[54] 3-Hydroxy-3-methylglutaryl-CoA reductase from yeast

[54]

YEAST HMG-CoA

REDUCTASE

[54] 3 - H y d r o x y - 3 - M e t h y l g l u t a r y l - C o A

455

Reductase from Yeast

By NILOFER QURESHI, SUKANYA NIMMANNIT, and JOHN W. PORTER CHs HOOC--CH2--C--CH2--CO--SCoA

+

2NADPH

+

2 H+

1

OH

CHs I H O O C - - CH~--C--CH2-- C H 2 O H

+

2NADP +

+

2CoA

OH

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase catalyzes the reduction by NADPH of D-HMG-CoA to mevalonic acid. The stoichiometry of this reaction is shown above. In yeast, this enzyme is present in the mitochondrial matrix. 1"2 Originally, this enzyme was solubilized by prolonged autolysis a and then purified approximately 200-fold to a specific activity of 1.4 /zmol of NADPH oxidized per minute per milligram of protein. 4 Later, this enzyme was purified to a specific activity of approximately 20/zmol of NADPH oxidized per minute per milligram of protein. 5 This procedure yields a homogeneous preparation of yeast HMG-CoA reductase. ~

Assay Method Principle. The HMG-CoA reductase activity can be conveniently determined by a modification of the spectrophotometric method of Kirtley and Rudney, 4 which measures the oxidation of NADPH at 340 nm.

Reagents Potassium phosphate buffer, ! M, pH 7.0 Dithiothreitol, 0.2 M i I. Shimizu, J. Nagai, H. H a t a n a k a , and H. Katsuki, Biochim. Biophys. Acta 296, 310 (1973). 2 p. T. T r o c h a and D. B. Sprinson, Arch. Biochem. Biophys. 174, 45 (1976). 3 I. F. Durr a n d H. R u d n e y , J. Biog. Chem. 235, 2572 (1960). 4 M. E. Kirtley and H. R u d n e y , Biochemistry 6, 230 (1967). 5 N. Qureshi, R. E. D u g a n , S. N i m m a n n i t , W.-H. Wu, and J. W. Porter, Biochemistry 15, 4185 (1976).

METHODS IN ENZYMOLOGY,VOL. 71

Copyright © 1981by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181971-X

456

H Y D R O X Y M E T H Y L G L U T A R Y L - C o A ENZYMES

[54]

HMG-CoA, ~ 18 mM NADPH, 1.6 mM Procedure. The complete assay mixture 5 contains 0.1 mmol of potassium phosphate buffer, pH 7.0, 5 /xmol of dithiothreitol, 300 nmol of HMG-CoA, 0.16/zmol of NADPH, and HMG-CoA reductase, in a total volume of 1.0 ml. Following a 10-min preliminary incubation at 30° to establish a base line rate of absorbance change at 340 nm, the reaction is initiated by the addition of HMG-CoA. The rate of the reaction is monitlem : 6.22 x tored by following the oxidation of NADPH at 340 nm t~' ~340nm 103).

Assay for Protein. Protein assays are carried out by the biuret method 7 and by the method of Lowry et al. 8 after the protein solution is dialyzed against water to remove dithiothreitol. Units. One unit of HMG-CoA reductase is defined as the amount of enzyme that catalyzes the oxidation of I nmol of NADPH per minute per milliliter of incubation mixture. Specific activity is expressed as units per milligram of protein. Purification Procedure The results of a typical purification of yeast HMG-CoA reductase are summarized in the table. Unless otherwise indicated, all steps are carried out at 4° . Solubilization of Enzyme. HMG-CoA reductase is solubilized from Fleischmann's active dry yeast for bakers by a modification of the autolysis procedure of Kirtley and Rudney. 4 Twelve pounds of dry yeast are suspended in 12 liters of dibasic phosphate, 0.3 M, containing 1 mM dithiothreitol and 1 mM EDTA and then stirred for 12 hr at 4°. This suspension is centrifuged for 15 min at 20,000 g or by continuous flow centrifugation. The supernatant is discarded, and the gummy precipitate is resuspended in 9 liters of the same buffer and stirred another 48 hr at 4°. The suspension is centrifuged as before, and the precipitate is resuspended in 9 liters of the same buffer and stirred for 40 hr. The supernatant solution obtained after centrifugation is retained. This contains the major portion of the solubilized enzyme. Heat Treatment. The crude supernatant solution (7130 ml) is heated (in e HMG-CoA was prepared as described by S. Goldfarb and H. C. Pitot, J. Lipid Res. 12, 512 (1971). It was then purified by paper chromatography as described by J. Brodie and J. W. Porter, Biochem. Biophys. Res. Commun. 3, 173 (1960). 7 A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949). s o . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall,'J. Biol. Chem. 193, 265 (1951).

