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[64] Lactate-oxaloacetate transhydrogenase from Veillonella alcalescens
[64]
LACTATE-OXALOACETATE TRANSHYDROGENASE
367
undergoes a slow unfolding and dissociation that is seen in sedimentation velocity patterns as two incompletely resolved forms of the enzyme. At pH 9.0 in triethanolamine-hydrochloride buffer, the protein exists as a species of molecular weight 70,000 that is enzymically inactive. The substrate NADH and the activator FDP partially stabilize the 140,000 molecular weight form of the enzyme against dissociation in triethanolamine-hydrochloride buffer at pH 8.0. z The active enzyme consists of four identical subunits of molecular weight 35,000. At pH 6.0, each subunit has the ability to bind 1 molecule of FDP (with a mixture of positive and negative cooperativity) and 1 molecule of NADH (noncooperative binding) .3.5 The extinction coefficient of the pure enzyme in dilute phosphate buffer is r-l~ L~lem = 11.3 at 280 nm at pH 7.0. 5 The amino acid composition of the enzyme and the amino acid sequence of the 20 N-terminal amino acids and that of a 53-residue tryptic polypeptide containing the single cysteine residue, have all been determined. The isoelectric point of the enzyme is 4.3. 6
[64] L a c t a t e - O x a l o a c e t a t e T r a n s h y d r o g e n a s e f r o m Veillonella alcalescens By S. H. GEORGE ALLEN L-Lactate + oxaloacetate -~- pyruvate + L-malate
Thus far, the lactate-oxaloacetate transhydrogenase or malatelactate transhydrogenase (EC 1.1.99.7) has been found in only one genus, Veillonella. The transhydrogenase was originally isolated from Micrococcus lactilyticus, 1~ which was subsequently renamed Veillonella alcalescens. This bacterium has been isolated from the rumen of sheep 3 as well as from the oral cavity of man.4 The transhydrogenase is also present in Veillonella parvula. 5 The Veillonella are obligate anaerobes and are found in environments rich in lactic and other organic acids produced by other microorganisms. Rogosa and co-workers 6 have shown that the Veillonella lack M. I. Dolin, E. F. Phares, and M. V. Long, Biochem. Biophys. Res. Commun. 21, 303 (1965). 2 S. H. G. Allen, J. Biol. Chem, 241, 5266 (1966). a A. T. Johns, J. Gen. Microbiol. 5, 317 (1951). 4 R. J. Gibbons and J. van Houte, Annu. Rev. Microbiol. 29, 19 (1975). 5 S. K. C. Ng and L. R. Hamilton, J. Bacteriol. 105, 999 (1971). M. Rogosa, M. I. Krichevsky, and F. S. Bishop, J. Bacteriol. 90, 164 (1965).
METHODS IN ENZYMOLOGY, VOL. 89
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181989-2
368
OXIDATION--REDUCTION ENZYMES
[64]
h e x o k i n a s e a n d , in f a c t , c a n n o t m e t a b o l i z e a n y e x o g e n o u s c a r b o h y d r a t e . Furthermore, these bacteria do not contain lactate dehydrogenase activity. I n s t e a d t h e l a c t a t e - o x a l o a c e t a t e t r a n s h y d r o g e n a s e f u n c t i o n s to o x i d i z e l a c t a t e to p y r u v a t e p r o b a b l y b y c o u p l i n g w i t h a n L - m a l a t e d e h y d r o g e n a s e p r e s e n t in Veillonella. L-Lactate + oxaloacetate ~ pyruvate + L-malate L-Malate + NAD + ~ oxaloacetate + NADH H + Net: L-Lactate + NAD + ~ pyruvate + NADH H + T h u s , l a c t a t e e n t e r i n g t h e cell w o u l d b e c o n v e r t e d to p y r u v a t e w h i c h is m e t a b o l i z e d e i t h e r to a c e t i c o r to p r o p i o n i c a c i d s v i a p a t h w a y s s i m i l a r to these described for the propionic acid bacteria. 6
Assay M e t h o d Principle. The transhydrogenase is readily reversible, and thus enzyme activity can bc assayed in either direction.1'2 The specific activity of the enzyme is 5 0 % higher with L-malate and pyruvate as substrates than it is with L-lactate and oxaloacctate. Routinely, because of the higher activity and the relative instability of oxaloacetate solutions, the assay is performed with L-malate and pyruvatc as substrates. T w o assay procedures are used. The direct assay depends upon the increase in absorbance at 258 n m due to oxaloacetate formation. A molar extinction coefficient for oxaloacetate was determined experimentally to bc E2~, = 8.4 × 10" M -I c m -I. Initialrates with this assay are linear with time and enzyme concentration. The reverse reaction, i.e.,the disappearance of oxaloacetatc, can also be measured in this type of assay. The indirect assay, employing NADH oxidation, which was approximately 7 times more sensitive than thc direct assay, measures the formation of oxaloacctate from L-malate and pyruvate by coupling the transhydrogenase with the malate dehydrogenase. L-Malate + pyruvate
transhydrogenase
oxaloacetate + L-lactate
malate dehydrogenase
Oxaloacetate + NADH + H + L-malate + NAD + Net: Pyruvate + NADH + H + ~ L-lactate + NAD ÷ I n i t i a l r a t e s w i t h this a s s a y w e r e a l s o l i n e a r w i t h t i m e a n d e n z y m e c o n c e n tration. The reverse reaction, i.e., the appearance of pyruvate from o x a l o a c e t a t e a n d L - l a c t a t e , c a n a l s o b e m e a s u r e d b y this t y p e o f a s s a y , e x c e p t t h a t l a c t a t e d e h y d r o g e n a s e r a t h e r t h a n m a l a t e d e h y d r o g e n a s e is u s e d . W i t h all t h e s e a s s a y s , o n e unit o f e n z y m e is d e f i n e d as t h a t a m o u n t c a t a l y z i n g t h e o x i d a t i o n o f 1 /xmol o f e i t h e r L - m a l a t e o r L - l a c t a t e p e r minute.
