 to 30% saturated ammonium sulfate fraction (290ug); and III, 126-foldpurified enzyme (175 ug) obtained after Sephadex G-100 column chromatography.
5.0 6.0 70 80 90 Axs
10 B.
40-° °- 180l ~40
0~~~~~~~ 50 ~- 5.0 6.0 7.0 8.0 9.0 30 10 °C pH FIG. 3. Effect of pH (A) and temperature (B) on the activity of the purified V. parvula M4 pyruvate kinase.
NG AND HAMILTON
1278
J. BACTERIOL.
the enzyme in the presence of 1.3 mM ATP (33.9 U). Increasing the Mg2+ concentration to 12 mM only increased the rate to 1.17 times that at 6 mM Mg2+, whereas the rates above 12 mM decreased progressively, such that at 20 Mg2+ \ C> mM Mg2+ and above the rates were less than that with 6 mM Mg2+. Thus, Mg2+ ions had only a slight effect on reversing the ATP inhibib Lo tion of the V. parvula enzyme. Activity of the purified V. parvula pyruvate [PEP] M kinase was also inhibited to varying degrees by 3-phosphoglycerate, 2,3-diphosphoglycerate, 0 8 4 and malate (Fig. 6). Although inhibition was PEP mM FIG. 4. Effect of PEP concentration on the activity significant only at high concentrations of the of the purified pyruvate kinase from V. parvula M4 in inhibitor, plotting the data (not shown) by the method of Hunter and Downs (11) showed that the presence of 5 mM ADP and 10 mM Mg2+. the inhibition by all three compounds was TABLE 2. Slope and S.., values obtained from Hill competitive, with Kg values ranging from 4.4 to plots of the effect of varying concentrations of PEP, 7.5 mM (Table 3). In all cases, the S0., value for ADP, and Mg2+ ions on the purified V. parvula M4 PEP in the presence of each inhibitor was pyruvate kinase increased slightly. Activators. A variety of metabolites is known (mM) n value to activate pyruvate kinase from various sources (17, 18, 23, 28). The purified pyruvate kinase 1.5 4.4 from V. parvula M4 was also activated by 2.0 2.8 various cellular metabolites. 2.4 2.0
060
X
40
I.-
~ ~
~
~
~
~
~
~
20 CPE]
TABLE 3. Kinetic constants of the inhibitors of the purified pyruvate kinase from V. parvula M4 Inhibitor
ATP 3-phospho-glycerate Malate 2,3-Diphospho-
Uf)
-
(PEP) (MM)
(mM) (M
Type of inhibition
1.4 1.9
3.4 4.4
Noncompetitive Competitive
1.7 1.8
5.5 7.5
Competitive
Competitive
glycerate 4-
No inhibitor
av UL) (n
10 5 mM PEP
co
0
TABLE 4. Effect of magnesium on the inhibition of the purified pyruvate kinase from V. parvula M4 by ~~1.32
c,) a)
7.5 mM PEP
a.
4 mM PEP
.0
1.4
Magnesium concn 6 (- ATP) 6 10 12 15 18 20 30
mM ATP
Ratea
Relative activity5
63.0
1.86
33.9 34.9 39.7 38.6 34.6 31.9 23.7
1.0 1.03 1.17 1.14
1.02 4 5 3 2 0.94 ATP (mM) 0.70 FIG. 5. Inhibitory effect of ATP on the activity of a Micromoles of pyruvate formed/milligram of prothe purified pyruvate kinase from V. parvula M4. Assayed in the presence of 5 mM PEP, 5 mM ADP tein per minute. "Activity relative to that obtained with 1.3 mM and 10 mM MgSO4 by the spectrophotometric ATP and 6 mM MgSO4 (line 2). method. 0
1.0+3-PGA
>
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PYRUVATE KINASE FROM V. PARVULA
VOL. 122, 1975
enzyme was not significantly affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate,
lactate, fumarate, succinate, or cyclic AMP when incubated with the enzyme at a concentration of 5 mM.
0.80.62.3 D-PGA
a)
0.4-
DISCUSSION z 0.2The results presented herein represent the first definitive information on pyruvate kinase -r----T-- --v-- 1 from any member of the genus Veillonella. As 4 8 mentioned previously, Rogosa and co-workers (26) produced indirect evidence for pyruvate Inhibitor (mM) FIG. 6. Inhibition of the V. parvula M, pyruvate kinase in extracts of an oral strain (VH-11) of V. kinase by 3-phosphoglycerate (3-PGA), 2,3-diphos- alcalescens but did not assay specifically for the phoglycerate (2,3-DPGA) and malate. Assayed as in enzyme. Michaud and Delwiche (19) did assay for pyruvate kinase by conventional procedures Fig. 5. but failed to detect the enzyme in their strain As shown in Fig. 7, the addition of 5 mM (Cl) of V. alcalescens. They suggested that the glucose-6-P to the assay containing 5 mM PEP, presence or absence of pyruvate kinase may 5 mM ADP, and 10 mM MgSO, resulted in represent a difference in strains. While one may activation of the enzyme and a shift of the PEP saturation curve to the hyperbolic form. The 120addition of glucose-6-P increased the maximum activity from 63 to 94 U; a plot of 1/va-v. versus (1/glucose-6-P) with 4 mM PEP (not shown) gave a value of 5.3 mM for the activation constant (Ka). A hill plot of the data (Fig. 8) shows that the degree of cooperativity (n = 2.2) and the S0o5 value of PEP were reduced in the presence of glucose-6-P (Table 5). Although somewhat similar results were obtained with 5 mM fructose-6-P and dihydrox00 yacetone-P (Table 5), activation by only 0.5 -6th-.P arl M yu mMglcs-6P(-6P mM AMP (Fig. 7) produced a significant increase (86%) in the maximum activity and an almost fourfold decrease in the So. for PEP (Fig. 8, Table 5). Again, hyperbolic kinetics was obtained with PEP in the presence of AMP, with a Ka for AMP of 1.1 mM. It should be noted that both glucose-6-P and AMP (Fig. 7), as well as fructose-6-P and dihydroxyacetone-P (not vate kinase. Assayed as in Fig. 5. shown), did not prevent substrate inhibition. The addition 5 mM fructose-1,6-P2 to pyru- TABLE 5. Kinetic constants of the activators of the kinase from Ml V. parvula purified pyrCvate vate kinase assays also resulted in a shift to hyperbolic kinetics; however, depending on the 80.5 Maximum concentration of PEP, fructose-1,6-P2 was both n Activator (PEP) K. activitya an inhibitor and activator (Fig. 9). The effector was inhibitory between 1.2 and 3 mM, but an 0
+
Malate
U)
0
0)
activator above and below this narrow range and did prevent substrate inhibition at high PEP concentrations. Significantly, while the maximum activity obtained in the presence of fructose-1,6-P2 was 62% higher than the control, the So., for PEP was increased to 2.3 mM. Again the degree of cooperativity for PEP was reduced in the presence of the effector. The activity of the purified V. parvula
Glucose-6-P Fructose-6-P
Fructose-1,6-P2 Dihydroxyacetone-P AMP
2.6 1.8 1.5 2.1 2.0
0.9 1.1 2.3 1.2 0.36
No activator
4.4
1.4
5.3
0.32 1.1
94 82 102 95 117
63
a Micromoles of pyruvate formed/milligram of protein per minute.