[54]

YEAST H M G - C o A

REDUCTASE

457

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o

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458

HYDROXYMETHYLGLUTARYL-CoA ENZYMES

[54]

centrifuge cups) in a water bath (65°) until the temperature of the enzyme solution is 65°, which takes approximately 30 min, and then for an additional 5 min. This mixture is immediately cooled on ice and centrifuged at 20,000 g for 20 min. Ammonium Sulfate Fractionation. The supernatant solution (6620 ml) is brought to 60% saturation with solid ammonium sulfate (390 g/liter); after stirring for 30 min at 4°, the precipitated protein is collected by centrifugation. The pellet is resuspended in a minimum volume of 0.1 M potassium phosphate buffer, pH 7.0, containing 1 mM dithiothreitol and 1 mM EDTA. (The latter components are present in all buffers used in subsequent steps.) The enzyme is then dialyzed against the same buffer for 3 hr, with a change of buffer after 1.5 hr. Calcium Phosphate Gel Adsorption and Ammonium Sulfate Precipitation. The dialyzed protein (345 ml) is diluted four times with water containing 1 mM dithiothreitol and 1 mM EDTA to a final concentration of 0.025 M potassium phosphate, and then the protein concentration is adjusted to 7 mg/ml with 0.025 M potassium phosphate buffer. This solution is mixed with calcium phosphate gel prepared as described by Tsuboi and Hudson 9 at a ratio of protein (in milligrams) to gel (dry weight) of 1 : 1. After stirring for 15 min, the gel is centrifuged at 8000 g for 5 min and the pellet is washed twice with one-tenth the original volume of 0.1 M potassium phosphate buffer. The HMG-CoA reductase is eluted from the gel by washing three or four times with 0.4 M potassium phosphate buffer. The enzymatically active eluate fractions are then combined (2000 ml), and the enzyme protein is concentrated by precipitation with solid ammonium sulfate (0-65% saturation, 430 g/liter). The precipitate is dissolved in a minimum volume of 0.1 M potassium phosphate buffer and then dialyzed against the same buffer for 3 hr at 4°, with a change of buffer at 1.5 hr. DEAE-Cellulose Column Chromatography. Standard DEAE-cellulose ion exchanger is purchased from Schwarz-Mann and then prepared for enzyme purification by washing with acid, followed by base and water, according to the method of Peterson and Sober. 10The dialyzed protein (82 ml) is diluted to a concentration of 0.01 M potassium phosphate buffer, and about 190 mg of protein are applied to a DEAE-cellulose column (3.5 x 28 cm) previously equilibrated by washing overnight with 0.01 M monobasic potassium phosphate and then with 250 ml of 0.01 M potassium phosphate buffer, pH 7.0. Protein is applied to the column, and the latter is washed with 0.01 M potassium phosphate buffer. The HMG-CoA reductase is eluted from the column with 450 ml of a linear concentration gradient of 0.01-0.50 M potassium phosphate buffer, pH 7.0, at room a K. K. Tsuboi and P. B. Hudson, J. Biol. Chem. 224, 879 (1957). 10 E. A. Peterson and H. A. Sober, this series, Vol. 5, p. 6.

[54]

YEAST H M G - C o A REDUCTASE

459

temperature. Most of the HMG-CoA reductase is eluted when the concentration of potassium phosphate is approximately 0.125 M. Ten-milliliter fractions are collected, and enzymatically active fractions are pooled and concentrated by ultrafiltration in an Amicon cell with a PM-10 membrane. Affinity Chromatography. DEAE-cellulose-purified enzyme (15 x 104 units and 72 mg of protein) in 20 ml of 0.125 M potassium phosphate buffer, pH 7.0, is diluted fivefold with water containing EDTA, 1 mM, and dithiothreitol, 5 mM, and then applied to a 0.75 x 3.4 cm column (1.5 ml of gel) of agarose-hexane-CoA (type V) gel supplied by P-L Biochemicals. The gel is previously washed with 0.025 M potassium phosphate buffer, pH 7.0, containing 1 mM EDTA and 5 mM dithiothreitol. Nonadsorbed protein is washed from the column at 22° with l0 ml of the pH 7.0 buffer used to equilibrate the column, and then the column is washed with 50 ml of 0.075 M potassium phosphate buffer containing 5 mM dithiothreitol. The HMG-CoA reductase is eluted from the column with a KC1 gradient (0 to 2 M, 14 ml) in 0.025 M potassium phosphate buffer, pH 7.0, containing 5 mM dithiothreitol. The rate of elution is I ml/min. Ten-milliliter fractions are collected before the start of the gradient, and 1-ml fractions are collected thereafter. Most of the protein passes directly through the column without binding, and then the bound reductase is removed on gradient elution with KC1 (Fig. 1). A range in specific activity of 15,000 to 22,000 nmol of N A D P H oxidized per minute per milligram of protein is normally obtained in the purifications of this enzyme. The recovery of enzyme from the gel is usually 40-45%, but it may vary from 20 to 70%. In order to obtain a good yield, careful regulation of the ratio of gel to protein is necessary. Maximum recovery of the active enzyme is obtained when the column is almost saturated with enzyme. After elution, the protein concentration is very low and the enzyme loses activity unless it is concentrated immediately. Dialysis with collodion tubes (Schleicher and Schuell) is used to concentrate the enzyme and to remove salt from the protein.

