[53] Isopropylmalate dehydratase from yeast

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ISOPROPYLMALATE DEHYDRATASE FROM YEAST

423

short forms of isoenzyme I have been studied using t~-isopropylmalate synthase (yeast)-fl-galactosidase (E. coli) fusion proteins) ~ Much of the long form of the fusion protein is imported into the mitoehondria, the short form stays in the cytoplasm, suggesting that the first 30 amino acids of the long form contain all of the information necessary for mitochondrial import. The native short form of isoenzyme I is functional in leucine biosynthesis and is inhibited by leucine with an apparent Ki of about 4 × 10-4 M a t pH 7.2. I~ t~-Isopropylmalate Synthase from Other Sources Purification procedures as well as structural and kinetic analyses have been published for a-isopropylmalate synthase from Salmonella typhimurium,19- 22 Neurospora crassa, t t,23a4 and Alcaligenes eutrophus. 2s 2o G. B. Kohlhaw, T. R. Leary, and H. E. Umbarger, J. Biol. Chem. 244, 2218 (1969). 21G. B. Kohlhaw and T. R. Leafy, this series, Vol. 17, p. 771. 22 j. C. Bartholomew and J. M. Calvo, Biochim. Biophys. Acta 250, 577 (1971). 23 R. E. Webster and S. R. Gross, Biochemistry 4, 2309 (1965). 24 R. E. Webster, C. A. Nelson, and S. R. Gross, Biochemistry 4, 2319 (1965). 2s j. Wiegel and H. Schlegel, Arch. Microbiol. 112, 239 (1977), 112, 247 (1977), and 114, 203 (1977).

[53]

Isopropylmalate

Dehydratase

from Yeast

B y G U N T E R B. K O H L H A W

Isopropylmalate dehydratase (EC 4.2.1.33, isopropylmalate isomerase) catalyzes the second pathway-specific reaction in leucine biosynthesis, the interconversion between tx-isopropylmalate and fl-isopropylmalate. Yeast isopropylmalate dehydratase is encoded by LEU1. The enzyme is located in the cytosol. 1 It is unstable when removed from the cells, but can be stabilized by a variety of conditions. The specific activity of isopropylmalate dehydratase decreases more than 10-fold when cells are grown in minimal medium supplemented with 2 m M leucine2 or 0.5% tryptone.3 i E. D. Ryan, J. W. Tracy, and G. B. Kohlhaw, J. Bacteriol. 116, 222 (1973).

2 V. R. Baichwal,T. S. Cunningham,P. R. Gatzek,and G. B. Kohlhaw,Curt. Genet. 7, 369 (1983). 3 T. Satyanarayana, H. E. Umbarger, and G. Lindegren, J. Bacteriol. 96, 2012 and 2018

(1968). METHODSIN ENZYMOLOGY,VOL. 166

Copyright© 1988by AcademicPreL Inc. Allrisht*ofr~productionin any formreserved.

424

ENZYMES

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This regulation appears to take place, at least in part, at the transcriptional level. 4 LEU1 is coregulated with LEU2, the gene encoding fl-isopropylmalate dehydrogenase, even though the two genes reside on separate chromosomes.

Assay M e t h o d Isopropylmalate dehydratase catalyzes the following reactions: a-Isopropylmalate ~ (Dimethylcitraconate) ~ fl-Isopropylmalate

The equilibrium of the reaction favors the formation of a-isopropylmalate2 ,6 The reaction mixture contains dimethylcitraconate when the reaction is at equilibrium, 5 but it is uncertain whether the compound exists as a free intermediate in vivo.

Principle of Assay Use is made of the double bond character of dimethylcitraconate, which causes the compound to absorb in the ultraviolet range (190250 nm; E235= 4 5 3 0 M -~ cm-l). Measurements are routinely made at 235 nm. Since this wavelength lies on a steeply ascending branch of the absorption curve (2m~, is close to 200 nm), exact adjustment of the spectrophotometer is important.

