JOURNAL OF BACrERIOLOGY, Feb. 1975, p. 600-607 Copyright 0 1975 American Society for Microbiology
Vol. 121, No. 2 Printed in U.SA.
Production of Racemic Lactic Acid in Pediococcus cerevisiae Cultures by Two Lactate Dehydrogenases GEOFFREY L. GORDON AND H. W. DOELLE* Department of Microbiology, University of Queensland, St. Lucia, Queensland 4067, Australia Received for publication 22 November 1974
Nicotinamide adenine dinucleotide (NAD)-dependent D(-)- and L(+)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for D(-)-lactate dehydrogenase and 136,000 to 139,000 for L(+)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by D(-)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, L(+)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey MichaelisMenten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. a-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevistae. By definition, all lactic acid bacteria produce some lactic acid during fermentation of carbohydrates. In most cases this lactic acid is either levorotatory or dextrorotatory, but occasionally a mixture of both isomers can be formed. Lactobacillus sake has been found to form L( +)-lactic acid by the action of a nicotinamide adenine dinucleotide (NAD)-dependent L( + )-lactate dehydrogenase (EC 1.1.1.27; L+LDH), which in the presence of lactate racemase (EC 5.1.2.1) results in the production of a racemic mixture of lactic acid in the culture (14). A similar situation has also been reported recently in L. curvatus (20). However, certain other members of this group of organisms produce racemic mixtures of lactic acid by the simultaneous action of NAD-dependent D(-)lactate dehydrogenase (EC 1.1.1.28; D-LDH) as well as L+LDH (4, 5, 10, 17). Since pediococci are traditionally regarded as organisms that produce racemic lactic acid as a major end product of glucose catabolism (1), the question arises: how is this racemic mixture actually formed? Preliminary studies by Hiyama et al. (14) indicated that production of 60(
racemic lactate by Pediococcus lindneri and P. hennebergi is brought about by the presence of two different lactate dehydrogenases within their cells. The present investigation was under-
taken in an attempt to provide information at the enzyme level on the mechanism of production of racemic lactate mixtures in cultures of P. cerevisiae. MATERIALS AND METHODS Materials. All cofactors, substrates, nucleotides, glycolytic intermediates, as well as dithiothreitol (DTT) and tris(hydroxymethyl)aminomethane (Tris), were purchased from Sigma Chemical Co. (St. Louis, Mo.). Lactate dehydrogenases from rabbit muscle and L. leichmannii were purchased from Boehringer-Mannheim GmbH (Mannheim, Germany). Diethylaminoethyl-Sephadex A-25 and Sepharose 6B were products of Pharmacia Fine Chemical Company (Uppsala, Sweden); hydroxylapatite Bio Gel HTP was obtained from Bio-Rad Laboratories (Richmond, Ca.). Ammonium sulfate was special enzyme grade from Schwarz/Mann (Orangeburg, N.Y.). All other chemicals were analytical grade. Organisms and culture conditions. P. cerevisiae 813 and P. pentosaceus 9206 were obtained from the
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National Collection of Dairy Organisms (National Institute for Research in Dairying, Shinfield, Reading, Berkshire, England) and from the National Collection of Industrial Bacteria (Torry Research Station, Aberdeen, Scotland), respectively. Both were maintained by monthly subculture on MRS agar (3) slopes, which were stored at 4 C after growth for 24 h at 28 C. Large-scale growth of P. cerevisiae was undertaken in a 10-liter carboy of MRS medium containing only 1.0% (wt/vol) glucose. The complete medium without glucose was sterilized by autoclaving at 115 C for 20 min; a 40% glucose solution, sterilized separately by autoclaving, was then added aseptically to a final concentration of 1.0%. Bacterial growth in the carboy was initiated with a 1% (vol/vol) inoculum of a 24-h culture grown in the same medium. The culture was incubated at 28 C for 18 h, with constant but slow magnetic stirring. The cells were harvested by centrifugation, washed with cold 0.9% (wt/vol) NaCl, and stored as a frozen cell paste. Cells for lactate racemase studies were grown in 100-ml amounts of modified MRS, which was prepared by omitting sodium acetate and ammonium citrate and by reducing the glucose concentration to 0.1%. Preparation of cell-free extracts. Ten grams of thawed cell paste was suspended in about 200 ml of 0.05 M sodium phosphate buffer (pH 7.0) containing 2 mM DTT and 2 mM sodium-DL-lactate, and disrupted in 50-ml fractions with 50 g of glass beads (0.10- to 0.11-mm diameter) at 4,000 rpm in a Braun cell homogenizer (type MSK; Melsungen, Germany) for 15 min. During disruption, the glass vessel was cooled by a continuous stream of liquid CO2. The opaque, straw-colored liquid was decanted and then clarified by centrifugation at 37,000 x g for 30 min. The resultant supernatant was then used for all enzymatic procedures. Enzyme assays. Both lactate dehydrogenases were assayed spectrophotometrically at 340 nm by using a Pye-Unicam SP 8000 recording spectrophotometer with 3.0-ml, 1.0-cm path length quartz cuvettes. Lactate oxidation was measured in a system containing 25 mM Tris-maleate buffer (pH 8.2), 2.0 mM sodium NAD, and 10 mM lithium L(+)- or 10 mM D(-)-lactate in a final volume of 3.0 ml. Pyruvate reduction was measured in a 3.0-ml system containing 25 mM Tris-maleate buffer (pH 5.4 or 7.4) and concentrations of sodium pyruvate and sodium reduced NAD, as indicated in Table 1. Dehydrogenase
601
units were expressed as micromoles of NADH produced or oxidized per minute at 30 C. Specific activity is defined as units per milligram of enzyme protein. The assay system used for lactate racemase and the defimition for lactate racemizing units of activity were those used by Hiyama et al. (14). Analytical methods. Residual glucose in the medium was determined by a mixed enzyme-dye method (A. St. G. Huggett and D. A. Nixon, Biochem. J. 66: 12P, 1957). Isomer production of lactic acid in culture supernatants, as well as in assays of lactate racemase, was determined enzymatically by the method of Hohorst (15), using L+LDH from rabbit muscle and D-LDH from L. leichmannii. Dry weights of the cells were determined in preweighed glass vials after addition of a washed-cell suspension and drying at 105 C to constant weight. Protein concentration in cell-free extracts was measured by the biuret method (12), with crystalline bovine serum albumin as standard, whereas protein concentrations of all other purification steps were estimated by using their absorbance at 260 and 280 nm (21). Molecular weight estimations. Native molecular weights were estimated by gel filtration through a Sephadex G-200 column (2.6 by 65 cm) eluted with 0.05 M sodium phosphate buffer (pH 7.0) containing 2 mM DTT. Standard proteins and methods for their assay have been described previously (11). The native molecular weight of D-LDH was estimated electrophoretically, by the method of Hedrick and Smith (13), in an alkaline polyacrylamide gel system (2) as described previously (11). However, an estimate for L+LDH was not possible in this system owing to the extreme lability of this enzyme at alkaline pH. Therefore, the native molecular weight of L+LDH was estimated electrophoretically at pH 7.0 by the procedure of Weber and Osborn (22) modified by the omission of sodium dodecyl sulfate. Five polyacrylamide gel concentrations (5 to 9%). were used for the estimation by the method of Hedrick and Smith (13). Subunit molecular weights of the two enzymes were estimated in gels containing 0.1% sodium dodecyl sulfate by the method of Weber and Osborn (22) as described previously (11).
RESULTS Growth characteristics and lactate production by P. cerevisiae. The growth characTABLE 1. Summary of assay conditions for pyruvate teristics of P. cerevisiae grown in standing batch reduction in P. cerevisiae culture at 28 C in a 10-liter carboy are shown in 1. A cell yield of 4 g (dry weight per liter) Fig. WSodium Sodium pyrAssay pH corresponds to about 7 g (wet weight) of cells/ uvate (mM) NADH (mM) liter. The cells were harvested early in the sta0.04 2.0 5.4 tionary phase after 18 h of growth, as lactate 0.1 10 7.4 production appeared to have ceased by this 5.4 7.4
0.3 1.0
0.02 0.02
stage. During the fermentation, L( + ) -lactic acid was the first isomer to appear in the culture me-
GORDON AND DOELLE
602
6
TL
I_HoursI0 FIG. 1. Growth characteristics of P. cerevisiae NCDO 813 in MRS broth (3) at 28 C. Symbols: 0, bacterial dry weight; 0, L(+)-lactate; 0, D(-)-lactate; V, culture pH; A, glucose in medium.