[64]
LACTATE-OXALOACETATE TRANSHYDROGENASE
369
Direct Assay Reagents Tris-HC1 buffer, 0.5 M, pH 7.8 Sodium pyruvate, 0.1 M (Sigma Chemical Company, St. Louis, Missouri) Tris-L-malate, 0.2 M (Sigma) Procedure. The reagents listed above are added to a 0.5-ml quartz spectrophotometric cell (1-cm light path) in the following order and amounts: Tris-HC1 buffer, 0.05 ml; sodium pyruvate, 0.10 ml; Tris-Lmalate, 0.05 ml; distilled water, 0.04 ml; lactate-oxaloacetate transhydrogenase, 0.01 ml of an appropriate dilution. Usually larger but proportional volumes of the reagents are combined to form a mixture that could be added as a single volume of 0.20 ml. All reactants except the transhydrogenase are kept at room temperature or in a bath at the same temperature as the spectrophotometric cell chamber (usually 25°) prior to adding the enzyme that initiates the reaction. The cell contents are mixed well, and the reaction at 258 nm is measured. Since pyruvate itself absorbs some light at this wavelength, a cuvette with all the reagents except the enzyme can be used as a reagent blank. In measuring the rate in the reverse direction, 0.05 ml of 0.2 M DLlithium lactate (Sigma Chemical Company) and 0.01 ml of 10 mM Trisoxaloacetate, pH 6.5, are substituted for pyruvate and malate. All other conditions are the same as described above. Oxaloacetic acid is adjusted to pH 6.5 with Tris base using bromocresol green indicator, and this solution is made fresh daily.
Indirect Assay Reagents Tris-HC1 buffer, 0.5 M, pH 7.8 NADH, 4 mM (Sigma Chemical Company) Sodium pyruvate, 0.1 M (Sigma Chemical Company) Tris-L-malate, 0.2 M (Sigma Chemical Company) Malate dehydrogenase (Boehringer-Mannheim); dilution: 0.01 ml contains 0.1 unit (dilution of the commercial preparation is made in 1% bovine serum albumin) Procedure. The reagents listed above are added to a 0.50-ml cuvette (1-cm light path) in the following order and amounts: Tris-HCl buffer, 0.05 ml; NADH, 0.01 ml; sodium pyruvate, 0.08 ml; Tris-L-malate, 0.05 ml; malate dehydrogenase, 0.01 ml; distilled water, 0.03 ml; lactateoxaloacetate transhydrogenase, 0.01 ml of an appropriate dilution.
370
OXIDATION--REDUCTION ENZYMES
[64]
Usually, larger but proportional volumes of the reagents are combined to form a mixture that can be added as a single volume of 0.20 ml. The enzyme is added to initiate the reaction, which is measured at 340 nm. In the reverse direction, 0.05 ml of 0.2 M DL-lithium lactate (Sigma Chemical Company), 0.01 ml of 10 mM Tris-oxaloacetate, pH 6.5 (Sigma Chemical Company), and 0.01 ml of lactate dehydrogenase (Boehringer-Mannheim) containing 0.1 unit are substituted for the pyruvate, malate, and malate dehydrogenase. All other conditions are the same as described above. Since oxaloacetate tends to decarboxylate at a slow rate, the rate of pyruvate formation should be measured in a cuvette containing all the reagents except transhydrogenase, at each level of oxaloacetate used. Purification Procedure Growth o f Cells. Veillonella alcalescens, ATCC 12641 or 12642, is grown on thioglycolate medium without dextrose (Baltimore Biological Laboratories) supplemented with 0.4% yeast extract, 1.7% sodium lactate, and 0.1 mg% resazurin. Cultures are grown at 37° under strict anaerobic conditions in an atmosphere of 100% CO2 in serum bottles as described by Miller and Wolin. r Large-scale cultivation is carried out in 25-liter carboys containing 15 liters of a medium consisting of 1% yeast extract, 1% tryptone, and 2% sodium lactate as described by Delwiche et al. s In this preparation, the culture medium contains 6 /xCi per liter of [7~4CJnicotinamide (New England Nuclear), which has a specific radioactivity of 43 /.~Ci/nmol. With freshly autoclaved medium and a 5% active culture as inoculum, no further anaerobic precautions are needed to obtain vigorous growth at 30° . In both media, the presence of lactate is required in order to obtain maximal transhydrogenase production. After 3 days of cultivation the cells are harvested with a Sharpies centrifuge. Approximately 50 g wet weight of cells are obtained per 15 liters of medium. Crude Extract. A 30% wet weight to volume suspension of cells is made in 0.2 M potassium phosphate buffer, pH 7.0. Pyrex beads (120 ~M) equal to the weight of cells (204 g) are added to this suspension, which is then placed in an Eppenbach mill (Gifford-Wood Co., Hudson, New York), and the cells are ruptured at 0-5 °. For smaller preparations, cells can be ruptured by sonication at 0-5 °. The cells and cell debris are removed by centrifugation at 20,000 g for 20 min. A clear brown extract is obtained as previously described. 9 Specific activities of 20-80 /zmol/ r T. L. Miller and M. J. Wolin, Appl. Microbiol. 27, 985 (1974). s E. A. Delwiehe~ E. F. Phares, and S. F. Carson, J. Bacteriol. 71, 598 (1956). 9 S. H. G. Allen and J. H. Patil, J. Biol. Chem. 247, 909 (1972).
[64]
LACTATE-OXALOACETATE TRANSHYDROGENASE
371
minute per milligram of protein are usually found in crude extracts of cells. Since large dilutions of these extracts must be made in order to assay the enzyme, direct spectrophotometric assays are possible without interference with other reactions. The extract contains 11.5 g of protein, as measured with the biuret test, and approximately l0 Gunits of transhydrogenase, which is assayed with malate and pyruvate as substrates and coupled to malate dehydrogenase as previously described.l°
Ion-Exchange Chromatography Purification to homogeneity requires only a two-step procedure, using DEAE-cellulose batch treatment followed by QAE-Sephadex chromatography as described by Allen and Patil) DEAE-Celhdose. The crude extract (560 ml) is dialyzed against 10 liters of 0.05 M phosphate buffer, pH 7.5, for 15 hr, with three changes of buffer. Dilute the dialyzed extract to 3000 ml with cold distilled water. Add 1000 ml of packed DEAE-cellulose (Schleicher and Schuell, type 40, 0.84 meg/g) that have been washed and equilibrated with 0.005 M potassium phosphate, pH 7.5. Stir at 4° for 20 min, remove a 1.0-ml sample, centrifuge, and test the clear supernatant for transhydrogenase activity. If more than 5% of the total activity remains in the supernatant, add more packed DEAE-cellulose and cold water. When the enzyme is absorbed, filter on a Biichner funnel with Whatman No. 4 paper. Resuspend the DEAE-cellulose in 5 liters of 0.005 M potassium phosphate buffer, pH 7.4. Stir at 4 ° for 20 min, then filter and discard the colloidal, yellow filtrate. Resuspend the DEAE-cellulose in 5 liters of 0.20 M potassium phosphate buffer, and stir at 4° for at least 3 hr. Filter and assay the clear yellow solution for transhydrogenase activity. Table I shows that 74% of the starting activity is recovered in this eluate and that the specific activity of the enzyme is twice that in the crude. Solid ammonium sulfate (Schwarz-Mann) is added to a final concentration of 3.7 M, and the precipitate is collected by centrifugation at 16,000g for 45 min. This precipitate can be stored at - 2 0 ° without loss of activity. QAE-Sephadex. The ammonium sulfate precipitate is dissolved in 200 ml of 0.05 M Tris-HC1, pH 7.5, and dialyzed against 4 liters of 0.05 M potassium phosphate, pH 7.4, for 15 hr with three changes of buffer. The preparation is absorbed onto a QAE-Sephadex (Pharmacia) column (5 × 88 cm) that has been equilibrated with 0.05 M potassium phosphate pH 7.4. Wash the absorbed protein with 1 liter of the same buffer, and then elute with a linear gradient of 0.05 to 0.40 M potassium phosphate, pH 7.4. Ten-milliliter samples are collected at a flow rate of 0.2 ml/min. A single major protein peak, which contains the transhydrogenase, is eluted. ~o S. H. G. Allen, this series, Vol. 13, p. 265.