NG AND HAMILTON
1280 100+ G-6-P
10-
+AMP
>1 1.0-
+FDP
1.0
0.1
10
PEP (mM) FIG. 8. Hill plots for AMP, glucose-6-P (G-6-P), dihydroxyacetone-P (DHAP) and fructose-1-6-P2 (FDP). 120 .t
80-
+FDP
0
i
Q)
Control
0
400
0
0
4
8
PEP (mM) FIG. 9. Effect of fructose-1,6-P2 (5 mM) on V. parvula M4 pyruvate kinase activity. Assayed as in Fig. 5.
question the function of pyruvate kinase in such nonglycolytic organisms such as the Veillonella, the very existence of the enzyme implies that it plays a significant role in the control of gluconeogensis in these bacteria (20, 22). The allosteric nature of the V. parvula M, pyruvate kinase is evident from the kinetic data (Fig. 4) and, in this respect, is similar to other microbial pyruvate kinases (7, 15, 17, 23). However, from the results of this study, the V. parvula M, enzyme exhibited a much higher Hill slope value for PEP (4.4) than that observed with the enzyme from yeast (2.0), E. coli (1.3) B. flavum (3.0), and A. vinelandii (1.8). Liao and Atkinson (15) have reported that the activity of pyruvate kinase from A. vinelandii was activated by glucose-6-P, fructose-6-P, and
J. BACTERIOL.
fructose-1,6-P2, as well as by 3-phosphoglycerate. The fact that 3-phosphoglycerate, 2,3diphosphoglycerate, and malate competitively inhibited the enzyme from V. parvula M4 (Fig. 6) differentiates it from the enzyme found in A. vinelandii, although the effect of former two compounds is probably of limited physiological significance. The enzyme from B. flavum was also competitively inhibited by malate. Malate inhibition of the V. parvula M, enzyme suggests that it may play a minor role in the regulation of gluconeogenesis in this organism, since inhibition of pyruvate kinase must occur before gluconeogenesis can proceed. The presence of malic enzyme in the organism (22) indicates that during gluconeogenesis pyruvate could be converted directly to malate by CO2 fixation, with the resultant malate inhibiting pyruvate kinase. This assumes that malate can accumulate to the appropriate intracellular concentration to cause inhibition. This may be the limiting factor in such a regulatory mechanism since this compound is also an intermediate in the pathway of energy metabolism in this organism (20). Of the compounds inhibiting the V. parvula M, pyruvate kinase, ATP would probably be the most readily available and thus exert the greatest effect on the enzyme under in vivo conditons. Opposing the effect of this compound would be activating effects of AMP, glucose-6-P, fructose-6-P, fructose-1,6-P2, and dihydroxyacetone-P, the steady-state concentration of which must be kept low for gluconeogenesis to occur. All of these positive modifiers altered the sigmoid shape of the PEP saturation curve to that of a hyperbole, and all increased the maximum activity of the enzyme at saturating PEP concentrations. Furthermore, all of these compounds except fructose-1,6-P2 and AMP had little or no effect on the affinity of the enzyme for PEP. With fructose-1,6-P2, the affinity was decreased, whereas with AMP it was increased (Table 5). Although the sigmoidal nature of the curves was changed by these effectors, positive cooperativity for PEP still existed since the Hill coefficients were between 1.5 and 2.3. AMP and the hexose phosphates had a somewhat different effect on the kinetic constants of the pyruvate kinase from A. vinelandii (15). Although these effectors (with the exception of FDP) altered the sigmoidal nature of the PEP response curve, as they did with the V. parvula M, enzyme, the maximum activity was not affected, but the affinity of the enzyme for PEP was increased. On the other hand, the enzyme from A. xylinum (3) was insensitive to both of Ithese metabolites, whereas pyruvate kinase
VOL. 122, 1975
PYRUVATE KINASE FROM V. PARVULA
from B. flavum (23) was not activated by fructose-1,6-P2 but was activated by AMP in a manner that did not alter the sigmoidal kinetics observed in the absence of AMP. Two allosteric pyruvate kinases are known to exist in E. coli: one activated by fructose-1,6-P2 (30) and the other by AMP (18). Recently, Liao and Atkinson (15) have shown that the properties of the A. vinelandii pyruvate kinase are dependent on interaction between the energy charge of the cell (1) and the concentration of hexose phosphates. Such a control system appears also to apply to the regulation of the V. parvula M, pyruvate kinase since the enzyme is inhibited by ATP (and malate) and is activated by AMP and various hexose phosphates. Probably of crucial importance to the regulatory process in this organism is the regulation of ATP production because the organism, being a strict anaerobe, cannot generate ATP by oxidative phosphorylation and cannot metabolize carbohydrates. Therefore, with the growth of the organism on lactate or pyruvate, a balance must be struck between catabolism and biosynthesis, particularly that occurring during gluconeogenesis via amphibolic pathways. This balance is probably effected in V. parvula M, by the control of critical enzymes, such as pyruvate kinase, that must partition PEP between synthesis and energy metabolism. With excess energy charge (high ATP), gluconeogenesis would be allowed to proceed because of the inhibition of pyruvate kinase by ATP, whereas on the other hand, at low energy charge (high AMP), synthesis of hexoses and other compounds would be slowed since any PEP formed would undoubtedly be converted to pyruvate and ATP by the AMPactivated enzyme. Thus, ATP would be a major cellular signal for gluconeogenesis to occur, whereas AMP and hexose phosphates can be seen as the primary signal responsible for stopping this process. ACKNOWLEDGMENT This research was supported by a grant (A-4426) from the National Research Council of Canada.