J. BACTERIOL.
sodium phosphate buffer (pH 7.0) containing 2 mM DTT. This solution was layered onto a Sepharose 6B column (2.8 by 70 cm), which had been equilibrated with the same buffer, and the column was eluted at a flow rate of 30 ml/h. L+LDH was eluted first and was followed closely by D-LDH, with a slight overlap of activities. Fractions with more than 50% of the activity of each peak fraction were pooled (Fig. 2). The first peak was concentrated to about 5 ml in an Amicon ultrafiltration cell fitted with a UM-50 membrane. The concentrated solution was diluted to 50 ml with 0.05 M sodium phosphate buffer (pH 6.0) containing 2 mM DTT and reconcentrated to about 5 ml. This solution was then applied to an hydroxylapatite column (1.0 by 30 cm), which had been equilibrated with the same buffer. The column was eluted at a flow rate of 3.6 ml/h with a 200-ml linear gradient of 0.05 to 0.5 M sodium phosphate buffer (pH 6.0). The single activity peak fractions were pooled and concentrated by ultrafiltration (UM-50 membrane), diluted with 0.05 M sodium phosphate buffer (pH 7.0) containing 2 mM DTT, and reconcentrated as
dium, whereas the D(-) isomer did not appear until several hours later when the pH had fallen to about 5. At this point, further L(+) acid production ceased. Lactate racemng activity in cell-free extracts of pediococci. Cell-free extracts prepared from cells of P. cerevisiae and P. pentosaceus, grown in the absence of acetate and citrate, exhibited considerable lactate racemizing activities of 4.1 and 3.6 U/mg of protein, respectively. However, after overnight Sepharose 65 rI I dialysis of the extracts against the buffer system used for cell-free extract preparation, this activity was completely lost, while specific activities of both lactate dehydrogenases were essentially constant. Partial purification of NAD-dependent L+LDH and D-LDH from P. cerevisiae. Enzyme activities were monitored by lactate oxidation throughout the purification procedure. All operations were carried out between 0 and 4 C, except the ammonium sulfate fractionation, which was performed at room temperature (about 20 C). After centrifugation at 4 C, the cell-free extract was equilibrated at room temperature, after which it was adjusted to 45% ammonium sulfate saturation by the slow addition of the solid chemical (277 g/liter) together with slow magnetic stirring while a constant, gentle stream of dry nitrogen was played over the liquid surface. The pellet formed after centrifugation (20 min at 37,000 x g) was discarded, whereas the ammonium sulfate concentration of the supernatant was increased to 70% saturation (by addition of 171 g of solid chemical per liter to the 45% saturated-ammonium sulfate Elution Volume (ml) supernatant, with identical precautions). CenFIG. 2. Elution profiles of D-LDH and L+LDH trifugation (20 min at 37,000 x g) produced a activity during purification. Symbols: *, L+LDH; 0, pellet which was suspended in 5 ml of 0.05 M D-LDH; A, absorbancy at 280 nm.
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before. Sufficient glycerol was added to the partially purified L+LDH preparation to give final concentration of 30% (vol/vol), with the preparation then being stored at 4 C without appreciable loss of activity over a period of several months. The second peak eluted from the Sepharose 6B column was concentrated, diluted with 0.05 M Tris-hydrochloride buffer (pH 8.0) containing 2 mM DTT and 0.05 M NaCl, and reconcentrated to about 5 ml. This preparation was then applied to diethylaminoethyl-Sephadex A-25 column (2.0 by 30 cm), which had been equilibrated with the same buffer. The column was eluted at a flow rate of 100 ml/h with a 500-ml linear gradient of 0.05 to 0.5 M NaCl. The single activity peak fractions were pooled and concentrated by ultrafiltration (UM-50 membrane), diluted with 0.05 M Tris-maleate buffer (pH 8.0) containing 2 mM DTT, reconcentrated, and stored frozen without appreciable loss of D- LDH activity over a period of several months. A summary of a typical purification procedure appears in Table 2; representative elution profiles are displayed in Fig. 2. Both enzyme preparations were examined for homogeneity by polyacrylamide gel electrophoresis, but in both cases the preparations possessed a major protein band, which corresponded to the activity band, contaminated with two or three other faint protein bands. pH optima and reversibility of the reactions. Hydrogen ion concentration was found to have different effects on lactate oxidation by
603
L+LDH and D-LDH from P. cerevisiae. Maximum activity for L+LDH was found at pH 7.6, whereas that for D-LDH was at pH 9.6. Pyruvate reduction by D-LDH demonstrated an activity peak at pH 8.0 (Fig. 3), which shifted to a sharp peak at pH 3.6 when the pyruvate concentration was lowered from 5.0 to 0.5 mM. On the other hand, pyruvate reduction by L+LDH exhibited maximum activity in the pH range 4.5 to 6.0 at either pyruvate concentration. In 1963 it was estimated that the internal pH of actively metabolizing cells of L. plantarum is 5.4 (19). This value, as well as the pH value normally regarded as physiological (7.4), was used in an estimation of the reversibility of both enzymes. Reaction velocities for lactate oxidation and pyruvate reduction of both enzymes at these pH values were estimated from the pH profiles for lactate oxidation and those shown in Fig. 3. From this data it was calculated that lactate oxidation was 6.1% and 1.1% of pyruvate reduction at pH 7.4 and 5.4, respectively, for D-LDH and 2.7% at pH 7.4 for L+LDH. L+LDH was not reversible at pH 5.4. Michaelis constants for substrates. Michaelis constants (Ki) for all substrates of both enzymes are given in Table 3. Values for lactate and NAD were estimated for the purified enzymes from linear double-reciprocal plots at one substrate concentration. Michaelis constants for pyruvate and NADH were estimated at pH 5.4 and 7.4 by plotting saturation curves at four concentrations of the second substrate. The reciprocals for apparent maximum veloci-
TABLE 2. Summary of the purification of lactate dehydrogenases from P. cerevisiae Purification step
1. Cell-free extract
2. Ammonium sulfate fractionation, 45 to 70% saturation 3. Sepharose 6B chromatography: Peak I
Total Ttlnucleic acid |(mg)
Vol (ml) protein l(mg)
168 5.0
D-LDH
L+LDH Sp act
Total
(U/mg) activity (U)
To t
Yield (%)
Sp act (U/mg)
activ(ty (U)
Yield
1008
722
0.0257
25.97
100
0.0177
17.86 100
285
119
0.0610
17.39
67
0.0457
13.04
73
18.5 18.0
77.7 41.4
4.3 3.1
0.2093 0.0294
16.27 1.22
63 5
0.0127 0.3067
0.99 12.70
6 71
4. Hydroxylapatite chromatography of peak I at pH 6.0
16.5
3.9
0.1
1.8116
7.17
28
0
0
O
5. DEAE-Sephadex chromatography of peak II at pH 8.0a
27.0
2.2
0
0
0
0
1.5700
3.39
19
PeakII
-
DEAE, Diethylaminoethyl.
604
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GORDON AND DOELLE
ties for the four different second substrate concentrations were then plotted against the reciprocal of the substrate concentration to give the Km of the second substrate (Fig. 4). All substrate saturation curves for bo'th enzymes were hyperbolic, double-reciprocal plots which were linear and gave Km values for both substrates which increased with increasing pH. Effect of pyruvate analogues. Of the four analogues tested (a-ketobutyric, oxamic, oxalic,
E TO
4020
O-
E
-8
4
8
'/Pyruvote (mM'4) 'C
0
60
-
FIG. 4. Example for determination of Km value for substrates of pyruvate reduction. The reaction mixtures contained 25 mM Tris-maleate buffer (pH 5.4), partially purified D-LDH (2.7 ,gg of protein), NADH as indicated, and the following concentrations of pyruvate: A, 0.1 mM; A, 0.2 mM; 0, 0.3 mM; *, 0.5
~
80-
mM.