Reagents Potassium phosphate buffer, pH 7.0, 0.2 M Dimethylcitraconate (DMC), 0.1 M fl-Isopropylmalate (fl-IPM), 0.1 M (Citraconate, 0.2 M )

Procedure Either DMC or fl-IPM can be used as substrate (see Comments on Assay Method below for the use of citraconate), recording either the decrease or the increase in absorption at 235 nm. A 0.5-ml reaction mixture typically contains 0.2 ml potassium phosphate buffer, 0.01 ml substrate solution, 0.27 ml H20, and 0.02 ml enzyme solution. To reduce total absorption, a cuvette with a 2 mm path length is used. Buffer and water are mixed and equilibrated at the desired temperature, usually 30 ° . Enzyme solution is added and the background rate recorded for about 30 sec. The 4 y..p Hsu and P. Schimmel, J. Biol. Chem. 259, 3714 (1984). 5 S. R. Gross, this series, Vol. 17, p. 786. 6 R. Bigelis, Ph.D. thesis. Purdue University, West Lafayette, Indiana, 1974.

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ISOPROPYLMALATE DEHYDRATASE FROM YEAST

425

reaction is then started by adding the substrate solution, and the rate is recorded for several minutes. With crude extract, the rate is stable for at least 2 min. Initial rate is calculated after subtracting the background rate. A unit of activity is defined as the amount of enzyme catalyzing the disappearance (formation) of 1 pmol of substrate (product) per minute. Specific activity is units per milligram of protein. Protein concentration is determined by the biuret procedure or by the dye-binding method of Bradford. 7 To avoid interference from high concentrations of glycerol or a m m o n i u m sulfate, the protein is precipitated with trichloroacetic acid (final concentration 10%). Comments on Assay Method

Neither dimethylcitraconate nor fl-isopropylmalate are commercially available. The natural isomer offl-isopropylmalate can be isolated from the culture medium of overproducing organismsS,9; dimethylcitraconate has to be commercially synthesized. ~° However, for routine determinations of isopropylmalate dehydratase activity, a commercially available substrate analog, citraconate, may be used: H a - - c o 2-

H a - - c o 2-

/ C H - - C - - C O 2-

H 3 C - - C - - C O 2-

H3C\

II

II

H3C Dimethylcitraconate

Citra¢onate

The UV absorption spectra of the two compounds are virtually superimposable, which means that the same extinction coefficient can be employed. The apparent Km value for citraconate is approximately twice that for dimethylcitraconate 1~ (see below, Properties). Since a cell-free extract from a leul mutant fails to act on citraconate (under routine assay conditions; P. R. Brisco and G. B. Kohlhaw, unpublished observations), it is very likely that it is indeed isopropylmalate dehydratase and not another enzyme that catalyzes the conversion of the analog. Purification P r o c e d u r e for Yeast Isopropylmalate D e h y d r a t a s e Yeast isopropylmalate dehydratase can be purified from wild-type cells (strain $288c) if stabilizing agents such as p-isopropylmalate, glycerol, 7 M. M. Bradford, Anal. Biochem. 72, 248 (1976). s j. M. Calvo and S. R. Gross, this series, Vol. 17, p. 791. 9 p. N. Fultz, K. L. Choung, and J. Kemper, J. Bacteriol. 142, 513 (1980). lo j. V. Schloss, R. Magolda, and M. Emptage, this volume [12]. ,i W. Chen, M.S. thesis. Purdue University, West Lafayette, Indiana, 1984.

426

ENZYMES

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and/or ammonium sulfate are utilized. ~2The cells are grown aerobically at 30 ° in a synthetic medium 3 containing salts, succinic acid (5.8 g/liter), trace elements, vitamins, and glucose (20 g/liter). They are harvested in late log phase, washed once with cold 0.05 M potassium phosphate buffer, pH 6.8 (buffer A), and stored at - 2 0 °.

Step 1. Preparation of Crude Extract About 100 g of frozen cell paste is thawed and suspended in 100 ml of 0.1 M potassium phosphate buffer, pH 6.8, containing 1 mMfl-isopropylmalate. Aliquots of 35 ml of the suspension are then disrupted for four consecutive 30-sec treatments with a Bronson $75 Sonifier set at 5 amp. This step, like all subsequent steps, is carried out at 0 - 5 °.