372
OXIDATION--REDUCTION ENZYMES
[64]
TABLE I PREPARATION OF laG-LABELED TRANSHYDROGENASE a
Step Crude extract dialyzed DEAE-cellulose, 0.2 m eluate QAE-Sephadex chromatography fractions 327-354
Total protein (mg)
Specific activity (units/mg protein)
11,536
79
1,011,344
811
100
4,504
166
747,662
1276
74
1,110
368
405,000
3920
40
Total Radiounits activity (/~mol/min) (cpm/mg)
Recovery of units (%)
a Units refer to the micromoles of product formed per minute. Specific activities and radioactive activities are constant across the center o f the peak. Fractions with the highest activity are pooled (Table I) and the protein is precipitated at 3.7 M a m m o n i u m sulfate. Centrifuge at 24,000 g for 45 rain, suspend in 0.05 M Tris-HC1, p H 7.4, and store at - 2 0 °" Figure ! shows a typical elution profile in which D E A E - S e p h a d e x has been used instead of Q A E - S e p h a d e x . The profiles obtained f r o m either resin are similar; h o w e v e r , the specific activity of the e n z y m e obtained f r o m the Q A E - S e p h a d e x columns is a p p r o x i m a t e l y 2 times higher. Thus, Q A E - S e p h a d e x is the resin o f choice. Properties Properties o f the Pure Enzyme. The colorless protein was h o m o g e n e o u s as m e a s u r e d in the analytical ultracentrifuge 11 and by disc gel electrophoresis. It had a m a x i m u m specific activity of a p p r o x i m a t e l y 400 units/rag. During storage at - 2 0 ° the specific activity fell to a b o u t 200, but the preparation a p p e a r e d to be stable at this activity level indefinitely, when stored at - 2 0 °. An a b s o r b a n c e coefficient at 280 nm, b a s e d on dry weight of the pure protein was 1.27 cm 2 mg -1. An S~o,w c o r r e c t e d for protein concentration was found to be 5.26 S, and the diffusion coefficient, D~0,w was 6.57 Fick units. 11 Calculation of the m o l e c u l a r weight b a s e d on the S v e d b e r g equation and using a partial specific v o l u m e of 0.726 mg/g was found to be 71,000 -+ 3600. A frictional ratio was 1.19, which corresponds to a prolate ellipsoid with an axial ratio of a p p r o x i m a t e l y 4. The molecular weight was also determined with the high speed (miniscus11 s. H. G. Allen, Eur. J. Biochem. 35, 338 0973).
[64]
LACTATE-OXALOACETATE TRANSHYDROGENASE
2.0. zoo
~J':~
I
~l!il'
1,5
~o-o.25
~,/
tso I 16o I "
8 0,20
I~;=
~ 6-o.ts~
r,'. ', /I
I //
40
20 0
,'l'
;', 0
,!/
.......... " '-"I Ib
2'0
50
4b
50
373
× k
,.~ "~
, ."
',\ '" ~ | '9, k / "~-" ...... , ~o 6'0 Yb
8'0 90
FRACTION NUMBER
FiG. 1. The chromatographic purification of lactate-oxaloacetate transhydrogenase. After desalting on a Sephadex G-25 column (2.5 x 18 cm), 132 mg of protein (as measured by absorbance at 280 nm) containing 15,850 units of transhydrogenase were absorbed onto a DEAE-Sephadex (A-50) column (2 x 50 cm) that had been equilibrated with 0.005 M potassium phosphate buffer, pH 7.0. The approximate flow rate was 0.10 ml/min; 7.5-ml fractions were collected. The protein was eluted with a phosphate buffer (pH 7.0) ~adient of 0.05 to 0.4 M, with 500 ml in the mixing bottle ( ) . Protein (---) was determined by 280 nm absorbance and specific activity ( - - - ) using the spectrophotometric assay coupled with malate dehydrogenase and NADH. Radioactivity ( . . . . . . . ) was measured with a Nuclear Mark I scintillation spectrometer (Nuclear Chicago). Ninety-six milligrams of protein containing 7700 units of transhydrogenase activity were recovered from the column.
depletion) sedimentation-equilibrium method at four different protein concentrations. A value of 69,700 ___ 2500 was obtained from these results, n Amino acid analysis is given in Table II; the partial specific volume was calculated from the amino acid content as described by Cohn and Edsal112 to b e 0.726 mg/g. Molecular Weight ofSubunits. S e d i m e n t a t i o n - d i f f u s i o n a n a l y s i s in a 5 M g u a n i d i n e - H C l a n d 5 m M 2 - m e r c a p t o e t h a n o l r e v e a l e d a single s e d i m e n t ing species. A value of 1.87 S was obtained when the s~0,w was extrapolated to zero protein concentration. Diffusion experiments revealed D20,w of 4.13 - 10 Fick units. Assuming a partial specific volume of 0.726 mg/g, the molecular weight was calculated to be 40,000 _+ 1000. The frictional ratio was calculated at 2.28, which corresponds to a prolate ellipsoid having an axial ratio of approximately 28. Molecular weight values obtained by sucrose gradient centrifugation and SDS-polyacrylamide gel 1~ E. J. Cohn and J. T. Edsall, "Proteins, Amino Acids and Peptides," p. 374. Van Nostrand-Reinhold, Princeton, New Jersey, 1943.