LITERATURE CITED 1. Atkinson, D. E. 1969. Regulation of enzyme function. Annu. Rev. Microbiol. 23:47-68. 2. Atkinson, D. E., J. A. Hathaway, E. C. Smith. 1965. Kinetics of regulatory enzymes. Kinetic order of the yeast diphosphopyridine nucleotide isocitrate dehydrogenase reaction and a model for the reaction. J. Biol. Chem. 240:2682-2690. 3. Benziman, M. 1969. Factors affecting the activity of pyruvate kinase of Acetobacter xylinum. Biochem. J. 112:631-636. 4. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum protein. Ann. N.Y. Acad.
1281
Sci. 121:404-427. 5. Foubert, E. L., and H. C. Douglas. 1948. Studies on the
anaerobic micrococci. II. The fermentation of lactate by Micrococcus lactilyticus. J. Bacteriol. 56:35-36. 6. Hamilton, I. R., R. H. Burris, and P. W. Wilson. 1965. Pyruvate metabolism by a nitrogen-fixing bacterium. Biochem. J. 96:383-389. 7. Hess, B., R. Haeckel, K. Brand. 1966. FDP-activation of yeast pyruvate kinase. Biochem. Biophys. Res. Commun. 24:824-831. 8. Holmsen, H., and E. Storm. 1969. The adenosine triphosphate inhibition of the pyruvate kinase reaction and its dependence on the total magnesium ion concentration. Biochem. J. 112:303-316. 9. Hunsley, J. R., and C. H. Suelter. 1969. Yeast pyruvate kinase. I. Purification and some chemical properties. J. Biol. Chem. 244:4815-4818. 10. Hunsley, J. R., and C. H. Suelter. 1969. Yeast pyruvate kinase. II. Kinetic properties. J. Biol. Chem. 244:4819-4822. 11. Hunter, A., and C. E. Downs. 1945. The inhibition of arginase by amino acids. J. Biol. Chem. 157:427-446. 12. Johns, A. T. 1951. The mechanism of propionic acid formation by Veillonella gazogenes. J. Gen. Microbiol. 5:326-336. 13. Koshland, D. E., G. Nemethy, and D. Filmer. 1966. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5:365-385. 14. Layne, E. 1951. Spectrophotometric and turbidometric methods for measuring proteins, p. 447-454. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 15. Liao, C. L., and D. E. Atkinson. 1971. Regulation at the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: pyruvate kinase. J. Bacteriol. 106:37-44. 16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 17. Maeba, P., and B. D. Sarwal. 1968. The regulation of pyruvate of Escherichia coli by fructose diphosphate and adenylic acid. J. Biol. Chem. 243:448-450. 18. Malcovati, M., and H. L. Kornberg. 1969. Two type of pyruvate kinase in Escherichia coli K12. Biochim. Biophys. Acta 178:420. 19. Michaud, R. N., and E. A. Delwiche. 1970. Multiple impairment of glycolysis in Veillonella alcalescens. J. Bacteriol. 101:138-140. 20. Ng, K. C., and I. R. Hamilton. 1971. Lactate metabolism by Veillonella parvula M4. J. Bacteriol. 105:999-1005. 21. Ng, K. C., and I. R. Hamilton. 1973. Carbon dioxide fixation by Veillonella parvula M4 and its relation to propionic acid formation. Can. J. Microbiol. 19:715-723. 22. Ng, K. C., and I. R. Hamilton. 1974. Gluconeogenesis by Veillonella parvula M4: evidence for the indirect conversion of pyruvate to P-enolpyruvate. Can. J. Microbiol. 20:19-27. 23. Oxaki, H., and L. Shiio. 1969. Regulation of the TCA and glyoxylate cycles in Brevibacterium flavum. II. Regulation of phosphoenolpyruvate carboxylase and pyruvate kinase. J. Biochem. 66:297-311. 24. Rogosa, M. 1964. The genus Veillonella. I. General cultural, ecological, and biochemical considerations. J. Bacteriol. 87:162-170. 25. Rogosa, M. 1965. The genus Veillonella. IV. Serological groupings and species emendations. J. Bacteriol. 90:704-709. 26. Rogosa, M., M. I. Krichevsky, and F. S. Bishop. 1965. Truncated glycolytic system in Veillonella. J. Bacteriol. 90:164-171. 27. Touminen, F. W., and R. W. Bernlohr. 1971. Pyruvate kinase of the spore-forming bacterium, Bacillus
1282
NG AND HAMILTON
picheniformis. I. Purification, stability, regulation of synthesis and evidence of multiple molecular states. J. Biol. Chem. 246:1733-1745. 28.