20~~~~
and malonic acids), only a-ketobutyric could be used for the oxidation of NADH. The Km values at pH 5.4 and 7.4 were 22 and 30 mM, respec2 4 8 6 citrate phosphate tris maleate tively, for D-LDH and 32 and 86 mM, respectively, for L+LDH. FIG. 3. Effect of pH on D-LDH and L+LDH acWhen the same analogues were tested as tivities. The enzyme reactions were assayed by pyru- inhibitors of pyruvate reduction, only 5.0 mM vate reduction with 25 mM buffer, 0.1 mM NADH and either 0.5 mM (A) or 5.0 mM pyruvate (B). Sym- oxamatt caused 50% inhibition of D-LDH at pH 5.4, whereas 0.5 mM oxamate and 0.5 mM bols: 0 and A, L+LDH; 0 and A, D-LDH. oxalate at pH 5.4 and 7.0 mM oxamate at pH TABLE 3. Michaelis constants (K.) for substrates of 7.4 resulted in 50% inhibition of L+LDH. Differential properties of the enzymes. L+LDH and D-LDH While studying the lactate dehydrogenases of L. Km (mM substrate) plantarum, Dennis and Kaplan (4) found sevVariable substrate pH L+LDH D-LDH eral chemical procedures that initiated markedly different responses from the D-LDH and the L+LDH of this organism. First, lactate Pyruvate 5.4 1.0 0.15 7.4 oxidation by L+LDH was severely inhibited by 8.0 0.67 8 mM oxamate, whereas D-LDH was not afNADH 5.4 0.02 0.0083 fected at all by the equivalent oxamate concen7.4 0.04 0.01 tration. Second, the D-LDH was totally inactivated by incubation at 50 C for 3 min, whereas D(-)- or L(+)-lactate 8.2 67 30 L+LDH was not denatured until a temperature of 80 C was reached. However, in the present NAD 8.2 5.0 1.05 study both enzymes from P. cerevisiae were pH
inactivated by incubation at 55 C for 3 min, and 8 mM oxamate was not inhibitory to lactate oxidation by either enzyme. Regulatory properties. Many glycolytic and hexosemonophosphate pathway intermediates, as well as D(-)- and L(+)-lactate, were tested on pyruvate reduction at both pH values by both enzymes, and not one had any effect. On the other hand, adenosine triphosphate, adenosine diphosphate, guanosine triphosphate, and NAD inhibited both enzymes to varying degrees (Table 4). Adenosine triphosphate appears to be the most potent inhibitor, and its action was found to be competitive with respect to NADH and noncompetitive with respect to pyruvate for D-LDH at both pH values and at pH 5.4 for L+LDH.
Effect of divalent metal ions and sulfhydryl-binding reagents. When the effect of six divalent metal ions was tested on D-LDH and L+LDH, only HgCl2 and CuSO, inhibited pyruvate reduction, with HgCl2 generally causing the more potent inhibition (Table 4). Chlorides of calcium, cobalt, magnesium, and manganese showed no effect, either positive or negative, at a final concentration of 10 mM. Sulfhydryl-binding reagents, p-hydroxymercuribenzoate and iodoacetamide, were tested as inhibitors of pyruvate reduction. Whereas iodoacetamide was without effect, p-hydroxymercuribenzoate inhibited both enzymes (Table 4). Molecular weights. The molecular weights of P. cerevisiae D-LDH and L+LDH were estimated by gel filtration, by electrophoresis in TABLE 4. Inhibition of D-LDH and L+LDH from P. cerevisiae by compounds of regulatory significance and by divalent metal ions and Concn causing 50% inhibition (mM) Determinationa
D-LDH
pH 5.4
ATP .......... 0.8 ADP .......... 3.0 GTP .......... 3.0 NAD .......... 10 (41)c HgCl2 ......... 0.01 CuSO4 ........ 10 (34)c
p-Hydroxymercuribenzoate
0.26
.
L+LDH
pH 7.4
pH 5.4
0.6 2.7 3.3 3.0 1.6 0.5
1.2 1.0 10 (44)c 5.0 0.02 0.2
0.12
0.6
nondenaturing polyacrylamide gels, and by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate (Table 5). Figure 5 is the calibration curve for estimation of the native molecular weights by electrophoresis in neutral polyacrylamide gels. Other calibration curves have already been published (11). DISCUSSION A previous study of the production of D(-)and L(+)-lactic acid in cultures of P. cerevisiae has shown that the ratio of L(+) acid to total lactic acid is high initially, but decreases as the cultures grow (9). This finding was verified during the present investigation, and it presented the problem of how this L(+)-lactate is produced before the D(-) isomer during the growth cycle. The total absence of any lactate racemizing activity in crude extracts of P. cerevisiae, together with reports of NADdependent D- and L-lactate dehydrogenases in other pediococci (6, 14) clearly reveal that two lactate dehydrogenases are the vehicles for racemic lactate production by this organism. Kinetic and physical properties of NADdependent D- and L-lactate dehydrogenases from P. cerevisiae can be compared with those from other organisms producing racemic lactic acid, as studied by other workers. Michaelis constants for lactate and NAD, as well as pH optima for lactate oxidation for both enzymes, are remarkably similar to those previously reported for P. pentosaceus (6) and L. plantarum (4). Comparison of the D-LDH can proceed further, with the enzyme from P. cerevisiae exhibiting heat lability and resistance to oxamate inhibition similar to this enzyme from TABLE 5. Estimated molecular weights of P. cerevisiae lactate dehydrogenases Mol wt Method
pH 7.4
NIb 4.5 NI 3.5 0.03 1.5
0.6 (43)C
a ATP, Adenosine 5-triphosphate; ADP, adenosine 5-'-diphosphate; GTP, guanosine 5'-diphosphate. b NI, Not inhibitory at a final concentration of 10
mM.
in parentheses indicates percent inhibition caused by the listed inhibitor concentration. c Figure
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Gel filtration on Sephadex G-200 Polyacrylamide gel electrophoresis A. Nondenaturing gels by method of Hedrick and Smith (13) i. Gel system of Davis (2) ii. Modified gel system of Weber and Osborn (22)b B. Denaturing gels by method and gel system of Weber and
L+LDH
D-LDH
139,000
71,000
136,000
72,000 73,000
37,000
39,000
-a
Osbom (22) aCould not be determined since L+LDH rapidly loses activity at alkaline pH. b Gel system modified by omitting sodium dodecyl sulfate.
606
GORDON AND DOELLE
Bovine serum albumin-dimer
8
A
7 CL 0
4-I
6[
-
51 B
a
z
Hexokinase
4 3
Bovine serum
albumin-monomer Ovalbumin I.
100 120 60 80 40 Molecular Weight (x 103)
140
FIG. 5. Standard curve for the estimation of the native molecular weights of P. cerevisiae D-LDH and L+LDH by electrophoresis in nondenaturing polyacrylamide gels (pH 7.0) by the method of Hedrick and Smith (13). A, L+LDH; B, D-LDH. Standard protein molecular weights: ovalbumin, 45,000; bovine serum albumin, 67,000; dimer, 134,000; hexokinase, 99,000.