374
OXIDATION--REDUCTION ENZYMES
[64]
TABLE II AMINO ACID COMPOSITION OF LACTATE-OXALOACETATE TRANSHYDROGENASE
Number of residues (moles/70,000 g of protein) Amino acid residue
15 Hours
23 Hours
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
47 17 14 62 28 33 62 28 69 54 12 32 25 30 38 18 27 7
47 16 14 49 27 30 59 26 67 53 12 33 22 31 38 18 25 8
electrophoresis yielded molecular weight values of 30,000 and 43,000, respectively. 9 T h e s e results also agree with the m i n i m u m molecular weight values of b e t w e e n 30,000 and 40,000 obtained by Dolin et al., 1 which w e r e b a s e d on the total pyridine nucleotide content. Thus, the e n z y m e is c o m p o s e d o f two identical subunits. Nature o f N A D / N A D H Binding. The pyridine nucleotide is v e r y firmly b o u n d to the protein and cannot be r e m o v e d by charcoal absorption or b y a m m o n i u m sulfate fractionation. It appears that the r e m o v a l of the N A D requires p r o f o u n d changes in the structure of the protein. T r e a t m e n t with 35% perchloric acid (final concentration) at r o o m t e m p e r a t u r e and heating at 100° at p H 10 both release the prosthetic group. Concentrations of u r e a of 3.5 M and higher, 0.1% SDS, and 5 M guanidine HCI also r e m o v e the bound pyridine nucleotide. T r e a t m e n t o f the t r a n s h y d r o g e n a s e with crude N A D a s e (Sigma Chemical C o m p a n y ) had been r e p o r t e d 9 to r e m o v e the [14C]nicotinamide from the transhydrogenase. Subsequent e x p e r i m e n t s revealed that p r o t e a s e activity in this N A D a s e preparation caused breakd o w n of the protein and subsequent liberation o f the prosthetic group.
[64]
375
LACTATE-OXALOACETATE TRANSHYDROGENASE
E • NADH • L a c t a t e
E • NAD • L a c t a t e
E • NADH • P y r u v a t e
E • NAD • O x a l o a c e t a t e
lactate~//± oxaloacetate
E - NAD
E • NADH
malat e / / ~ ± E • NADH • M a l a t e
malate//~±
oxaloacetate
E • NAD. Malate
E • NADH • O x a l o acetate
pyruvate E • NAD • P y r u v a t e
SCHEME
Purified NADase '3 did not cause release of nicotinamide or loss of transhydrogenase '4 activity. NADH can be removed by succinylation or citraconylation of the transhydrogenase? 1 In all cases studied, removal of the prosthetic group is accompanied by dissociation of the transhydrogenase into subunits and complete loss of enzymic activity. Reassociation of the subunits in the presence of either the isolated, purified prosthetic group or authentic NADH has not been possible. Mechanism of Action of the Transhydrogenase. The transhydrogenase has been shown to catalyze a "Ping-Pong" type of reaction mechanism. 9 Evidence for this is based on the fact that the two half-reactions: L - L a c t a t e + E - N A D + ~- p y r u v a t e + E • N A D + H + L-Malate + E • NAD ~ oxaloacetate + E. NAD + H ÷
can be carried out separately and stoichiometrically. Second, kinetic analysis at low substrate concentrations, yields the parallel lines on Lineweaver-Burk plots that are diagnostic of the "Ping-Pong" mechanism. 9 At higher substrate concentrations, inhibition is observed, and the full mechanism is more complicated. All substrates form abortive complexes with the enzyme ~5 and evidence has been presented to support the participation of ternary complexes in the mechanism. The significance of these is not clear. A simplified mechanism can be written as shown in the reaction scheme. A puzzling aspect of the mechanism is the apparent ability of the enzyme to maintain about 40% of the bound pyridine nucleotide prosthetic groups in the reduced state, i.e., as NADH. Treatment of the native enzyme with pyruvate or oxaloacetate quenches the NADH fluorescence 13 F. J. F e h r e n b a c h , Eur. J. Biochem. 18, 94 (1971). T h e N A D ÷ g l y c o h y d r o l a s e ( E C 3 . 2 . 2 . 5 ) w a s a k i n d gift f r o m D r . F e h r e n b a c h , U n i v e r s i t y o f F r e i b e r g , F e d e r a l R e p u b l i c o f G e r many. ~4 S. H . G . A l l e n , u n p u b l i s h e d d a t a , 1972, '~ M . I. D o l i n , J. Biol. Chem. 244, 5273 (1969).
376
OXIDATION--REDUCTION ENZYMES
[65]
and yields either lactate or malate, which can be recovered stoichiometrically after either gel filtration or dialysis to remove the enzyme. Upon standing at 4°, the enzyme-bound NAD slowly becomes reduced 1'' until about 40% of the total prosthetic group are as NADH. Furthermore, the native enzyme has been shown to contain covalently linked pyruvate2 The exact nature of the attachment of the pyruvate to the protein as well as its role, if any, in the mechanism are unknown. Treatment of the native transhydrogenase with either hydroxylamine or sodium borohydride have little effect on the enzymic activity. Yet reduction with [3H]sodium borohydride results in incorporation of radioactivity into the protein, which after repeated lyophilization and acid hydrolysis can be identified chromatography as [all]lactate.9 Dolin 15 has also shown the presence of tightly bound pyruvate using the lactate dehydrogenase assay. The possibility exists that these molecules act as a reservoir of reducing equilalents that interact with the bound NAD, keeping a constant N A D : N A D H ratio in the native enzyme. This may be necessary for keeping the transhydrogenase in the proper configuration or important in allowing the enzyme to react more readily with keto acid substrates. The amount of bound pyruvate present on each enzyme molecule has not been determined, and the role of this bound keto acid has not been ascertained.
[65] P y r u v a t e D e h y d r o g e n a s e C o m p l e x f r o m Bovine Kidney and Heart
By
FLORA
H.
PETTIT
and
LESTER
J. REED
Pyruvate + CoA + NAD÷~ acetyl-CoA+ CO2 + NADH + H + In eukaryotic cells the pyruvate dehydrogenase complex is located in mitochondria, within the inner membrane-matrix compartment. The complex consists of three catalytic components: pyruvate dehydrogenase (El) (EC 1.2.4.1), dihydrolipoyl transacetylase (E2) (EC 2.3.1.12), and dihydrolipoyl dehydrogenase (E3) (EC 1.6.4.3). These three enzymes, acting in sequence, catalyze the above overall reaction.