Touminen, F. W., and R. W. Bernlohr. 1971. Pyruvate
kinase of the spore-forming bacterium, Bacillus picheniformis. II. Kinetic properties. J. Biol. Chem. 246:1746:1755.
J. BACTERIOL.
29. Washio, W., and Y. Mano. 1960. Studies on pyruvate kinase from Baker's yeast. J. Biochem. (Tokyo) 48:874-885. 30. Waygood, E. B., and B. D. Sanwal. 1974. The control of pyruvate kinases of Escherichia coli. I. Physiochemical and regulatory properties of the enzyme activated by fructose 1,6-diphosphate. J. Biol. Chem. 249:265-274.
Vol. 122, No. 3 Printed in U.S.A.
Purification and Regulatory Properties of Pyruvate Kinase from Veillonella parvula STEPHEN K. C. NG' AND IAN R. HAMILTON* Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3 Received for publication 6 March 1975
The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations of phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg2+ ions with S... values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 mM PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (K1 = 3.4 mM), and this inhibition was only slightly affected by increasing concentrations of Mg2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP > fructose-1,6-P2 > dihydroxyacetone-P > glucose-6-P > fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was signiflcantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M. plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP. Abundant information is available on pyruvate kinase (EC 2.7.1.40) in carbohydrate-fermenting microorganisms such as yeast (9, 29), Escherichia coli (17, 30), Brevibacterium flavum (23), Azotobacter vinelandii (15), Acetobacter xylinum (3), and Bacillus lichenformis (27, 28). However, there are conflicting reports as to the presence of pyruvate kinase in members of the obligate anaerobic genus Veillonella, shown to be incapable of metabolizing carbohydrates as an energy source (5, 12, 20, 25). Rogosa et al. (26) showed that V. alcalescens VH-11 and V. parvula BYR-2 were unable to ferment carbohydrates because they lacked hexokinase and, hence, were incapable of converting hexoses to the corresponding hexose' Prent addres: Department of Biochemistry, University of Westm Ontario, London, Canada.
phosphates. However, when crude extracts of V. alcalescens VH-11 were supplemented with yeast hexokinase, [IC ]pyruvate was produced from [14C ]glucose, suggesting that these extracts contained all of the remaining enzymes in the glycolytic pathway, including pyruvate kinase. This was confirmed when it was shown that lactic acid was produced after the incubation of glucose-6-phosphate (P) with extracts supplemented with commercial lactic dehydrogenase. Contrary to these findings are the more recent results by Michaud and Delwiche (19), obtained with V. alcalescens Cl isolated from sheep rumen. These authors were unable to detect hexokinase, phosphoglyceromutase, or pyruvate kinase activity in this organism. These workers proposed that the earlier observations by Rogo-
11274
VOL. 122, 1975
PYRUVATE KINASE FROM V. PARVULA
sa's group (26) did not actually verify the presence of pyruvate kinase, but rather indicated a pathway to pyruvate that effectively circumvented this enzyme. In view of the controversy over the presence of pyruvate kinase in species of Veillonella and the importance of this enzyme in microbial gluconeogenesis, we undertook to examine this question in detail with the oral strain V. parvula M,. Since we had shown previously that crude, dialyzed extracts of this organism contained an active pyruvate kinase (22), purification and characterization of the enzyme were undertaken. The results indicate that, although V. parvula M4 cannot ferment carbohydrates, the pyruvate kinase isolated from this nonglycolytic organism is regulated in a manner similar to those found in glycolytic organisms. MATERIALS AND METHODS Bacteriological. Cells of V. parvula M4 were grown in Rogosa 1% lactate broth (24) and harvested in the late exponential phase by centrifugation at 13,000 x g for 15 min. The cells were washed once with phosphate buffer (50 mM, pH 6.5) containing 20 mM mercaptoethanol and finally suspended in this buffer at a cell concentration of 20 to 30 mg (dry weight) per ml. Preparation of crude extract. Crude, cell-free extracts, prepared from the above suspensions by anaerobic sonic disruption in a Branson Sonifier (Heat Systems, Plainview, N.Y.) for 20 min, were dialyzed overnight at 4 C against 2 liters of the above buffer (21). The dialyzed extracts were then concentrated to 5 ml by the Diaflo ultrafiltration method (Amicon Ultrafiltration, Lexington, Ky.) using a PM 30 filter with pure nitrogen gas (65 lb/in2). The concentrated extract was then diluted to 50 to 100 mg/ml with 0.1 M phosphate buffer (pH 7.0) containing 20 mM mercaptoethanol and stored at 4 C under nitrogen until used. Crude extracts were dark reddishbrown in color, which, on storage in the absence of a reducing agent, rapidly faded to light pink. Loss of the reddish-brown color was coincident with the loss of pyruvate kinase activity. However, if the extracts were stored at a minimum concentration of 50 mg/ml under nitrogen at 4 C in phosphate buffer (50 mM, pH 7.0) with 20 mM 2-mercaptoethanol, activity was preserved with a loss of only 5.1% of the original activity in 2 months. The samples were not frozen, since freezing and thawing always resulted in a rapid decrease in enzyme activity. Purification of the enzyme. The purification of pyruvate kinase in V. parvula M4 was initiated by removing the nucleic acids from the concentrated sonic extract by dropwise addition of protamine sulfate (0.02% final concentration). After mixing for 30 min at 4 C, the suspension was centrifuged at 35,000 x g for 20 min and the supernatant was purified further. Attempts were made to eliminate deoxyribonucleic acid by incubating the extract with crystalline deoxyribonuclease (1 Ag/ml) for 1 h at 37 C; however,
1275