other lactic acid bacteria (4, 8, 11, 16, 19). However, in regard to these characters, the L+LDH from P. cerevisiae appears to resemble other D-LDH in that it is heat labile and oxamate does not inhibit lactate oxidation. This is in contradiction to the behavior of L+LDH from various lactobacilli (4, 10), and only further investigation will show whether these charare common to most lactic acid bacteria if they are confined solely to the lactobacilli. The effect of pyruvate analogues on pyruvate reduction by both enzymes is worthy of mention. a-Ketobutyrate can be utilized as a reducible substrate by both D-LDH and L+LDH from P. cerevisiae, which is also the case in L. plantarum (4). However, oxamate is a potent inhibitor of pyruvate reduction by L. plantarum L+LDH with 50% inhibition resulting at 0.15 mM oxamate, whereas D-LDH is unaffected. Under similar circumstances neither D-LDH nor L+LDH from P. cerevisiae is inhibited. The slight inhibition of L+ LDH from P. cerevisiae caused by p-hydroxymercuribenzoate is unusual since the presence of essential sulfhydryl groups in the enzyme protein is indicated by its absolute requirement for reducing compounds during purification. Perhaps the DTT added to the partially purified preparation or the relatively high substrate levels used for
acters
or
J. BACTERIOL.
assay were sufficient to protect the enzyme from the sulfhydryl inhibitor. Molecular size and arrangement of both lactate dehydrogenases from P. cerevisiae are of interest since they conform to the general pattern that is gradually becoming apparent for all lactic acid bacteria. The dimeric arrangement for D-LDH from P. cerevisiae with a molecular weight of about 70,000 shows a close similarity to the enzymes from Leuconostoc lactis (11, 16) and Lactobacillus leichmannii (11), whereas the tetrameric arrangement for L-+ LDH from P. cerevisiae with a molecular weight of about 140,000 shows a close similarity to the enzyme from Streptococcus cremoris (7). Native molecular weights of both of these enzymes from P. cerevisiae are comparable to values reported for many other lactic acid bacteria (5, 8, 10). Metabolic control of D-LDH and L+LDH from P. cerevisiae appears to be similar to the mechanisms described previously for L. lactis (11), with pyruvate reduction by both enzymes being subject to product inhibition by NAD and to energy-dependent inhibition by adenosine triphosphate. Although these inhibitory effects are not as profoundly affected by pH as they are in leuconostocs, it seems that they still form the basis for the regulation of total lactate production by P. cerevisiae. However, this regulatory phenomenon fails to explain the reason for the sequential production of the two isomers of lactic acid in cultures of P. cerevisiae. It is our belief that culture pH is the determining factor in this production. In the present study it has been found that D-LDH and L+LDH from P. cerevisiae react differently to change in pH depending upon the pyruvate concentration in the assay (Fig. 3). Although Mizushima and Kitahara (18) reported that the intracellular pyruvate concentration in L. plantarum is approximately 5.2 mM, it would appear that the actual concentration in P. cerevisiae is much lower than this. Since the specific activity of pyruvate reduction in a crude extract of P. cerevisiae is 8.7 U/mg of protein while pyruvate kinase (EC 2.7.1.40) is extremely difficult to detect (G. Gordon and H. Doelle, unpublished observations), it seems unlikely that pyruvate would be able to accumulate in these cells and thus reach an intracellular concentration as high as 5 mM. Therefore, the pH profile most likely to correspond to physiological conditions is that obtained using low pyruvate concentrations (0.5 mM). During the growth cycle of P. cerevisiae, L( +)-lactate production commences with initiation of growth when the pH of the medium is about 6.7 (Fig. 1), whereas this production
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607
8. Garland, R. C. 1973. Purification and properties of D( )-lactate dehydrogenase from Leuconostoc mesenteroides. Arch. Biochem. Biophys. 157:36-43. 9. Garvie, E. I. 1967. The production of L(+) and D(-) lactic acid in cultures of some lactic acid bacteria, with a special study of Lactobacillus acidophilus NCDO2. J. Dairy Res. 34:31-38. 10. Gasser, F., M. Doudoroff, and R. Contopoulos. 1970. Purification and properties of NAD-dependent lactic dehydrogenases of different species of Lactobacillus. J. Gen. Microbiol. 62:241-250. 11. Gordon, G. L., and H. W. Doelle. 1974. Molecular aspects for the metabolic regulation of the nicotinamide adenine dinucleotide-dependent D( )-lactate dehydrogenase from Leuconostoc. Microbios 9:199-215. 12. Gornall, A., C. Bardowell, and M. David. 1949. Determination of serum proteins by means of the biuret reagent. J. Biol. Chem. 177:751-756. 13. Hedrick, J. L., and A. J. Smith. 1968. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126:155-164. 14. Hiyama, T., S. Fukui, and K. Kitahara. 1968. Purification and properties of lactate racemase from Lactobacillus sake. J. Biochem. (Tokyo) 64:99-107. 15. Hohorst, H.-J. 1963. L(+)-Lactate determination with lactic dehydrogenase and DPN, p. 266-270. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis. Academic Press Inc., New York. 16. Hontebeyrie, M., and F. Gasser. 1973. Separation et purification de la D-lactico-deshydrogenase et de la glucose-6-phosphate deshydrogenase de Leuconostoc lactis. Etude de quelques propietes. Biochimie 55: ACKNOWLEDGMENTS 1047-1056. This work was carried out with finances from the Australian 17. Mizushima, S., T. Hiyama, and K. Kitahara. 1964. Quantitative studies on glycolytic enzymes in LactobaUniversities Commission and with support from a Commoncillus plantarum. III. Intracellular activities of reverse wealth Postgraduate Research Award (G.L.G.). reaction of D- and L-lactate dehydrogenases during glucose fermentation. J. Gen. Appl. Microbiol. (Tokyo) CITED LITERATURE 10:33-44. 1. Breed, R. S., E. D. G. Murray, and N. R. S. Smith (ed.). 18. Mizushima, S., and K. Kitahara. 1964. Quantitative studies on glycolytic enzymes in Lactobacillus 1957. Bergey's manual of determinative bacteriology, plantarum. II. Intracellular concentrations of glycolytic 7th ed. The Williams & Wilkins Co., Baltimore. intermediates in glucose-metabolizing washed cells. J. 2. Davis, B. J. 1964. Disc electrophoresis. II. Method and Bacteriol. 87:1429-1435. application to serum proteins. Ann. N.Y. Acad. Sci. 19. Mizushima, S., Y. Machida, and K. Kitahara. 1963. 121:404-427. Quantitative studies on glycolytic enzymes in Lac3. DeMan, J. C., M. Rogosa, and M. E. Sharpe. 1960. A tobacillus plantarum. I. Concentration of inorganic medium for the cultivation of lactobacilli. J. Appl. ions and coenzymes in fermenting cells. J. Bacteriol. Bacteriol. 23:130-135. 86:1295-1300. 4. Dennis, D., and N. 0. Kaplan. 1960. D- and L-lactic acid dehydrogenases in Lactobacillus plantarum. J. Biol. 20. Stetter, K. O., and 0. Kandler. 1973. Untersuchungen zur Entstehung von DL-Milchsiiure bei Lactobacillen Chem. 235:810-818. und Characterisierung einer Milchsiiureracemase bei 5. Dennis, D., M. Reichlin, and N. 0. Kaplan. 1965. Lactic einigen Arten der Untergattung Streptobacterium. acid racemization. Ann. N.Y. Acad. Sci. 119:868-876. Arch. Mikrobiol. 94:221-247. 6. Doelle, H. W. 1971. Nicotinamide adenine dinucleotidedependent and nicotinamide adenine dinucleotide- 21. Warburg, O., and W. Christian. 1942. Isolierung und Kristallisation des Garungsferments Enolase. Bioindependent lactate dehydrogenases in homofermentachem. Z. 310:384-421. tive and heterofermentative lactic acid bacteria. J. 22. Weber, K., and M. Osborn. 1969. The reliability of Bacteriol. 108:1284-1289. molecular weight determinations by dodecylsulphate7. Dynon, M. K., G. R. Jago, and B. E. Davidson. 1972. The polyacrylamide gel electrophoresis. J. Biol. Chem. subunit structure of lactate dehydrogenase from Strep244:4406-4412. tococcus cremoris US3. Eur. J. Biochem. 30:348-353.
D(-)-lactate pH is about production 5.0). Actively fermenting L. plantarum cells have an intemal pH of 5.4 (19) and, in the absence of any contrary evidence, it seems reasonable to assume that the intracellular pH is similar to that of the medium. By reference to Fig. 3A, it can be seen that, as the pH falls from about 6.5 to 5.0, L+LDH attains maximum activity while D -LDH activity is still relatively low. At this pH in the culture, D(-)-lactate production commences with the pH falling to about 4.0 as a result, which can be explained by the activity maximum of D-LDH at a pH of 3.6. However, this apparently carefully regulated total production of one isomer of lactic acid followed by a switch to the total production of the other does not appear to be common among all racemic lactate-producing Lactobacillaceae. For instance, the percentage of L(+) acid in the culture changes little during growth of L. plantarum (9), which is reflected in the fact that both lactate dehydrogenases from this organism display an optimum for pyruvate reduction at pH 6.0 (17). ceases
same time as commences (when the
at almost the
Vol. 121, No. 2 Printed in U.SA.