Assay Method Principle. Activity of this multienzyme complex is assayed spectrophotometrically by measurement of NADH production. The method is
METHODS IN ENZYMOLOGY, VOL. 89
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181989-2
LACTATE-OXALOACETATE TRANSHYDROGENASE
367
undergoes a slow unfolding and dissociation that is seen in sedimentation velocity patterns as two incompletely resolved forms of the enzyme. At pH 9.0 in triethanolamine-hydrochloride buffer, the protein exists as a species of molecular weight 70,000 that is enzymically inactive. The substrate NADH and the activator FDP partially stabilize the 140,000 molecular weight form of the enzyme against dissociation in triethanolamine-hydrochloride buffer at pH 8.0. z The active enzyme consists of four identical subunits of molecular weight 35,000. At pH 6.0, each subunit has the ability to bind 1 molecule of FDP (with a mixture of positive and negative cooperativity) and 1 molecule of NADH (noncooperative binding) .3.5 The extinction coefficient of the pure enzyme in dilute phosphate buffer is r-l~ L~lem = 11.3 at 280 nm at pH 7.0. 5 The amino acid composition of the enzyme and the amino acid sequence of the 20 N-terminal amino acids and that of a 53-residue tryptic polypeptide containing the single cysteine residue, have all been determined. The isoelectric point of the enzyme is 4.3. 6
[64] L a c t a t e - O x a l o a c e t a t e T r a n s h y d r o g e n a s e f r o m Veillonella alcalescens By S. H. GEORGE ALLEN L-Lactate + oxaloacetate -~- pyruvate + L-malate
Thus far, the lactate-oxaloacetate transhydrogenase or malatelactate transhydrogenase (EC 1.1.99.7) has been found in only one genus, Veillonella. The transhydrogenase was originally isolated from Micrococcus lactilyticus, 1~ which was subsequently renamed Veillonella alcalescens. This bacterium has been isolated from the rumen of sheep 3 as well as from the oral cavity of man.4 The transhydrogenase is also present in Veillonella parvula. 5 The Veillonella are obligate anaerobes and are found in environments rich in lactic and other organic acids produced by other microorganisms. Rogosa and co-workers 6 have shown that the Veillonella lack M. I. Dolin, E. F. Phares, and M. V. Long, Biochem. Biophys. Res. Commun. 21, 303 (1965). 2 S. H. G. Allen, J. Biol. Chem, 241, 5266 (1966). a A. T. Johns, J. Gen. Microbiol. 5, 317 (1951). 4 R. J. Gibbons and J. van Houte, Annu. Rev. Microbiol. 29, 19 (1975). 5 S. K. C. Ng and L. R. Hamilton, J. Bacteriol. 105, 999 (1971). M. Rogosa, M. I. Krichevsky, and F. S. Bishop, J. Bacteriol. 90, 164 (1965).
METHODS IN ENZYMOLOGY, VOL. 89
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181989-2
368
OXIDATION--REDUCTION ENZYMES
[64]
h e x o k i n a s e a n d , in f a c t , c a n n o t m e t a b o l i z e a n y e x o g e n o u s c a r b o h y d r a t e . Furthermore, these bacteria do not contain lactate dehydrogenase activity. I n s t e a d t h e l a c t a t e - o x a l o a c e t a t e t r a n s h y d r o g e n a s e f u n c t i o n s to o x i d i z e l a c t a t e to p y r u v a t e p r o b a b l y b y c o u p l i n g w i t h a n L - m a l a t e d e h y d r o g e n a s e p r e s e n t in Veillonella. L-Lactate + oxaloacetate ~ pyruvate + L-malate L-Malate + NAD + ~ oxaloacetate + NADH H + Net: L-Lactate + NAD + ~ pyruvate + NADH H + T h u s , l a c t a t e e n t e r i n g t h e cell w o u l d b e c o n v e r t e d to p y r u v a t e w h i c h is m e t a b o l i z e d e i t h e r to a c e t i c o r to p r o p i o n i c a c i d s v i a p a t h w a y s s i m i l a r to these described for the propionic acid bacteria. 6
Assay M e t h o d Principle. The transhydrogenase is readily reversible, and thus enzyme activity can bc assayed in either direction.1'2 The specific activity of the enzyme is 5 0 % higher with L-malate and pyruvate as substrates than it is with L-lactate and oxaloacctate. Routinely, because of the higher activity and the relative instability of oxaloacetate solutions, the assay is performed with L-malate and pyruvatc as substrates. T w o assay procedures are used. The direct assay depends upon the increase in absorbance at 258 n m due to oxaloacetate formation. A molar extinction coefficient for oxaloacetate was determined experimentally to bc E2~, = 8.4 × 10" M -I c m -I. Initialrates with this assay are linear with time and enzyme concentration. The reverse reaction, i.e.,the disappearance of oxaloacetatc, can also be measured in this type of assay. The indirect assay, employing NADH oxidation, which was approximately 7 times more sensitive than thc direct assay, measures the formation of oxaloacctate from L-malate and pyruvate by coupling the transhydrogenase with the malate dehydrogenase. L-Malate + pyruvate
transhydrogenase
oxaloacetate + L-lactate
malate dehydrogenase
Oxaloacetate + NADH + H + L-malate + NAD + Net: Pyruvate + NADH + H + ~ L-lactate + NAD ÷ I n i t i a l r a t e s w i t h this a s s a y w e r e a l s o l i n e a r w i t h t i m e a n d e n z y m e c o n c e n tration. The reverse reaction, i.e., the appearance of pyruvate from o x a l o a c e t a t e a n d L - l a c t a t e , c a n a l s o b e m e a s u r e d b y this t y p e o f a s s a y , e x c e p t t h a t l a c t a t e d e h y d r o g e n a s e r a t h e r t h a n m a l a t e d e h y d r o g e n a s e is u s e d . W i t h all t h e s e a s s a y s , o n e unit o f e n z y m e is d e f i n e d as t h a t a m o u n t c a t a l y z i n g t h e o x i d a t i o n o f 1 /xmol o f e i t h e r L - m a l a t e o r L - l a c t a t e p e r minute.