pyruvate kinase activity was lost during this procedure. The supernatant obtained after protamine sulfate treatment was subjected to ammonium sulfate fractionation at 4 C. Since more than 75% of the pyruvate kinase activity was precipitated between 10 and 30% saturation with ammonium sulfate while removing 50% of the total protein, this salt concentration was used during purification and routinely resulted in almost a twofold increase in specific activity. Column chromatography. The 10 to 30% ammonium sulfate fraction (1 to 4 ml) was subjected to gel filtration on a column (5 by 100 cm) of Sephadex G-200. The protein (40 to 80 mg) was eluted from the column with tris(hydroxymethyl)aminomethanehydrochloide buffer (50 mM, pH 7.0) and monitored continuously at 280 nm. The fractions (3 ml) containing enzyme activity were pooled and concentrated by the Diaflo ultrafiltration method. All procedures were carried out at 4 C. Further purification of the enzyme was undertaken by passing the Sephadex G-200 concentrate through a column (1.5 by 90 cm) of Sephadex G-100. The protein was eluted with phosphate buffer (50 mM, pH 6.5), and the fractions containing enzyme activity were pooled and concentrated as described previously. The preparation obtained at this stage was purified 126-fold (see Table 1) and constituted the purified enzyme used in the studies to be reported here. Pyruvate kinase assays. Pyruvate kinase was assayed by the conversion of the pyruvate formed from phosphoenolpyruvate (PEP) and adenosine 5'-diphosphate (ADP) to lactate with commercial lactic dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH). The oxidation of the NADH was monitored in a Unicam SP800 dual-beam spectrophotometer at 340 mn as described previously (22). Unless otherwise specified, each cuvette contained (millimolar): PEP, 4; ADP, 5; MgSO4, 10; NADH, 5; phosphate buffer (pH 7.0), 50; with 3.6 U of commercial lactic dehydrogenase, and extract preparation in 1 ml. Kinetic experiments routinely employed 20 Ag of purified pyruvate kinase. When dialyzed crude extracts were employed, 5 mM sodium arsenite was added to inhibit the degradation of pyruvate to acetate and CO2 by pyruvate dehydrogenase present in these preparations (22). Pyruvate kinase was assayed in the direction of pyruvate formation in the above assay without lactic dehydrogenase and NADH by measuring the amount of the pyruvate hydrozone formed upon the addition of 0.2 ml of 0.1% 2,4-dinitrophenylhydrazine in 2 N HCI (6). Pyruvate kinase was also assayed in the direction of PEP formation by measuring the formation of [32P]PEP and [32PJADP after incubations with pyruvate and [y-32Pladenosine 5'-triphosphate (ATP) and [a-32P]ATP, respectively (22). Other enzymes. The above [a-32P]ATP assay was also used to test for the presence of PEP synthetase and pyruvate dikinase activity in enzyme preparations of V. parvula M4. The presence of enolase activity in purified pyruvate kinase preparations was measured in the pyruvate-orthophosphate kinase spectrophotometric assay by determining the increased oxidation of NADH with 2-phosphoglycerate (5 mM)
1276
J. BACTERIOL.
NG AND HAMILTON
as the substrate instead of PEP. PEP carboxykinase and PEP carboxylase were assayed by converting to malate the oxaloacetate formed from PEP with commercial malate dehydrogenase, and NADH. The reaction mixture for PEP carboxykinase contained (millimolar): PEP, 5; ADP, 5; NADH, 0.5; NaHCOa, 5; MgSO4, 5; 3.6 U of malate dehydrogenase; and 20 pg of V. parvula M4 enzyme preparation. For the PEP carboxylase assay, ADP was omitted from the reaction mixture. Pyruvate carboxylase was assayed by converting to malate the oxaloacetate formed from radioactive pyruvate and isolating the labeled malate by paper chromatography. The assay contained (millimolar): sodium [3-"4Cjpyruvate (5.4 x 104 dpm/pmol), 5; ATP and NADH, 0.5; NaHCO3, 5; 3.6 U of commercial malate dehydrogenase; and enzyme preparation in 1 ml of phosphate buffer (50 mM, pH 7.0). The reaction was stopped by adding 0.5 ml of 0.1% 2,4-dinitrophenylhydrazine in 2 N HCl. The a-keto acid hydrozone precipitate was sedimented by centrifugation at 35,000 x g for 15 min at 4 C, and the ["4C]malate was isolated from the supematant by paper chromatography as described previously (21). Pyruvate dehydrogenase was determined by measuring the evolution of 14CJC02 from [1-4C lpyruvate. The reaction mixture contained (millimolar): sodium [1-14Cjpyruvate (5.8 x 104 dpm/pmol), 5; MgSO4, 5; and 20 pg of isolated enzyme in 2 ml of phosphate buffer (50 mM, pH 7.0). Assays were carried out in tubes containing tight-fitting serum stoppers through which polyethylene cups were suspended. Reaction was allowed to proceed at 37 C for 30 min and then were stopped by injecting 0.2 ml of 4 N HCI by syringe. After an additional 30-min incubation period, 0.2 ml of 1 N hyamine hydroxide was added by syringe to the polyethene cup to absorb ["4C]CO, produced during the reaction. The tubes were again incubated for 30 min, the contents of the cup was washed into a counting vial with 3 ml of methanol and 10 ml of scintillation liquid, and the radioactivity was determined. In all cases, one unit of enzyme is defined as the formation of 1 Amol of product per min. Enzyme specific activity is defined as enzyme units per milligram of protein. Electrophoresis. Acrylamide gel electrophoresis was carried by the procedures outlined by Davis (4). The gels were stained for 1 h in a solution of 1% amido shwartz stain in 7% acetic acid. Analyses. Protein was assayed by the methods of
et al. (16) and Layne (14). The latter method employed with samples containing 2-mercaptoethanol because this reducing agent interfered with the Lowry method. Materials. All radioactive materials were purchased either from NEN Ltd. (Montreal, Canada) or from the Radiochemical Centre (Amersham, England). Commerical enzymes and metabolites were obtained from Boehringer-Mannheim Corp. (New York). Column materials were purchased from Pharmacia (Montreal).