Production of Racemic Lactic Acid in Pediococcus cerevisiae Cultures by Two Lactate Dehydrogenases GEOFFREY L. GORDON AND H. W. DOELLE* Department of Microbiology, University of Queensland, St. Lucia, Queensland 4067, Australia Received for publication 22 November 1974
Nicotinamide adenine dinucleotide (NAD)-dependent D(-)- and L(+)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for D(-)-lactate dehydrogenase and 136,000 to 139,000 for L(+)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by D(-)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, L(+)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey MichaelisMenten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. a-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevistae. By definition, all lactic acid bacteria produce some lactic acid during fermentation of carbohydrates. In most cases this lactic acid is either levorotatory or dextrorotatory, but occasionally a mixture of both isomers can be formed. Lactobacillus sake has been found to form L( +)-lactic acid by the action of a nicotinamide adenine dinucleotide (NAD)-dependent L( + )-lactate dehydrogenase (EC 1.1.1.27; L+LDH), which in the presence of lactate racemase (EC 5.1.2.1) results in the production of a racemic mixture of lactic acid in the culture (14). A similar situation has also been reported recently in L. curvatus (20). However, certain other members of this group of organisms produce racemic mixtures of lactic acid by the simultaneous action of NAD-dependent D(-)lactate dehydrogenase (EC 1.1.1.28; D-LDH) as well as L+LDH (4, 5, 10, 17). Since pediococci are traditionally regarded as organisms that produce racemic lactic acid as a major end product of glucose catabolism (1), the question arises: how is this racemic mixture actually formed? Preliminary studies by Hiyama et al. (14) indicated that production of 60(
racemic lactate by Pediococcus lindneri and P. hennebergi is brought about by the presence of two different lactate dehydrogenases within their cells. The present investigation was under-
taken in an attempt to provide information at the enzyme level on the mechanism of production of racemic lactate mixtures in cultures of P. cerevisiae. MATERIALS AND METHODS Materials. All cofactors, substrates, nucleotides, glycolytic intermediates, as well as dithiothreitol (DTT) and tris(hydroxymethyl)aminomethane (Tris), were purchased from Sigma Chemical Co. (St. Louis, Mo.). Lactate dehydrogenases from rabbit muscle and L. leichmannii were purchased from Boehringer-Mannheim GmbH (Mannheim, Germany). Diethylaminoethyl-Sephadex A-25 and Sepharose 6B were products of Pharmacia Fine Chemical Company (Uppsala, Sweden); hydroxylapatite Bio Gel HTP was obtained from Bio-Rad Laboratories (Richmond, Ca.). Ammonium sulfate was special enzyme grade from Schwarz/Mann (Orangeburg, N.Y.). All other chemicals were analytical grade. Organisms and culture conditions. P. cerevisiae 813 and P. pentosaceus 9206 were obtained from the
LACTATE DEHYDROGENASES FROM P. CEREVISIAE
VOL. 121, 1975
National Collection of Dairy Organisms (National Institute for Research in Dairying, Shinfield, Reading, Berkshire, England) and from the National Collection of Industrial Bacteria (Torry Research Station, Aberdeen, Scotland), respectively. Both were maintained by monthly subculture on MRS agar (3) slopes, which were stored at 4 C after growth for 24 h at 28 C. Large-scale growth of P. cerevisiae was undertaken in a 10-liter carboy of MRS medium containing only 1.0% (wt/vol) glucose. The complete medium without glucose was sterilized by autoclaving at 115 C for 20 min; a 40% glucose solution, sterilized separately by autoclaving, was then added aseptically to a final concentration of 1.0%. Bacterial growth in the carboy was initiated with a 1% (vol/vol) inoculum of a 24-h culture grown in the same medium. The culture was incubated at 28 C for 18 h, with constant but slow magnetic stirring. The cells were harvested by centrifugation, washed with cold 0.9% (wt/vol) NaCl, and stored as a frozen cell paste. Cells for lactate racemase studies were grown in 100-ml amounts of modified MRS, which was prepared by omitting sodium acetate and ammonium citrate and by reducing the glucose concentration to 0.1%. Preparation of cell-free extracts. Ten grams of thawed cell paste was suspended in about 200 ml of 0.05 M sodium phosphate buffer (pH 7.0) containing 2 mM DTT and 2 mM sodium-DL-lactate, and disrupted in 50-ml fractions with 50 g of glass beads (0.10- to 0.11-mm diameter) at 4,000 rpm in a Braun cell homogenizer (type MSK; Melsungen, Germany) for 15 min. During disruption, the glass vessel was cooled by a continuous stream of liquid CO2. The opaque, straw-colored liquid was decanted and then clarified by centrifugation at 37,000 x g for 30 min. The resultant supernatant was then used for all enzymatic procedures. Enzyme assays. Both lactate dehydrogenases were assayed spectrophotometrically at 340 nm by using a Pye-Unicam SP 8000 recording spectrophotometer with 3.0-ml, 1.0-cm path length quartz cuvettes. Lactate oxidation was measured in a system containing 25 mM Tris-maleate buffer (pH 8.2), 2.0 mM sodium NAD, and 10 mM lithium L(+)- or 10 mM D(-)-lactate in a final volume of 3.0 ml. Pyruvate reduction was measured in a 3.0-ml system containing 25 mM Tris-maleate buffer (pH 5.4 or 7.4) and concentrations of sodium pyruvate and sodium reduced NAD, as indicated in Table 1. Dehydrogenase
601
units were expressed as micromoles of NADH produced or oxidized per minute at 30 C. Specific activity is defined as units per milligram of enzyme protein. The assay system used for lactate racemase and the defimition for lactate racemizing units of activity were those used by Hiyama et al. (14). Analytical methods. Residual glucose in the medium was determined by a mixed enzyme-dye method (A. St. G. Huggett and D. A. Nixon, Biochem. J. 66: 12P, 1957). Isomer production of lactic acid in culture supernatants, as well as in assays of lactate racemase, was determined enzymatically by the method of Hohorst (15), using L+LDH from rabbit muscle and D-LDH from L. leichmannii. Dry weights of the cells were determined in preweighed glass vials after addition of a washed-cell suspension and drying at 105 C to constant weight. Protein concentration in cell-free extracts was measured by the biuret method (12), with crystalline bovine serum albumin as standard, whereas protein concentrations of all other purification steps were estimated by using their absorbance at 260 and 280 nm (21). Molecular weight estimations. Native molecular weights were estimated by gel filtration through a Sephadex G-200 column (2.6 by 65 cm) eluted with 0.05 M sodium phosphate buffer (pH 7.0) containing 2 mM DTT. Standard proteins and methods for their assay have been described previously (11). The native molecular weight of D-LDH was estimated electrophoretically, by the method of Hedrick and Smith (13), in an alkaline polyacrylamide gel system (2) as described previously (11). However, an estimate for L+LDH was not possible in this system owing to the extreme lability of this enzyme at alkaline pH. Therefore, the native molecular weight of L+LDH was estimated electrophoretically at pH 7.0 by the procedure of Weber and Osborn (22) modified by the omission of sodium dodecyl sulfate. Five polyacrylamide gel concentrations (5 to 9%). were used for the estimation by the method of Hedrick and Smith (13). Subunit molecular weights of the two enzymes were estimated in gels containing 0.1% sodium dodecyl sulfate by the method of Weber and Osborn (22) as described previously (11).
RESULTS Growth characteristics and lactate production by P. cerevisiae. The growth characTABLE 1. Summary of assay conditions for pyruvate teristics of P. cerevisiae grown in standing batch reduction in P. cerevisiae culture at 28 C in a 10-liter carboy are shown in 1. A cell yield of 4 g (dry weight per liter) Fig. WSodium Sodium pyrAssay pH corresponds to about 7 g (wet weight) of cells/ uvate (mM) NADH (mM) liter. The cells were harvested early in the sta0.04 2.0 5.4 tionary phase after 18 h of growth, as lactate 0.1 10 7.4 production appeared to have ceased by this 5.4 7.4
0.3 1.0
0.02 0.02
stage. During the fermentation, L( + ) -lactic acid was the first isomer to appear in the culture me-
GORDON AND DOELLE
602
6
TL
I_HoursI0 FIG. 1. Growth characteristics of P. cerevisiae NCDO 813 in MRS broth (3) at 28 C. Symbols: 0, bacterial dry weight; 0, L(+)-lactate; 0, D(-)-lactate; V, culture pH; A, glucose in medium.