Step 2. Ammonium Sulfate Fractionation The suspension obtained after sonification is treated with solid ammonium sulfate to give 50% saturation. The pellet obtained after centdfugation at 27,000 g for 10 min is discarded. Enough solid ammonium sulfate is added to the supernatant solution to give 65% saturation. The precipitate is collected by centrifugation and dissolved in 2 ml of buffer A containing 1.24 M ammonium sulfate.

Step 3. High Ionic Strength Hydrophobic Chromatography on ValineSepharose The solution obtained in the previous step is applied to a valine-Sepharose column (2 cm × 21.5 cm) prepared by coupling L-valine to cyanogen bromide-activated Sepharose 4B 13 and equilibrated with buffer A containing 1.24 M ammonium sulfate. The same buffer is used initially to elute excluded and loosely adsorbed proteins; a shift to buffer A containing 30% glycerol and no ammonium sulfate then elutes isopropylmalate dehydratase.

Step 4. High Ionic Strength Hydrophobic Chromatography on LeucineSepharose Fractions from step 3 containing most of the isopropylmalate dehydratase activity are pooled and protein is precipitated by 70% saturation with ammonium sulfate. After standing for 1 hr, the precipitate is collected by

~2R. Bigelis and H. E. Umbarger, J. Biol. Chem. 250, 4315 (1975). ~3p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).

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427

ISOPROPYLMALATE DEHYDRATASE FROM YEAST TABLE I PURIFICATIONOF ISOPROPYLMALATEDEHYDRATASE

Step

Total activity (units)"

Total protein (mg)

Specific activity (units/rag)

Purification (-fold)

Recovery (%)

Extract Ammonium sulfate Valine-Sepharose Leucine-Sepharose

71 57 41 21

2285 160 40 3.4

0.031 0.356 1.025 6.176

1.0 11.5 33.1 199.2

100% 80.3 57.7 29.6

a Micromoles of substrate utilized per minute.

centrifugation and dissolved in buffer A containing 1.24 M a m m o n i u m sulfate. The solution is applied to a leucine-Sepharose column (1.2 cm X 10 cm; prepared the same way as the valine-Sepharose column 13) equilibrated with buffer A containing 1.24 M a m m o n i u m sulfate. Elution is accomplished by using a linear gradient of decreasing a m m o n i u m sulfate (1.24 M---, 0) and increasing glycerol concentrations (0 ---, 30%). The gradient (total volume 1 liter) is established in buffer A. Isopropylmalate dehydratase elutes around I. 1 M a m m o n i u m sulfate and 4% glycerol. The fractions containing most of the dehydratase activity are combined and treated with a m m o n i u m sulfate. Material precipitating between 54 and 62% saturation is collected. This precipitate is dissolved in buffer A containing 1.24 M a m m o n i u m sulfate and 30% (v/v) glycerol and stored at - 2 0 °. It should be homogeneous by the criterion of polyacrylamide disc. gel electrophoresis under denaturing conditions (0.1% sodium dodecyl sulfate). A sample purification is shown in Table I. Comments on Purification Procedure

Successful purification of isopropylmalate dehydratase depends to a large extent on the judicious use of the a m m o n i u m sulfate fractionations (step 2 and the concentration part of step 4). It is especially important that enough time be allowed for the precipitations to occur (no less than 1 hr). '2 The valine- and leucine-substituted Sepharoses are not interchangeable. Since neither valine nor leucine influences the activity of the dehydratase, the interaction between the substituents and the enzyme is believed to be strictly hydrophobic, resulting in different retentive abilities of the two types of Sepharose. Elevated concentrations of a m m o n i u m sulfate (> 30% saturation) increase retention, glycerol (30%) opposes this effect. At these

428

ENZYMES

[53]

concentrations, both agents stabilize isopropylmalate d e h y d r a t a s e , t4 allowing for a wide variety of retention-elution programs. Properties

Stability Yeast isopropylmalate dehydratase, purified through step 2, has a halflife of 2 - 3 hr when kept at 0 - 5 ° and at a protein concentration of 0.35 mg/ml in 0.05 M potassium phosphate buffer, pH 6.8? 4 Dimethyleitraconate (1 raM) has no effect on stability, but fl-isopropylmalate (1 raM) increases the half-life to 14 hr. Increasing concentrations of ammonium sulfate or glycerol increase stability; thus, the half-life of the enzyme is 27 hr in the presence of 1.24 M ammonium sulfate and 15 hr in the presence of 30% glycerol. A combination of these two agents at the indicated concentrations causes the half-life to increase to several months. In the absence of stabilizing agents, the enzyme is significantly less stable at pH 9.0 than at pH 7.0.