Lowry
was
RESULTS
Enzyme purification. Data on the purification of pyruvate kinase are given in Table 1. The single most useful step in the procedure was the chromatography of the 10 to 30% saturated ammonium sulfate fraction on Sephadex G-200, which resulted in a 25-fold increase in specific activity with no loss in total activity (Fig. 1). Subsequent column chromatography on Sephadex G-100 resulted in a doubling of the specific activity and produced a preparation containing only one major protein band after polyacrylamide disc-gel electrophoresis (Fig. 2). The minor contaminating bands were devoid of activity for enolase, PEP carboxylase, PEP carboxylkinase, pyruvate carboxylase, PEP synthetase, pyruvate-orthophosphate dikinase, and pyruvate dehydrogenase. From the Sephadex elution profiles, it was estimated that the molecular weight of the enzyme was approximately 150,000. Optimal conditions. Maximum activity of the purified enzyme in the forward direction was observed at pH 7.0 when assayed with 50 mM phosphate buffer (Fig. 3A). The optimum for the reverse reaction, when assayed in phosphate and glycine-NaOH buffers with pyruvate and [.y-_2PJATP, was pH 8.0. Furthermore, enzyme activity increased with increasing temperature up to 45 C (Fig. 3 B). All routine enzymes assays were carried out at 33 C and, at this temperature and at pH 7.0, PEP utilization was linear for at least 50 min with enzyme concentrations up to 200 ;tg.
TABLE 1. Purification of pyruvate kinase from V. parvula M. Step
Protein
1. Crude extract (40,000 x g) 2. Protamine sulfate 3. Ammonium sulfate (10-30%) 4. Sephadex G-200 5. Sephadex G-100
12,807 7,758 3,495
(mg)
139 70
a Micromoles of pyruvate formed/milligram of protein per minute.
|
Sp acta
Fold purification
activity Total(U)
0.50 0.70 1.20 30.0 63.0
1 1.4 2.4 60 126
6,400 5,400 4,200 4,200 4,400
I~~T 1.6.-1-6 -0.8 o
0805 -
C'
0
1277
PYRUVATE KINASE FROM V. PARVULA
VOL. 122, 1975
shows the effect of increasing concentrations of
MgSO, on the activity of pyruvate kinase in a
reaction mixture containing 4 mM PEP and 1.3 mM ATP. In the absence of ATP but with 6 mM MgSO,, the rate (63 U) was 1.86 times that of
Cs
10
20
30 40 50 6070
80
FRACTION FIG. 1. Profile of the gel filtration of the partially purified pyruvate kinase from V. parvula M4 on Sephadex G-200. Void volume was eluted prior to the collection of fractions. Enzyme activity was assayed
a
spectrophotometrically.
Enzyme kinetics. When the activity of the purified V. parvula pyruvate kinase was assayed at varying PEP concentrations (as high > 19 mM), 5 mM ADP, and 10 mM MgSO4, sigmoidal rate curves were observed (Fig. 4), indicating that they enzyme interacts with more than one molecule of PEP. Maximum activity of the enzyme was observed over a narrow range from 2 to 4 mM, with progressive substrate inhibition being observed at higher concentrations. Hill plots of the data (Fig. 4, inset) give a slope value (n) of 4.4 for PEP, suggesting that at least four molecules of PEP participate in the rate-determining step (2). With 4 mM PEP, similar sigmoidal plots (nat shown) were also observed for varying concentrations of ADP and Mg2+ ions, with slope values of 2.8 and 2.0, respectively (Table 2). Table 2 also shows that the saturation (S0.5) values (13) ranged from 1.4 mM for PEP to 2.0 and 2.4 mM for ADP and Mg2+, respectively. Effect of inhibitors. During the conversion of PEP to pyruvate in the presence of ADP, ATP is a product of the pyruvate kinase reaction and has been shown to be an inhibitor of various microbial pyruvate kinases (3, 23, 28). Thus, it was of interest to determine whether ATP had any effect on the activity of the purified V. parvula enzyme. As shown in Fig. 5A, progressive inhibition by ATP was observed at various concentrations of PEP in the presence of 5 mM Mg2+. Hill plot slope values were close to 1.0, which is indicative of an inhibitor that follows Michaelis-Menten kinetics (Fig. 5C). Inhibition by ATP was noncompetitive, with a K1 for ATP of 3.4 mM. The presence of ATP did not affect So.5 for PEP (Table 3). Holmsen and Storm (8) have observed that Mg2+ salts at concentrations above 4 mM abolished the ATP inhibition of rabbit skeletal muscle pyruvate kinase. Therefore, similar experiments were undertaken with the purified pyruvate kinase from V. parvula M,. Table 4
7 :.
Is
II
I
FIG. 2. Disc-gel electrophoresis patterns of: I, crude extract (275 Mg); II, 10 to 30% saturated ammonium sulfate fraction (290ug); and III, 126-foldpurified enzyme (175 ug) obtained after Sephadex G-100 column chromatography.
5.0 6.0 70 80 90 Axs
10 B.
40-° °- 180l ~40
0~~~~~~~ 50 ~- 5.0 6.0 7.0 8.0 9.0 30 10 °C pH FIG. 3. Effect of pH (A) and temperature (B) on the activity of the purified V. parvula M4 pyruvate kinase.