J. BACTERIOL.
sodium phosphate buffer (pH 7.0) containing 2 mM DTT. This solution was layered onto a Sepharose 6B column (2.8 by 70 cm), which had been equilibrated with the same buffer, and the column was eluted at a flow rate of 30 ml/h. L+LDH was eluted first and was followed closely by D-LDH, with a slight overlap of activities. Fractions with more than 50% of the activity of each peak fraction were pooled (Fig. 2). The first peak was concentrated to about 5 ml in an Amicon ultrafiltration cell fitted with a UM-50 membrane. The concentrated solution was diluted to 50 ml with 0.05 M sodium phosphate buffer (pH 6.0) containing 2 mM DTT and reconcentrated to about 5 ml. This solution was then applied to an hydroxylapatite column (1.0 by 30 cm), which had been equilibrated with the same buffer. The column was eluted at a flow rate of 3.6 ml/h with a 200-ml linear gradient of 0.05 to 0.5 M sodium phosphate buffer (pH 6.0). The single activity peak fractions were pooled and concentrated by ultrafiltration (UM-50 membrane), diluted with 0.05 M sodium phosphate buffer (pH 7.0) containing 2 mM DTT, and reconcentrated as
dium, whereas the D(-) isomer did not appear until several hours later when the pH had fallen to about 5. At this point, further L(+) acid production ceased. Lactate racemng activity in cell-free extracts of pediococci. Cell-free extracts prepared from cells of P. cerevisiae and P. pentosaceus, grown in the absence of acetate and citrate, exhibited considerable lactate racemizing activities of 4.1 and 3.6 U/mg of protein, respectively. However, after overnight Sepharose 65 rI I dialysis of the extracts against the buffer system used for cell-free extract preparation, this activity was completely lost, while specific activities of both lactate dehydrogenases were essentially constant. Partial purification of NAD-dependent L+LDH and D-LDH from P. cerevisiae. Enzyme activities were monitored by lactate oxidation throughout the purification procedure. All operations were carried out between 0 and 4 C, except the ammonium sulfate fractionation, which was performed at room temperature (about 20 C). After centrifugation at 4 C, the cell-free extract was equilibrated at room temperature, after which it was adjusted to 45% ammonium sulfate saturation by the slow addition of the solid chemical (277 g/liter) together with slow magnetic stirring while a constant, gentle stream of dry nitrogen was played over the liquid surface. The pellet formed after centrifugation (20 min at 37,000 x g) was discarded, whereas the ammonium sulfate concentration of the supernatant was increased to 70% saturation (by addition of 171 g of solid chemical per liter to the 45% saturated-ammonium sulfate Elution Volume (ml) supernatant, with identical precautions). CenFIG. 2. Elution profiles of D-LDH and L+LDH trifugation (20 min at 37,000 x g) produced a activity during purification. Symbols: *, L+LDH; 0, pellet which was suspended in 5 ml of 0.05 M D-LDH; A, absorbancy at 280 nm.
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LACTATE DEHYDROGENASES FROM P. CEREVISIAE
before. Sufficient glycerol was added to the partially purified L+LDH preparation to give final concentration of 30% (vol/vol), with the preparation then being stored at 4 C without appreciable loss of activity over a period of several months. The second peak eluted from the Sepharose 6B column was concentrated, diluted with 0.05 M Tris-hydrochloride buffer (pH 8.0) containing 2 mM DTT and 0.05 M NaCl, and reconcentrated to about 5 ml. This preparation was then applied to diethylaminoethyl-Sephadex A-25 column (2.0 by 30 cm), which had been equilibrated with the same buffer. The column was eluted at a flow rate of 100 ml/h with a 500-ml linear gradient of 0.05 to 0.5 M NaCl. The single activity peak fractions were pooled and concentrated by ultrafiltration (UM-50 membrane), diluted with 0.05 M Tris-maleate buffer (pH 8.0) containing 2 mM DTT, reconcentrated, and stored frozen without appreciable loss of D- LDH activity over a period of several months. A summary of a typical purification procedure appears in Table 2; representative elution profiles are displayed in Fig. 2. Both enzyme preparations were examined for homogeneity by polyacrylamide gel electrophoresis, but in both cases the preparations possessed a major protein band, which corresponded to the activity band, contaminated with two or three other faint protein bands. pH optima and reversibility of the reactions. Hydrogen ion concentration was found to have different effects on lactate oxidation by
603
L+LDH and D-LDH from P. cerevisiae. Maximum activity for L+LDH was found at pH 7.6, whereas that for D-LDH was at pH 9.6. Pyruvate reduction by D-LDH demonstrated an activity peak at pH 8.0 (Fig. 3), which shifted to a sharp peak at pH 3.6 when the pyruvate concentration was lowered from 5.0 to 0.5 mM. On the other hand, pyruvate reduction by L+LDH exhibited maximum activity in the pH range 4.5 to 6.0 at either pyruvate concentration. In 1963 it was estimated that the internal pH of actively metabolizing cells of L. plantarum is 5.4 (19). This value, as well as the pH value normally regarded as physiological (7.4), was used in an estimation of the reversibility of both enzymes. Reaction velocities for lactate oxidation and pyruvate reduction of both enzymes at these pH values were estimated from the pH profiles for lactate oxidation and those shown in Fig. 3. From this data it was calculated that lactate oxidation was 6.1% and 1.1% of pyruvate reduction at pH 7.4 and 5.4, respectively, for D-LDH and 2.7% at pH 7.4 for L+LDH. L+LDH was not reversible at pH 5.4. Michaelis constants for substrates. Michaelis constants (Ki) for all substrates of both enzymes are given in Table 3. Values for lactate and NAD were estimated for the purified enzymes from linear double-reciprocal plots at one substrate concentration. Michaelis constants for pyruvate and NADH were estimated at pH 5.4 and 7.4 by plotting saturation curves at four concentrations of the second substrate. The reciprocals for apparent maximum veloci-
TABLE 2. Summary of the purification of lactate dehydrogenases from P. cerevisiae Purification step
1. Cell-free extract
2. Ammonium sulfate fractionation, 45 to 70% saturation 3. Sepharose 6B chromatography: Peak I
Total Ttlnucleic acid |(mg)
Vol (ml) protein l(mg)
168 5.0
D-LDH
L+LDH Sp act
Total
(U/mg) activity (U)
To t
Yield (%)
Sp act (U/mg)
activ(ty (U)
Yield
1008
722
0.0257
25.97
100
0.0177
17.86 100
285
119
0.0610
17.39
67
0.0457
13.04
73
18.5 18.0
77.7 41.4
4.3 3.1
0.2093 0.0294
16.27 1.22
63 5
0.0127 0.3067
0.99 12.70
6 71
4. Hydroxylapatite chromatography of peak I at pH 6.0
16.5
3.9
0.1
1.8116
7.17
28
0
0
O
5. DEAE-Sephadex chromatography of peak II at pH 8.0a
27.0
2.2
0
0
0
0
1.5700
3.39
19
PeakII
-
DEAE, Diethylaminoethyl.
604
J. BACTERIOL.
GORDON AND DOELLE
ties for the four different second substrate concentrations were then plotted against the reciprocal of the substrate concentration to give the Km of the second substrate (Fig. 4). All substrate saturation curves for bo'th enzymes were hyperbolic, double-reciprocal plots which were linear and gave Km values for both substrates which increased with increasing pH. Effect of pyruvate analogues. Of the four analogues tested (a-ketobutyric, oxamic, oxalic,
E TO
4020
O-
E
-8
4
8
'/Pyruvote (mM'4) 'C
0
60
-
FIG. 4. Example for determination of Km value for substrates of pyruvate reduction. The reaction mixtures contained 25 mM Tris-maleate buffer (pH 5.4), partially purified D-LDH (2.7 ,gg of protein), NADH as indicated, and the following concentrations of pyruvate: A, 0.1 mM; A, 0.2 mM; 0, 0.3 mM; *, 0.5
~
80-
mM.