[64]
LACTATE-OXALOACETATE TRANSHYDROGENASE
369
Direct Assay Reagents Tris-HC1 buffer, 0.5 M, pH 7.8 Sodium pyruvate, 0.1 M (Sigma Chemical Company, St. Louis, Missouri) Tris-L-malate, 0.2 M (Sigma) Procedure. The reagents listed above are added to a 0.5-ml quartz spectrophotometric cell (1-cm light path) in the following order and amounts: Tris-HC1 buffer, 0.05 ml; sodium pyruvate, 0.10 ml; Tris-Lmalate, 0.05 ml; distilled water, 0.04 ml; lactate-oxaloacetate transhydrogenase, 0.01 ml of an appropriate dilution. Usually larger but proportional volumes of the reagents are combined to form a mixture that could be added as a single volume of 0.20 ml. All reactants except the transhydrogenase are kept at room temperature or in a bath at the same temperature as the spectrophotometric cell chamber (usually 25°) prior to adding the enzyme that initiates the reaction. The cell contents are mixed well, and the reaction at 258 nm is measured. Since pyruvate itself absorbs some light at this wavelength, a cuvette with all the reagents except the enzyme can be used as a reagent blank. In measuring the rate in the reverse direction, 0.05 ml of 0.2 M DLlithium lactate (Sigma Chemical Company) and 0.01 ml of 10 mM Trisoxaloacetate, pH 6.5, are substituted for pyruvate and malate. All other conditions are the same as described above. Oxaloacetic acid is adjusted to pH 6.5 with Tris base using bromocresol green indicator, and this solution is made fresh daily.
Indirect Assay Reagents Tris-HC1 buffer, 0.5 M, pH 7.8 NADH, 4 mM (Sigma Chemical Company) Sodium pyruvate, 0.1 M (Sigma Chemical Company) Tris-L-malate, 0.2 M (Sigma Chemical Company) Malate dehydrogenase (Boehringer-Mannheim); dilution: 0.01 ml contains 0.1 unit (dilution of the commercial preparation is made in 1% bovine serum albumin) Procedure. The reagents listed above are added to a 0.50-ml cuvette (1-cm light path) in the following order and amounts: Tris-HCl buffer, 0.05 ml; NADH, 0.01 ml; sodium pyruvate, 0.08 ml; Tris-L-malate, 0.05 ml; malate dehydrogenase, 0.01 ml; distilled water, 0.03 ml; lactateoxaloacetate transhydrogenase, 0.01 ml of an appropriate dilution.
370
OXIDATION--REDUCTION ENZYMES
[64]
Usually, larger but proportional volumes of the reagents are combined to form a mixture that can be added as a single volume of 0.20 ml. The enzyme is added to initiate the reaction, which is measured at 340 nm. In the reverse direction, 0.05 ml of 0.2 M DL-lithium lactate (Sigma Chemical Company), 0.01 ml of 10 mM Tris-oxaloacetate, pH 6.5 (Sigma Chemical Company), and 0.01 ml of lactate dehydrogenase (Boehringer-Mannheim) containing 0.1 unit are substituted for the pyruvate, malate, and malate dehydrogenase. All other conditions are the same as described above. Since oxaloacetate tends to decarboxylate at a slow rate, the rate of pyruvate formation should be measured in a cuvette containing all the reagents except transhydrogenase, at each level of oxaloacetate used. Purification Procedure Growth o f Cells. Veillonella alcalescens, ATCC 12641 or 12642, is grown on thioglycolate medium without dextrose (Baltimore Biological Laboratories) supplemented with 0.4% yeast extract, 1.7% sodium lactate, and 0.1 mg% resazurin. Cultures are grown at 37° under strict anaerobic conditions in an atmosphere of 100% CO2 in serum bottles as described by Miller and Wolin. r Large-scale cultivation is carried out in 25-liter carboys containing 15 liters of a medium consisting of 1% yeast extract, 1% tryptone, and 2% sodium lactate as described by Delwiche et al. s In this preparation, the culture medium contains 6 /xCi per liter of [7~4CJnicotinamide (New England Nuclear), which has a specific radioactivity of 43 /.~Ci/nmol. With freshly autoclaved medium and a 5% active culture as inoculum, no further anaerobic precautions are needed to obtain vigorous growth at 30° . In both media, the presence of lactate is required in order to obtain maximal transhydrogenase production. After 3 days of cultivation the cells are harvested with a Sharpies centrifuge. Approximately 50 g wet weight of cells are obtained per 15 liters of medium. Crude Extract. A 30% wet weight to volume suspension of cells is made in 0.2 M potassium phosphate buffer, pH 7.0. Pyrex beads (120 ~M) equal to the weight of cells (204 g) are added to this suspension, which is then placed in an Eppenbach mill (Gifford-Wood Co., Hudson, New York), and the cells are ruptured at 0-5 °. For smaller preparations, cells can be ruptured by sonication at 0-5 °. The cells and cell debris are removed by centrifugation at 20,000 g for 20 min. A clear brown extract is obtained as previously described. 9 Specific activities of 20-80 /zmol/ r T. L. Miller and M. J. Wolin, Appl. Microbiol. 27, 985 (1974). s E. A. Delwiehe~ E. F. Phares, and S. F. Carson, J. Bacteriol. 71, 598 (1956). 9 S. H. G. Allen and J. H. Patil, J. Biol. Chem. 247, 909 (1972).
[64]
LACTATE-OXALOACETATE TRANSHYDROGENASE
371
minute per milligram of protein are usually found in crude extracts of cells. Since large dilutions of these extracts must be made in order to assay the enzyme, direct spectrophotometric assays are possible without interference with other reactions. The extract contains 11.5 g of protein, as measured with the biuret test, and approximately l0 Gunits of transhydrogenase, which is assayed with malate and pyruvate as substrates and coupled to malate dehydrogenase as previously described.l°
Ion-Exchange Chromatography Purification to homogeneity requires only a two-step procedure, using DEAE-cellulose batch treatment followed by QAE-Sephadex chromatography as described by Allen and Patil) DEAE-Celhdose. The crude extract (560 ml) is dialyzed against 10 liters of 0.05 M phosphate buffer, pH 7.5, for 15 hr, with three changes of buffer. Dilute the dialyzed extract to 3000 ml with cold distilled water. Add 1000 ml of packed DEAE-cellulose (Schleicher and Schuell, type 40, 0.84 meg/g) that have been washed and equilibrated with 0.005 M potassium phosphate, pH 7.5. Stir at 4° for 20 min, remove a 1.0-ml sample, centrifuge, and test the clear supernatant for transhydrogenase activity. If more than 5% of the total activity remains in the supernatant, add more packed DEAE-cellulose and cold water. When the enzyme is absorbed, filter on a Biichner funnel with Whatman No. 4 paper. Resuspend the DEAE-cellulose in 5 liters of 0.005 M potassium phosphate buffer, pH 7.4. Stir at 4 ° for 20 min, then filter and discard the colloidal, yellow filtrate. Resuspend the DEAE-cellulose in 5 liters of 0.20 M potassium phosphate buffer, and stir at 4° for at least 3 hr. Filter and assay the clear yellow solution for transhydrogenase activity. Table I shows that 74% of the starting activity is recovered in this eluate and that the specific activity of the enzyme is twice that in the crude. Solid ammonium sulfate (Schwarz-Mann) is added to a final concentration of 3.7 M, and the precipitate is collected by centrifugation at 16,000g for 45 min. This precipitate can be stored at - 2 0 ° without loss of activity. QAE-Sephadex. The ammonium sulfate precipitate is dissolved in 200 ml of 0.05 M Tris-HC1, pH 7.5, and dialyzed against 4 liters of 0.05 M potassium phosphate, pH 7.4, for 15 hr with three changes of buffer. The preparation is absorbed onto a QAE-Sephadex (Pharmacia) column (5 × 88 cm) that has been equilibrated with 0.05 M potassium phosphate pH 7.4. Wash the absorbed protein with 1 liter of the same buffer, and then elute with a linear gradient of 0.05 to 0.40 M potassium phosphate, pH 7.4. Ten-milliliter samples are collected at a flow rate of 0.2 ml/min. A single major protein peak, which contains the transhydrogenase, is eluted. ~o S. H. G. Allen, this series, Vol. 13, p. 265.