NG AND HAMILTON
1278
J. BACTERIOL.
the enzyme in the presence of 1.3 mM ATP (33.9 U). Increasing the Mg2+ concentration to 12 mM only increased the rate to 1.17 times that at 6 mM Mg2+, whereas the rates above 12 mM decreased progressively, such that at 20 Mg2+ \ C> mM Mg2+ and above the rates were less than that with 6 mM Mg2+. Thus, Mg2+ ions had only a slight effect on reversing the ATP inhibib Lo tion of the V. parvula enzyme. Activity of the purified V. parvula pyruvate [PEP] M kinase was also inhibited to varying degrees by 3-phosphoglycerate, 2,3-diphosphoglycerate, 0 8 4 and malate (Fig. 6). Although inhibition was PEP mM FIG. 4. Effect of PEP concentration on the activity significant only at high concentrations of the of the purified pyruvate kinase from V. parvula M4 in inhibitor, plotting the data (not shown) by the method of Hunter and Downs (11) showed that the presence of 5 mM ADP and 10 mM Mg2+. the inhibition by all three compounds was TABLE 2. Slope and S.., values obtained from Hill competitive, with Kg values ranging from 4.4 to plots of the effect of varying concentrations of PEP, 7.5 mM (Table 3). In all cases, the S0., value for ADP, and Mg2+ ions on the purified V. parvula M4 PEP in the presence of each inhibitor was pyruvate kinase increased slightly. Activators. A variety of metabolites is known (mM) n value to activate pyruvate kinase from various sources (17, 18, 23, 28). The purified pyruvate kinase 1.5 4.4 from V. parvula M4 was also activated by 2.0 2.8 various cellular metabolites. 2.4 2.0
060
X
40
I.-
~ ~
~
~
~
~
~
~
20 CPE]
TABLE 3. Kinetic constants of the inhibitors of the purified pyruvate kinase from V. parvula M4 Inhibitor
ATP 3-phospho-glycerate Malate 2,3-Diphospho-
Uf)
-
(PEP) (MM)
(mM) (M
Type of inhibition
1.4 1.9
3.4 4.4
Noncompetitive Competitive
1.7 1.8
5.5 7.5
Competitive
Competitive
glycerate 4-
No inhibitor
av UL) (n
10 5 mM PEP
co
0
TABLE 4. Effect of magnesium on the inhibition of the purified pyruvate kinase from V. parvula M4 by ~~1.32
c,) a)
7.5 mM PEP
a.
4 mM PEP
.0
1.4
Magnesium concn 6 (- ATP) 6 10 12 15 18 20 30
mM ATP
Ratea
Relative activity5
63.0
1.86
33.9 34.9 39.7 38.6 34.6 31.9 23.7
1.0 1.03 1.17 1.14
1.02 4 5 3 2 0.94 ATP (mM) 0.70 FIG. 5. Inhibitory effect of ATP on the activity of a Micromoles of pyruvate formed/milligram of prothe purified pyruvate kinase from V. parvula M4. Assayed in the presence of 5 mM PEP, 5 mM ADP tein per minute. "Activity relative to that obtained with 1.3 mM and 10 mM MgSO4 by the spectrophotometric ATP and 6 mM MgSO4 (line 2). method. 0
1.0+3-PGA
>
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PYRUVATE KINASE FROM V. PARVULA
VOL. 122, 1975
enzyme was not significantly affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate,
lactate, fumarate, succinate, or cyclic AMP when incubated with the enzyme at a concentration of 5 mM.
0.80.62.3 D-PGA
a)
0.4-
DISCUSSION z 0.2The results presented herein represent the first definitive information on pyruvate kinase -r----T-- --v-- 1 from any member of the genus Veillonella. As 4 8 mentioned previously, Rogosa and co-workers (26) produced indirect evidence for pyruvate Inhibitor (mM) FIG. 6. Inhibition of the V. parvula M, pyruvate kinase in extracts of an oral strain (VH-11) of V. kinase by 3-phosphoglycerate (3-PGA), 2,3-diphos- alcalescens but did not assay specifically for the phoglycerate (2,3-DPGA) and malate. Assayed as in enzyme. Michaud and Delwiche (19) did assay for pyruvate kinase by conventional procedures Fig. 5. but failed to detect the enzyme in their strain As shown in Fig. 7, the addition of 5 mM (Cl) of V. alcalescens. They suggested that the glucose-6-P to the assay containing 5 mM PEP, presence or absence of pyruvate kinase may 5 mM ADP, and 10 mM MgSO, resulted in represent a difference in strains. While one may activation of the enzyme and a shift of the PEP saturation curve to the hyperbolic form. The 120addition of glucose-6-P increased the maximum activity from 63 to 94 U; a plot of 1/va-v. versus (1/glucose-6-P) with 4 mM PEP (not shown) gave a value of 5.3 mM for the activation constant (Ka). A hill plot of the data (Fig. 8) shows that the degree of cooperativity (n = 2.2) and the S0o5 value of PEP were reduced in the presence of glucose-6-P (Table 5). Although somewhat similar results were obtained with 5 mM fructose-6-P and dihydrox00 yacetone-P (Table 5), activation by only 0.5 -6th-.P arl M yu mMglcs-6P(-6P mM AMP (Fig. 7) produced a significant increase (86%) in the maximum activity and an almost fourfold decrease in the So. for PEP (Fig. 8, Table 5). Again, hyperbolic kinetics was obtained with PEP in the presence of AMP, with a Ka for AMP of 1.1 mM. It should be noted that both glucose-6-P and AMP (Fig. 7), as well as fructose-6-P and dihydroxyacetone-P (not vate kinase. Assayed as in Fig. 5. shown), did not prevent substrate inhibition. The addition 5 mM fructose-1,6-P2 to pyru- TABLE 5. Kinetic constants of the activators of the kinase from Ml V. parvula purified pyrCvate vate kinase assays also resulted in a shift to hyperbolic kinetics; however, depending on the 80.5 Maximum concentration of PEP, fructose-1,6-P2 was both n Activator (PEP) K. activitya an inhibitor and activator (Fig. 9). The effector was inhibitory between 1.2 and 3 mM, but an 0
+
Malate
U)
0
0)
activator above and below this narrow range and did prevent substrate inhibition at high PEP concentrations. Significantly, while the maximum activity obtained in the presence of fructose-1,6-P2 was 62% higher than the control, the So., for PEP was increased to 2.3 mM. Again the degree of cooperativity for PEP was reduced in the presence of the effector. The activity of the purified V. parvula
Glucose-6-P Fructose-6-P
Fructose-1,6-P2 Dihydroxyacetone-P AMP
2.6 1.8 1.5 2.1 2.0
0.9 1.1 2.3 1.2 0.36
No activator
4.4
1.4
5.3
0.32 1.1
94 82 102 95 117
63
a Micromoles of pyruvate formed/milligram of protein per minute.