Properties of Yeast HMG-CoA Reductase As shown in the table, yeast HMG-CoA reductase is purified approximately 5000-fold by the procedure outlined above. This preparation is stable for at least 3 months when stored at - 2 0 °. Purity. The enzyme purified as described is homogeneous, or nearly so, since it migrates as a single component on BioGel filtration and on a DEAE-cellulose column. When electrophoresed on 5% acrylamide gel, one major band of protein is observed, which coincides exactly with HMG-CoA reductase activity.

460

H Y D R O X Y M E T H Y L G L U T A R Y L - C o A ENZYMES

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ELUATIE Nml

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FIG. 1. Affinity chromatography of DEAE-ceUulose-purified HMG-CoA reductase obtained from autolyzed yeast. The details of this chromatographic separation are given in the text. Reductase activity (& . . . . &) and protein (o o) were assayed in the effluent. Reprinted, with permission, from Qureshi et al. 5

Molecular Weight. The molecular weight of the yeast H M G - C o A reductase is estimated to be approximately 2.6 × 105, based on its elution profile on a BioGel column. Electrophoresis of the dissociated e n z y m e on a sodium dodecyl sulfate-polyacrylamide (10%) disc gel showed one major staining protein band that corresponded to a subunit weight of approximately 60,000. This indicated that the native yeast H M G - C o A reductase is a tetramer that consists of subunits of the same molecular weight. Substrate Specificity. This enzyme catalyzes the reduction o f HMGCoA, mevaldic acid, 11 and mevaldic hemithioaceta112 to mevalonic acid in the presence o f N A D P H . It also catalyzes the oxidation o f mevaldic acid to HMG-CoA in the presence of N A D P ÷ and CoA. Kinetic Properties. Yeast H M G - C o A reductase is inactivated upon preincubation with CoA, 4 but this inhibition can be reversed by dialysis.la Without prior incubation, CoA is an activator for the reduction of mevaldate to mevalonic acid and it reduces the K m for mevaldate 20- to 30fold. 11The Km values for the substrates for this e n z y m e are as follows: for the overall reaction, H M G - C o A = 2 . 4 / x M 4 and N A D P H = 89/zM4; for 11 N. Qureshi, R. E. Dugan, W. W. Cleland, and J. W. Porter, Biochemistry 15, 4191 (1976). ,2 j. Rgtey, E. yon Stetten, U. Coy, and F. Lynen, Eur. J. Biochem. 15, 72 (1970). ,3 A. Tan-Wilson and G. B. Kohlhaw, Biochem. Biophys. Res. Commun. 85, 70 (1978).

[54]

YEAST HMG-CoA REDUCTASE

461

the second reductive step, mevaldate = 8 mM and 0.4 mM, 11 respectively, in the absence and in the presence of CoA, and NADPH = 24 /~M. 11 Irreversible Inhibition. Yeast HMG-CoA reductase is very susceptible to inhibition by low concentrations of certain thiol group reagents, e.g., iodoacetamide,4p-hydroxymercuribenzoate,4 and fungal products such as citrinin, TM ML236A, and ML 236B. T M It is also inhibited by ophenanthroline. 4 Mechanism of Reaction. Data from kinetic studies suggest that the oxidation of mevaldic acid to HMG-CoA and the reduction of mevaldic acid to mevalonic acid, catalyzed by the yeast HMG-CoA reductase in the presence or the absence of CoA, proceeds by a sequential mechanism. In terms of the overall reaction, HMG-CoA and NADPH bind to the enzyme in an ordered or random fashion. Reduction of HMG-CoA to mevaldate hemithioacetal then takes place, and NADPH replaces NADP +. The hemithioacetal then reverts to its components, mevaldate and CoA, and mevaldate is reduced to mevalonate. The products mevalonate and CoA, followed by NADP ÷, then leave the enzyme. 1~ The hydrogen used in this reduction is transferred from the A side of the pyridine ring of NADPH in both of the reactions in the conversion of HMG-CoA to mevalonate.lr Acknowledgments This work was supported in part by a research grant, HL 16364, from the National Heart and Lung Institute, National Institutes of Health, United States Public Health Service, and by the Medical Research Service of the Veterans Administration.

14 M. Kuroda, Y. Hazama-Shimada, and A. Endo, Biochim. Biophys. Acta 486, 254 (1977). 15 K. Tanzawa, M. Kuroda, and E. Endo, Biochim. Biophys. Acta 488, 97 (1977). 18 A. Endo, M. Kuroda, and K. Tanzawa, FEBS Lett. 72, 323 (1976). lr R. E. Dugan and J. W. Porter, J. Biol. Chem. 246, 5361 (1971).