372
OXIDATION--REDUCTION ENZYMES
[64]
TABLE I PREPARATION OF laG-LABELED TRANSHYDROGENASE a
Step Crude extract dialyzed DEAE-cellulose, 0.2 m eluate QAE-Sephadex chromatography fractions 327-354
Total protein (mg)
Specific activity (units/mg protein)
11,536
79
1,011,344
811
100
4,504
166
747,662
1276
74
1,110
368
405,000
3920
40
Total Radiounits activity (/~mol/min) (cpm/mg)
Recovery of units (%)
a Units refer to the micromoles of product formed per minute. Specific activities and radioactive activities are constant across the center o f the peak. Fractions with the highest activity are pooled (Table I) and the protein is precipitated at 3.7 M a m m o n i u m sulfate. Centrifuge at 24,000 g for 45 rain, suspend in 0.05 M Tris-HC1, p H 7.4, and store at - 2 0 °" Figure ! shows a typical elution profile in which D E A E - S e p h a d e x has been used instead of Q A E - S e p h a d e x . The profiles obtained f r o m either resin are similar; h o w e v e r , the specific activity of the e n z y m e obtained f r o m the Q A E - S e p h a d e x columns is a p p r o x i m a t e l y 2 times higher. Thus, Q A E - S e p h a d e x is the resin o f choice. Properties Properties o f the Pure Enzyme. The colorless protein was h o m o g e n e o u s as m e a s u r e d in the analytical ultracentrifuge 11 and by disc gel electrophoresis. It had a m a x i m u m specific activity of a p p r o x i m a t e l y 400 units/rag. During storage at - 2 0 ° the specific activity fell to a b o u t 200, but the preparation a p p e a r e d to be stable at this activity level indefinitely, when stored at - 2 0 °. An a b s o r b a n c e coefficient at 280 nm, b a s e d on dry weight of the pure protein was 1.27 cm 2 mg -1. An S~o,w c o r r e c t e d for protein concentration was found to be 5.26 S, and the diffusion coefficient, D~0,w was 6.57 Fick units. 11 Calculation of the m o l e c u l a r weight b a s e d on the S v e d b e r g equation and using a partial specific v o l u m e of 0.726 mg/g was found to be 71,000 -+ 3600. A frictional ratio was 1.19, which corresponds to a prolate ellipsoid with an axial ratio of a p p r o x i m a t e l y 4. The molecular weight was also determined with the high speed (miniscus11 s. H. G. Allen, Eur. J. Biochem. 35, 338 0973).
[64]
LACTATE-OXALOACETATE TRANSHYDROGENASE
2.0. zoo
~J':~
I
~l!il'
1,5
~o-o.25
~,/
tso I 16o I "
8 0,20
I~;=
~ 6-o.ts~
r,'. ', /I
I //
40
20 0
,'l'
;', 0
,!/
.......... " '-"I Ib
2'0
50
4b
50
373
× k
,.~ "~
, ."
',\ '" ~ | '9, k / "~-" ...... , ~o 6'0 Yb
8'0 90
FRACTION NUMBER
FiG. 1. The chromatographic purification of lactate-oxaloacetate transhydrogenase. After desalting on a Sephadex G-25 column (2.5 x 18 cm), 132 mg of protein (as measured by absorbance at 280 nm) containing 15,850 units of transhydrogenase were absorbed onto a DEAE-Sephadex (A-50) column (2 x 50 cm) that had been equilibrated with 0.005 M potassium phosphate buffer, pH 7.0. The approximate flow rate was 0.10 ml/min; 7.5-ml fractions were collected. The protein was eluted with a phosphate buffer (pH 7.0) ~adient of 0.05 to 0.4 M, with 500 ml in the mixing bottle ( ) . Protein (---) was determined by 280 nm absorbance and specific activity ( - - - ) using the spectrophotometric assay coupled with malate dehydrogenase and NADH. Radioactivity ( . . . . . . . ) was measured with a Nuclear Mark I scintillation spectrometer (Nuclear Chicago). Ninety-six milligrams of protein containing 7700 units of transhydrogenase activity were recovered from the column.
depletion) sedimentation-equilibrium method at four different protein concentrations. A value of 69,700 ___ 2500 was obtained from these results, n Amino acid analysis is given in Table II; the partial specific volume was calculated from the amino acid content as described by Cohn and Edsal112 to b e 0.726 mg/g. Molecular Weight ofSubunits. S e d i m e n t a t i o n - d i f f u s i o n a n a l y s i s in a 5 M g u a n i d i n e - H C l a n d 5 m M 2 - m e r c a p t o e t h a n o l r e v e a l e d a single s e d i m e n t ing species. A value of 1.87 S was obtained when the s~0,w was extrapolated to zero protein concentration. Diffusion experiments revealed D20,w of 4.13 - 10 Fick units. Assuming a partial specific volume of 0.726 mg/g, the molecular weight was calculated to be 40,000 _+ 1000. The frictional ratio was calculated at 2.28, which corresponds to a prolate ellipsoid having an axial ratio of approximately 28. Molecular weight values obtained by sucrose gradient centrifugation and SDS-polyacrylamide gel 1~ E. J. Cohn and J. T. Edsall, "Proteins, Amino Acids and Peptides," p. 374. Van Nostrand-Reinhold, Princeton, New Jersey, 1943.