Catalytic Properties The enzyme recognizes a-isopropylmalate, p-isopropylmalate, dimethylcitraconate, and also the analog citraconate, as indicated above; the trans isomer of citraconate, mesaconate, is not a substrate of the dehydratase. 1~ The apparent K , value of purified enzyme for dimethylcitraconate is 2.16 × 10-4 M under low-salt conditions and 2.37 × 10-4 M in the presence of 0.4 M ammonium sulfate.~4 With crude extract, an apparent K , value of 4.8 × 10-4 M has been observed for dimethylcitraconate; under identical conditions, the value for citraconate is 8.3 × 10-4 M. H The pH dependence of initial velocity, determined under standard (i.e., low ionic strength) assay conditions and with each of the three natural substrates, shows a sharp upturn at approximately pH 6 and a broad optimum between pH 7 and 9. The presence of chelators such as ethylenediaminetetraacetic acid ophenanthroline, or 8-hydroxyquinoline sulfonate in otherwise standard assay mixtures at pH 7.0 has only a very small effect on initial velocity?4 There is, however, a time- and concentration-dependent inactivation by KCN that is almost instantaneous and complete in the presence of 10 m M KCN. This effect is held back by high concentrations of ammonium sulfate. No metal requirement has been established. ,4 R. Bigelis and H. E. Umbarger, J. Biol. Chem. 251, 3545 (1976).

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fl-ISOPROPYLMALATE DEHYDROGENASE FROM YEAST

429

Structural Properties Sucrose density gradient centrifugation of native, stabilized isomerase purified through step 2 yields an apparent molecular weight of approximately 90,000, according to the procedure of Martin and Ames. ~4,m5The same value is obtained when highly purified, sodium dodecyl sulfate-denatured enzyme is subjected to polyacrylamide gel electrophoresis. 14 This suggests that yeast isopropylmalate dehydratase does not possess quaternary structure under the in vitro conditions tested; it does not necessarily reflect the in vivo situation. 16 Cloning and Sequencing By transformation of a leul auxotroph, a 3.5-kb yeast genomic fragment has been identified that complements the leul mutation? The sequence of over 600 bp of the 5'-flanking region and 144 bp of what apparently is a long open reading frame has been established. A major transcription start is seen at position --79 (relative to the beginning of the open reading frame), and Northern blotting has revealed the presence of a transcript about 2.9 kilonucleotides in length that is large enough to accommodate an open reading frame for a 90-kDa protein? Isopropylmalate Dehydratase from Other Sources Structure and regulation of isopropylmalate dehydratase from Neurospora crassa have been studied in considerable detail. 16,~7 The enzyme from Salmonella typhimurium has been studied with respect to stability and several other properties,6 but purification to homogeneity has apparently been elusive. 15 R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961). 16 V. E. Reichenbecher and S. R. Gross, J. Bacteriol. 133, 802 0978). 17 V. E. Reichenbecher, M. Fischer, and S. R. Gross, J. Bacteriol. 133, 794 0978).

[54] f l - I s o p r o p y l m a l a t e D e h y d r o g e n a s e

from Yeast

By GUNTER B. KOHLHAW fl-Isopropylmalate dehydrogenase (EC 1.1.1.85) catalyzes the third pathway-specific reaction in the biosynthesis ofleucine, an oxidative decarboxylation of fl-isopropylmalate (IPM) to yield a-ketoisocaproate and METHODS IN ENZYMOLOGY, VOL. 166

Copyright© 1988by AcademicPress,Inc. All rightsof reproduction in any form reserved.