20~~~~
and malonic acids), only a-ketobutyric could be used for the oxidation of NADH. The Km values at pH 5.4 and 7.4 were 22 and 30 mM, respec2 4 8 6 citrate phosphate tris maleate tively, for D-LDH and 32 and 86 mM, respectively, for L+LDH. FIG. 3. Effect of pH on D-LDH and L+LDH acWhen the same analogues were tested as tivities. The enzyme reactions were assayed by pyru- inhibitors of pyruvate reduction, only 5.0 mM vate reduction with 25 mM buffer, 0.1 mM NADH and either 0.5 mM (A) or 5.0 mM pyruvate (B). Sym- oxamatt caused 50% inhibition of D-LDH at pH 5.4, whereas 0.5 mM oxamate and 0.5 mM bols: 0 and A, L+LDH; 0 and A, D-LDH. oxalate at pH 5.4 and 7.0 mM oxamate at pH TABLE 3. Michaelis constants (K.) for substrates of 7.4 resulted in 50% inhibition of L+LDH. Differential properties of the enzymes. L+LDH and D-LDH While studying the lactate dehydrogenases of L. Km (mM substrate) plantarum, Dennis and Kaplan (4) found sevVariable substrate pH L+LDH D-LDH eral chemical procedures that initiated markedly different responses from the D-LDH and the L+LDH of this organism. First, lactate Pyruvate 5.4 1.0 0.15 7.4 oxidation by L+LDH was severely inhibited by 8.0 0.67 8 mM oxamate, whereas D-LDH was not afNADH 5.4 0.02 0.0083 fected at all by the equivalent oxamate concen7.4 0.04 0.01 tration. Second, the D-LDH was totally inactivated by incubation at 50 C for 3 min, whereas D(-)- or L(+)-lactate 8.2 67 30 L+LDH was not denatured until a temperature of 80 C was reached. However, in the present NAD 8.2 5.0 1.05 study both enzymes from P. cerevisiae were pH
inactivated by incubation at 55 C for 3 min, and 8 mM oxamate was not inhibitory to lactate oxidation by either enzyme. Regulatory properties. Many glycolytic and hexosemonophosphate pathway intermediates, as well as D(-)- and L(+)-lactate, were tested on pyruvate reduction at both pH values by both enzymes, and not one had any effect. On the other hand, adenosine triphosphate, adenosine diphosphate, guanosine triphosphate, and NAD inhibited both enzymes to varying degrees (Table 4). Adenosine triphosphate appears to be the most potent inhibitor, and its action was found to be competitive with respect to NADH and noncompetitive with respect to pyruvate for D-LDH at both pH values and at pH 5.4 for L+LDH.
Effect of divalent metal ions and sulfhydryl-binding reagents. When the effect of six divalent metal ions was tested on D-LDH and L+LDH, only HgCl2 and CuSO, inhibited pyruvate reduction, with HgCl2 generally causing the more potent inhibition (Table 4). Chlorides of calcium, cobalt, magnesium, and manganese showed no effect, either positive or negative, at a final concentration of 10 mM. Sulfhydryl-binding reagents, p-hydroxymercuribenzoate and iodoacetamide, were tested as inhibitors of pyruvate reduction. Whereas iodoacetamide was without effect, p-hydroxymercuribenzoate inhibited both enzymes (Table 4). Molecular weights. The molecular weights of P. cerevisiae D-LDH and L+LDH were estimated by gel filtration, by electrophoresis in TABLE 4. Inhibition of D-LDH and L+LDH from P. cerevisiae by compounds of regulatory significance and by divalent metal ions and Concn causing 50% inhibition (mM) Determinationa
D-LDH
pH 5.4
ATP .......... 0.8 ADP .......... 3.0 GTP .......... 3.0 NAD .......... 10 (41)c HgCl2 ......... 0.01 CuSO4 ........ 10 (34)c
p-Hydroxymercuribenzoate
0.26
.
L+LDH
pH 7.4
pH 5.4
0.6 2.7 3.3 3.0 1.6 0.5
1.2 1.0 10 (44)c 5.0 0.02 0.2
0.12
0.6
nondenaturing polyacrylamide gels, and by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate (Table 5). Figure 5 is the calibration curve for estimation of the native molecular weights by electrophoresis in neutral polyacrylamide gels. Other calibration curves have already been published (11). DISCUSSION A previous study of the production of D(-)and L(+)-lactic acid in cultures of P. cerevisiae has shown that the ratio of L(+) acid to total lactic acid is high initially, but decreases as the cultures grow (9). This finding was verified during the present investigation, and it presented the problem of how this L(+)-lactate is produced before the D(-) isomer during the growth cycle. The total absence of any lactate racemizing activity in crude extracts of P. cerevisiae, together with reports of NADdependent D- and L-lactate dehydrogenases in other pediococci (6, 14) clearly reveal that two lactate dehydrogenases are the vehicles for racemic lactate production by this organism. Kinetic and physical properties of NADdependent D- and L-lactate dehydrogenases from P. cerevisiae can be compared with those from other organisms producing racemic lactic acid, as studied by other workers. Michaelis constants for lactate and NAD, as well as pH optima for lactate oxidation for both enzymes, are remarkably similar to those previously reported for P. pentosaceus (6) and L. plantarum (4). Comparison of the D-LDH can proceed further, with the enzyme from P. cerevisiae exhibiting heat lability and resistance to oxamate inhibition similar to this enzyme from TABLE 5. Estimated molecular weights of P. cerevisiae lactate dehydrogenases Mol wt Method
pH 7.4
NIb 4.5 NI 3.5 0.03 1.5
0.6 (43)C
a ATP, Adenosine 5-triphosphate; ADP, adenosine 5-'-diphosphate; GTP, guanosine 5'-diphosphate. b NI, Not inhibitory at a final concentration of 10
mM.
in parentheses indicates percent inhibition caused by the listed inhibitor concentration. c Figure
605
LACTATE DEHYDROGENASES FROM P. CEREVISIAE
VOL. 121, 1975
Gel filtration on Sephadex G-200 Polyacrylamide gel electrophoresis A. Nondenaturing gels by method of Hedrick and Smith (13) i. Gel system of Davis (2) ii. Modified gel system of Weber and Osborn (22)b B. Denaturing gels by method and gel system of Weber and
L+LDH
D-LDH
139,000
71,000
136,000
72,000 73,000
37,000
39,000
-a
Osbom (22) aCould not be determined since L+LDH rapidly loses activity at alkaline pH. b Gel system modified by omitting sodium dodecyl sulfate.
606
GORDON AND DOELLE
Bovine serum albumin-dimer
8
A
7 CL 0
4-I
6[
-
51 B
a
z
Hexokinase
4 3
Bovine serum
albumin-monomer Ovalbumin I.
100 120 60 80 40 Molecular Weight (x 103)
140
FIG. 5. Standard curve for the estimation of the native molecular weights of P. cerevisiae D-LDH and L+LDH by electrophoresis in nondenaturing polyacrylamide gels (pH 7.0) by the method of Hedrick and Smith (13). A, L+LDH; B, D-LDH. Standard protein molecular weights: ovalbumin, 45,000; bovine serum albumin, 67,000; dimer, 134,000; hexokinase, 99,000.