374
OXIDATION--REDUCTION ENZYMES
[64]
TABLE II AMINO ACID COMPOSITION OF LACTATE-OXALOACETATE TRANSHYDROGENASE
Number of residues (moles/70,000 g of protein) Amino acid residue
15 Hours
23 Hours
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
47 17 14 62 28 33 62 28 69 54 12 32 25 30 38 18 27 7
47 16 14 49 27 30 59 26 67 53 12 33 22 31 38 18 25 8
electrophoresis yielded molecular weight values of 30,000 and 43,000, respectively. 9 T h e s e results also agree with the m i n i m u m molecular weight values of b e t w e e n 30,000 and 40,000 obtained by Dolin et al., 1 which w e r e b a s e d on the total pyridine nucleotide content. Thus, the e n z y m e is c o m p o s e d o f two identical subunits. Nature o f N A D / N A D H Binding. The pyridine nucleotide is v e r y firmly b o u n d to the protein and cannot be r e m o v e d by charcoal absorption or b y a m m o n i u m sulfate fractionation. It appears that the r e m o v a l of the N A D requires p r o f o u n d changes in the structure of the protein. T r e a t m e n t with 35% perchloric acid (final concentration) at r o o m t e m p e r a t u r e and heating at 100° at p H 10 both release the prosthetic group. Concentrations of u r e a of 3.5 M and higher, 0.1% SDS, and 5 M guanidine HCI also r e m o v e the bound pyridine nucleotide. T r e a t m e n t o f the t r a n s h y d r o g e n a s e with crude N A D a s e (Sigma Chemical C o m p a n y ) had been r e p o r t e d 9 to r e m o v e the [14C]nicotinamide from the transhydrogenase. Subsequent e x p e r i m e n t s revealed that p r o t e a s e activity in this N A D a s e preparation caused breakd o w n of the protein and subsequent liberation o f the prosthetic group.
[64]
375
LACTATE-OXALOACETATE TRANSHYDROGENASE
E • NADH • L a c t a t e
E • NAD • L a c t a t e
E • NADH • P y r u v a t e
E • NAD • O x a l o a c e t a t e
lactate~//± oxaloacetate
E - NAD
E • NADH
malat e / / ~ ± E • NADH • M a l a t e
malate//~±
oxaloacetate
E • NAD. Malate
E • NADH • O x a l o acetate
pyruvate E • NAD • P y r u v a t e
SCHEME
Purified NADase '3 did not cause release of nicotinamide or loss of transhydrogenase '4 activity. NADH can be removed by succinylation or citraconylation of the transhydrogenase? 1 In all cases studied, removal of the prosthetic group is accompanied by dissociation of the transhydrogenase into subunits and complete loss of enzymic activity. Reassociation of the subunits in the presence of either the isolated, purified prosthetic group or authentic NADH has not been possible. Mechanism of Action of the Transhydrogenase. The transhydrogenase has been shown to catalyze a "Ping-Pong" type of reaction mechanism. 9 Evidence for this is based on the fact that the two half-reactions: L - L a c t a t e + E - N A D + ~- p y r u v a t e + E • N A D + H + L-Malate + E • NAD ~ oxaloacetate + E. NAD + H ÷
can be carried out separately and stoichiometrically. Second, kinetic analysis at low substrate concentrations, yields the parallel lines on Lineweaver-Burk plots that are diagnostic of the "Ping-Pong" mechanism. 9 At higher substrate concentrations, inhibition is observed, and the full mechanism is more complicated. All substrates form abortive complexes with the enzyme ~5 and evidence has been presented to support the participation of ternary complexes in the mechanism. The significance of these is not clear. A simplified mechanism can be written as shown in the reaction scheme. A puzzling aspect of the mechanism is the apparent ability of the enzyme to maintain about 40% of the bound pyridine nucleotide prosthetic groups in the reduced state, i.e., as NADH. Treatment of the native enzyme with pyruvate or oxaloacetate quenches the NADH fluorescence 13 F. J. F e h r e n b a c h , Eur. J. Biochem. 18, 94 (1971). T h e N A D ÷ g l y c o h y d r o l a s e ( E C 3 . 2 . 2 . 5 ) w a s a k i n d gift f r o m D r . F e h r e n b a c h , U n i v e r s i t y o f F r e i b e r g , F e d e r a l R e p u b l i c o f G e r many. ~4 S. H . G . A l l e n , u n p u b l i s h e d d a t a , 1972, '~ M . I. D o l i n , J. Biol. Chem. 244, 5273 (1969).
376
OXIDATION--REDUCTION ENZYMES
[65]
and yields either lactate or malate, which can be recovered stoichiometrically after either gel filtration or dialysis to remove the enzyme. Upon standing at 4°, the enzyme-bound NAD slowly becomes reduced 1'' until about 40% of the total prosthetic group are as NADH. Furthermore, the native enzyme has been shown to contain covalently linked pyruvate2 The exact nature of the attachment of the pyruvate to the protein as well as its role, if any, in the mechanism are unknown. Treatment of the native transhydrogenase with either hydroxylamine or sodium borohydride have little effect on the enzymic activity. Yet reduction with [3H]sodium borohydride results in incorporation of radioactivity into the protein, which after repeated lyophilization and acid hydrolysis can be identified chromatography as [all]lactate.9 Dolin 15 has also shown the presence of tightly bound pyruvate using the lactate dehydrogenase assay. The possibility exists that these molecules act as a reservoir of reducing equilalents that interact with the bound NAD, keeping a constant N A D : N A D H ratio in the native enzyme. This may be necessary for keeping the transhydrogenase in the proper configuration or important in allowing the enzyme to react more readily with keto acid substrates. The amount of bound pyruvate present on each enzyme molecule has not been determined, and the role of this bound keto acid has not been ascertained.
[65] P y r u v a t e D e h y d r o g e n a s e C o m p l e x f r o m Bovine Kidney and Heart
By
FLORA
H.
PETTIT
and
LESTER
J. REED
Pyruvate + CoA + NAD÷~ acetyl-CoA+ CO2 + NADH + H + In eukaryotic cells the pyruvate dehydrogenase complex is located in mitochondria, within the inner membrane-matrix compartment. The complex consists of three catalytic components: pyruvate dehydrogenase (El) (EC 1.2.4.1), dihydrolipoyl transacetylase (E2) (EC 2.3.1.12), and dihydrolipoyl dehydrogenase (E3) (EC 1.6.4.3). These three enzymes, acting in sequence, catalyze the above overall reaction.
Assay Method Principle. Activity of this multienzyme complex is assayed spectrophotometrically by measurement of NADH production. The method is
METHODS IN ENZYMOLOGY, VOL. 89
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181989-2