NG AND HAMILTON
1280 100+ G-6-P
10-
+AMP
>1 1.0-
+FDP
1.0
0.1
10
PEP (mM) FIG. 8. Hill plots for AMP, glucose-6-P (G-6-P), dihydroxyacetone-P (DHAP) and fructose-1-6-P2 (FDP). 120 .t
80-
+FDP
0
i
Q)
Control
0
400
0
0
4
8
PEP (mM) FIG. 9. Effect of fructose-1,6-P2 (5 mM) on V. parvula M4 pyruvate kinase activity. Assayed as in Fig. 5.
question the function of pyruvate kinase in such nonglycolytic organisms such as the Veillonella, the very existence of the enzyme implies that it plays a significant role in the control of gluconeogensis in these bacteria (20, 22). The allosteric nature of the V. parvula M, pyruvate kinase is evident from the kinetic data (Fig. 4) and, in this respect, is similar to other microbial pyruvate kinases (7, 15, 17, 23). However, from the results of this study, the V. parvula M, enzyme exhibited a much higher Hill slope value for PEP (4.4) than that observed with the enzyme from yeast (2.0), E. coli (1.3) B. flavum (3.0), and A. vinelandii (1.8). Liao and Atkinson (15) have reported that the activity of pyruvate kinase from A. vinelandii was activated by glucose-6-P, fructose-6-P, and
J. BACTERIOL.
fructose-1,6-P2, as well as by 3-phosphoglycerate. The fact that 3-phosphoglycerate, 2,3diphosphoglycerate, and malate competitively inhibited the enzyme from V. parvula M4 (Fig. 6) differentiates it from the enzyme found in A. vinelandii, although the effect of former two compounds is probably of limited physiological significance. The enzyme from B. flavum was also competitively inhibited by malate. Malate inhibition of the V. parvula M, enzyme suggests that it may play a minor role in the regulation of gluconeogenesis in this organism, since inhibition of pyruvate kinase must occur before gluconeogenesis can proceed. The presence of malic enzyme in the organism (22) indicates that during gluconeogenesis pyruvate could be converted directly to malate by CO2 fixation, with the resultant malate inhibiting pyruvate kinase. This assumes that malate can accumulate to the appropriate intracellular concentration to cause inhibition. This may be the limiting factor in such a regulatory mechanism since this compound is also an intermediate in the pathway of energy metabolism in this organism (20). Of the compounds inhibiting the V. parvula M, pyruvate kinase, ATP would probably be the most readily available and thus exert the greatest effect on the enzyme under in vivo conditons. Opposing the effect of this compound would be activating effects of AMP, glucose-6-P, fructose-6-P, fructose-1,6-P2, and dihydroxyacetone-P, the steady-state concentration of which must be kept low for gluconeogenesis to occur. All of these positive modifiers altered the sigmoid shape of the PEP saturation curve to that of a hyperbole, and all increased the maximum activity of the enzyme at saturating PEP concentrations. Furthermore, all of these compounds except fructose-1,6-P2 and AMP had little or no effect on the affinity of the enzyme for PEP. With fructose-1,6-P2, the affinity was decreased, whereas with AMP it was increased (Table 5). Although the sigmoidal nature of the curves was changed by these effectors, positive cooperativity for PEP still existed since the Hill coefficients were between 1.5 and 2.3. AMP and the hexose phosphates had a somewhat different effect on the kinetic constants of the pyruvate kinase from A. vinelandii (15). Although these effectors (with the exception of FDP) altered the sigmoidal nature of the PEP response curve, as they did with the V. parvula M, enzyme, the maximum activity was not affected, but the affinity of the enzyme for PEP was increased. On the other hand, the enzyme from A. xylinum (3) was insensitive to both of Ithese metabolites, whereas pyruvate kinase
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PYRUVATE KINASE FROM V. PARVULA
from B. flavum (23) was not activated by fructose-1,6-P2 but was activated by AMP in a manner that did not alter the sigmoidal kinetics observed in the absence of AMP. Two allosteric pyruvate kinases are known to exist in E. coli: one activated by fructose-1,6-P2 (30) and the other by AMP (18). Recently, Liao and Atkinson (15) have shown that the properties of the A. vinelandii pyruvate kinase are dependent on interaction between the energy charge of the cell (1) and the concentration of hexose phosphates. Such a control system appears also to apply to the regulation of the V. parvula M, pyruvate kinase since the enzyme is inhibited by ATP (and malate) and is activated by AMP and various hexose phosphates. Probably of crucial importance to the regulatory process in this organism is the regulation of ATP production because the organism, being a strict anaerobe, cannot generate ATP by oxidative phosphorylation and cannot metabolize carbohydrates. Therefore, with the growth of the organism on lactate or pyruvate, a balance must be struck between catabolism and biosynthesis, particularly that occurring during gluconeogenesis via amphibolic pathways. This balance is probably effected in V. parvula M, by the control of critical enzymes, such as pyruvate kinase, that must partition PEP between synthesis and energy metabolism. With excess energy charge (high ATP), gluconeogenesis would be allowed to proceed because of the inhibition of pyruvate kinase by ATP, whereas on the other hand, at low energy charge (high AMP), synthesis of hexoses and other compounds would be slowed since any PEP formed would undoubtedly be converted to pyruvate and ATP by the AMPactivated enzyme. Thus, ATP would be a major cellular signal for gluconeogenesis to occur, whereas AMP and hexose phosphates can be seen as the primary signal responsible for stopping this process. ACKNOWLEDGMENT This research was supported by a grant (A-4426) from the National Research Council of Canada.
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