other lactic acid bacteria (4, 8, 11, 16, 19). However, in regard to these characters, the L+LDH from P. cerevisiae appears to resemble other D-LDH in that it is heat labile and oxamate does not inhibit lactate oxidation. This is in contradiction to the behavior of L+LDH from various lactobacilli (4, 10), and only further investigation will show whether these charare common to most lactic acid bacteria if they are confined solely to the lactobacilli. The effect of pyruvate analogues on pyruvate reduction by both enzymes is worthy of mention. a-Ketobutyrate can be utilized as a reducible substrate by both D-LDH and L+LDH from P. cerevisiae, which is also the case in L. plantarum (4). However, oxamate is a potent inhibitor of pyruvate reduction by L. plantarum L+LDH with 50% inhibition resulting at 0.15 mM oxamate, whereas D-LDH is unaffected. Under similar circumstances neither D-LDH nor L+LDH from P. cerevisiae is inhibited. The slight inhibition of L+ LDH from P. cerevisiae caused by p-hydroxymercuribenzoate is unusual since the presence of essential sulfhydryl groups in the enzyme protein is indicated by its absolute requirement for reducing compounds during purification. Perhaps the DTT added to the partially purified preparation or the relatively high substrate levels used for
acters
or
J. BACTERIOL.
assay were sufficient to protect the enzyme from the sulfhydryl inhibitor. Molecular size and arrangement of both lactate dehydrogenases from P. cerevisiae are of interest since they conform to the general pattern that is gradually becoming apparent for all lactic acid bacteria. The dimeric arrangement for D-LDH from P. cerevisiae with a molecular weight of about 70,000 shows a close similarity to the enzymes from Leuconostoc lactis (11, 16) and Lactobacillus leichmannii (11), whereas the tetrameric arrangement for L-+ LDH from P. cerevisiae with a molecular weight of about 140,000 shows a close similarity to the enzyme from Streptococcus cremoris (7). Native molecular weights of both of these enzymes from P. cerevisiae are comparable to values reported for many other lactic acid bacteria (5, 8, 10). Metabolic control of D-LDH and L+LDH from P. cerevisiae appears to be similar to the mechanisms described previously for L. lactis (11), with pyruvate reduction by both enzymes being subject to product inhibition by NAD and to energy-dependent inhibition by adenosine triphosphate. Although these inhibitory effects are not as profoundly affected by pH as they are in leuconostocs, it seems that they still form the basis for the regulation of total lactate production by P. cerevisiae. However, this regulatory phenomenon fails to explain the reason for the sequential production of the two isomers of lactic acid in cultures of P. cerevisiae. It is our belief that culture pH is the determining factor in this production. In the present study it has been found that D-LDH and L+LDH from P. cerevisiae react differently to change in pH depending upon the pyruvate concentration in the assay (Fig. 3). Although Mizushima and Kitahara (18) reported that the intracellular pyruvate concentration in L. plantarum is approximately 5.2 mM, it would appear that the actual concentration in P. cerevisiae is much lower than this. Since the specific activity of pyruvate reduction in a crude extract of P. cerevisiae is 8.7 U/mg of protein while pyruvate kinase (EC 2.7.1.40) is extremely difficult to detect (G. Gordon and H. Doelle, unpublished observations), it seems unlikely that pyruvate would be able to accumulate in these cells and thus reach an intracellular concentration as high as 5 mM. Therefore, the pH profile most likely to correspond to physiological conditions is that obtained using low pyruvate concentrations (0.5 mM). During the growth cycle of P. cerevisiae, L( +)-lactate production commences with initiation of growth when the pH of the medium is about 6.7 (Fig. 1), whereas this production
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8. Garland, R. C. 1973. Purification and properties of D( )-lactate dehydrogenase from Leuconostoc mesenteroides. Arch. Biochem. Biophys. 157:36-43. 9. Garvie, E. I. 1967. The production of L(+) and D(-) lactic acid in cultures of some lactic acid bacteria, with a special study of Lactobacillus acidophilus NCDO2. J. Dairy Res. 34:31-38. 10. Gasser, F., M. Doudoroff, and R. Contopoulos. 1970. Purification and properties of NAD-dependent lactic dehydrogenases of different species of Lactobacillus. J. Gen. Microbiol. 62:241-250. 11. Gordon, G. L., and H. W. Doelle. 1974. Molecular aspects for the metabolic regulation of the nicotinamide adenine dinucleotide-dependent D( )-lactate dehydrogenase from Leuconostoc. Microbios 9:199-215. 12. Gornall, A., C. Bardowell, and M. David. 1949. Determination of serum proteins by means of the biuret reagent. J. Biol. Chem. 177:751-756. 13. Hedrick, J. L., and A. J. Smith. 1968. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126:155-164. 14. Hiyama, T., S. Fukui, and K. Kitahara. 1968. Purification and properties of lactate racemase from Lactobacillus sake. J. Biochem. (Tokyo) 64:99-107. 15. Hohorst, H.-J. 1963. L(+)-Lactate determination with lactic dehydrogenase and DPN, p. 266-270. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis. Academic Press Inc., New York. 16. Hontebeyrie, M., and F. Gasser. 1973. Separation et purification de la D-lactico-deshydrogenase et de la glucose-6-phosphate deshydrogenase de Leuconostoc lactis. Etude de quelques propietes. Biochimie 55: ACKNOWLEDGMENTS 1047-1056. This work was carried out with finances from the Australian 17. Mizushima, S., T. Hiyama, and K. Kitahara. 1964. Quantitative studies on glycolytic enzymes in LactobaUniversities Commission and with support from a Commoncillus plantarum. III. Intracellular activities of reverse wealth Postgraduate Research Award (G.L.G.). reaction of D- and L-lactate dehydrogenases during glucose fermentation. J. Gen. Appl. Microbiol. (Tokyo) CITED LITERATURE 10:33-44. 1. Breed, R. S., E. D. G. Murray, and N. R. S. Smith (ed.). 18. Mizushima, S., and K. Kitahara. 1964. Quantitative studies on glycolytic enzymes in Lactobacillus 1957. Bergey's manual of determinative bacteriology, plantarum. II. Intracellular concentrations of glycolytic 7th ed. The Williams & Wilkins Co., Baltimore. intermediates in glucose-metabolizing washed cells. J. 2. Davis, B. J. 1964. Disc electrophoresis. II. Method and Bacteriol. 87:1429-1435. application to serum proteins. Ann. N.Y. Acad. Sci. 19. Mizushima, S., Y. Machida, and K. Kitahara. 1963. 121:404-427. Quantitative studies on glycolytic enzymes in Lac3. DeMan, J. C., M. Rogosa, and M. E. Sharpe. 1960. A tobacillus plantarum. I. Concentration of inorganic medium for the cultivation of lactobacilli. J. Appl. ions and coenzymes in fermenting cells. J. Bacteriol. Bacteriol. 23:130-135. 86:1295-1300. 4. Dennis, D., and N. 0. Kaplan. 1960. D- and L-lactic acid dehydrogenases in Lactobacillus plantarum. J. Biol. 20. Stetter, K. O., and 0. Kandler. 1973. Untersuchungen zur Entstehung von DL-Milchsiiure bei Lactobacillen Chem. 235:810-818. und Characterisierung einer Milchsiiureracemase bei 5. Dennis, D., M. Reichlin, and N. 0. Kaplan. 1965. Lactic einigen Arten der Untergattung Streptobacterium. acid racemization. Ann. N.Y. Acad. Sci. 119:868-876. Arch. Mikrobiol. 94:221-247. 6. Doelle, H. W. 1971. Nicotinamide adenine dinucleotidedependent and nicotinamide adenine dinucleotide- 21. Warburg, O., and W. Christian. 1942. Isolierung und Kristallisation des Garungsferments Enolase. Bioindependent lactate dehydrogenases in homofermentachem. Z. 310:384-421. tive and heterofermentative lactic acid bacteria. J. 22. Weber, K., and M. Osborn. 1969. The reliability of Bacteriol. 108:1284-1289. molecular weight determinations by dodecylsulphate7. Dynon, M. K., G. R. Jago, and B. E. Davidson. 1972. The polyacrylamide gel electrophoresis. J. Biol. Chem. subunit structure of lactate dehydrogenase from Strep244:4406-4412. tococcus cremoris US3. Eur. J. Biochem. 30:348-353.
D(-)-lactate pH is about production 5.0). Actively fermenting L. plantarum cells have an intemal pH of 5.4 (19) and, in the absence of any contrary evidence, it seems reasonable to assume that the intracellular pH is similar to that of the medium. By reference to Fig. 3A, it can be seen that, as the pH falls from about 6.5 to 5.0, L+LDH attains maximum activity while D -LDH activity is still relatively low. At this pH in the culture, D(-)-lactate production commences with the pH falling to about 4.0 as a result, which can be explained by the activity maximum of D-LDH at a pH of 3.6. However, this apparently carefully regulated total production of one isomer of lactic acid followed by a switch to the total production of the other does not appear to be common among all racemic lactate-producing Lactobacillaceae. For instance, the percentage of L(+) acid in the culture changes little during growth of L. plantarum (9), which is reflected in the fact that both lactate dehydrogenases from this organism display an optimum for pyruvate reduction at pH 6.0 (17). ceases
same time as commences (when the
at almost the
