[54] Aspartase

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R E A C T I O NLEADING S TO AND FROM THE CYCLE

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[54] Aspartase [EC 4.3.1.1

~-Aspartate ammonia-lyase]

By VmGINIA R. WILLIAMS and DON~D J. LARTmUE -OOC--CH2--NH3+--COO - ~- - O O C - - C H - ~ C H - - C O O - -b *NH4+ L-aspartate fumarate The enzyme which catalyzes the reversible conversion of L-asparate to fumarate and ammonium ion has been known for half a century. Its existence was first postulated by Harden 1 in 1901, but Quastel and Woolf2 in 1926 established the stoichiometry of the reaction and demonstrated that the enzyme was a deaminase rather than an oxidase. This deaminase was given the name "aspartase" by Woolf, 3 and it will be so designated throughout the discussion that follows. Assay Method

Principle. Aspartase can be assayed conveniently by measuring the production of either fumarate or ammonia. A unit of aspartase activity is that quantity of enzyme which produces 1 micromole of fumarate (or ammonia) per minute. Specific activity is defined as units per milligram of protein: These are not the traditional definitions, but they are consistent with the modern terminology for other enzymes. Method 1 A modification of the spectrophotometric fumarase assay developed by Racker 4 is used tor routine aspartase determination. This method has the advantages of analytical sensitivity and suitability for initial rate studies.

Reagents Tris-HCl buffer, 0.15 M, pH 7.0 Potassium L-aspartate, 0.5 M, pH 7.0 MgSO4, 30 mM EDTA, 3 mM, adjusted to pH 7.0 with Tris

Procedure. Absorbance is measured conveniently in a double-beam spectrophotometer equipped with a suitable recorder and constant temIA. Harden, J. Am. Chem. Soc. 79, 623 (1901). 2j. H. Quastel and B. Woolf, Biochem. J. 20, 545 (1926). ~B. Woolf, Biochem. J. 23, 472 (1929). • E. Racker, Biochim. Biophys. Acta 4, 211 (1950).

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perature housing. A temperature of 30 ° is recommended for routine assays, since it is easy to maintain. A pair of 1 cm silica cells is used with the reagent volumes given below. Into each cell pipette 1.0 ml of 0.15M Tris-HC1 buffer, 0.3 ml of 0.5 M L-aspartate, 0.1 ml of 30 mM MgSO~, 0.1 mI of 3 mM EDTA, and 1.4 ml water. Mix the solutions and adjust the spectrophotometer to zero absorbance at 240 mt~, then add to the sample cell 0.1 ml of a suitable (lilution of the enzyme; the change in absorbance is measured as a function of time. (With crude enzyme preparations, which contain large amounts of inert protein, the enzyme may be added to both the reference and sample cells and the substrate omitted from the reference cell.) The reaction rate should fall within 0.04-0.40 absorbance units per minute for a 1 cm light path cuvette. The rate remains constant for several minutes and is directly proportional to the enzyme concentration. The molar extinction coefficient of potassium fumarate under similar conditions is reported ~ to be 2.53 X 103 M -I cm-k Specific activity is calculated as follows:

&A240rain-1 Specific activity = 2.53 ml umole-VX mg protein ml -~ assay solution" Protein is determined by the method of Lowry et al., 6 using crystalline bovine serum albumin as standard. Method 2

Any method of ammonia measurement may be used. The simplest procedure is direct nesslerization7 of an aliquot of reaction mixture from method 1. It may be necessary to distill the ammonia by the Conway technique 8 prior to nesslerization. A number of substances interfere with direct determination, e.g., sulfhydryl compounds and some amino acids, such as histidine and arginine. This method is less sensitive than method l; consequently, assays should be incubated until the ammonia concentration reaches 1 raM. The reaction is stopped by the addition of 0.1 volume of 20% trichloroacetic acid to the assay tubes. Precipitated protein is centrifuged and the supernatant solution is analyzed for ammonia. A rate curve can be obtained from assays incubated for 0, 5, T. F. Emery, Biochemistry 2, 1041 (1963). 6 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). P. B. Hawk, B. L. Oser, and W. It. Summerson, "Practical Physiological Chemistry," 13th ed., p. 1329. Blakiston, New York, 1954. P. B. Hawk, B. L. Oser, and W. H. Summerson, "Practical Physiological Chemistry," 13th ed., p. 886. Blakistoa, New York, 1954.

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[$4]

I0, and 15 minutes. Since the reaction rate is not linear for longer periods, Method 1 should be used for precise results. Purification Procedure This purification method yields a preparation free of enzymes which might complicate the study of aspartic acid deamination, especially fumarase. Cells of Bacterium cadaveris Gale, 1944, ATCC-9760 (more appropriately, Enterobacter ha]nine), are grown in 6-liter batches of medium containing 1~ yeast extract, 1% tryptone, and 0.5~ K2HPO,. Growth from a 10~ inoculum is allowed to proceed for 48 hours at 30 ° without aeration or agitation. The cells are harvested, washed once with 80 mM KCI, and frozen until used. This procedure yields about 25 g of wet, packed cells from 18 liters of medium. Step 1. Sonication. Suspend approximately 50 g of cells (wet weight) in 100 ml of 0.1 M potassium phosphate buffer, pH 7.0; add 1.5 ml of 0.1 M mercaptoethanol, and divide the suspension into two portions. The cells are then disrupted by sonication in a Branson Model LS-75 Sonifier. Maintain the temperature below 10 ° for five 1 minute sonications for each portion of the suspension. Then centrifuge the sonicate in the No. 30 rotor of the Spinco Model L-2 ultracentrifuge at 105,000 g at 2 ° for 1 hour. Discard the precipitate. Step ~. Protamine Sul]ate Precipitation. Determine the protein content of the clear amber solution (110 ml) 9 and adjust to 30-45 mg/ml with 0.1 M potassium phosphate buffer, pH 7.0. Dissolve in potassium phosphate buffer (0.1 M, pH 7.0) a quantity of protamine sulfate equal to 15~ of the total weight of protein to form a 1 ~ solution (50-75 ml). Make the protamine sulfate solution to 1 mM mercaptoethanol, and then add it drop by drop to the enzyme solution, which is stirred in an ice bath. Stir for an additional 30 minutes after the addition is complete. During this and subsequent steps, do not allow the temperature to exceed 4 °. Centrifuge the suspension at 27,000 g for 30 minutes in a refrigerated centrifuge and discard the precipitate. Step 3. pH Fractionation. Adjust the supernatant solution from step 2 (about 170 ml) rapidly to pH 4.2 with 2 M acetic acid (about 15 ml). Centrifuge the solution immediately at 27,000 g for 10 minutes and discard the supernatant solution. Suspend the precipitate in 0.1 M potaso In the relatively crude fractions obtained in steps 1-5, protein may be determined by the rapid turbidimetric method of Exton [W. G. Exton, J. Lab. Clin. Med. 10, 722 (1925)], standardized against crystalline bovine serum albumin. The Lowry° method may also be used. Although more time-consuming, it has the advantage of greater accuracy.

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slum phosphate buffer, pH 7.0, (containing 1 mM mercaptoethanol and 10 t ~ / M g S 0 4 ) to a total volume equal to one-third to one-half the initial volume. Suspension of the thick, gummy precipitate is aided by one or two short sonications. Centrifuge the solution at 27,000 g for 15 minutes and discard the precipitate. Step 4. Ammonium Sul]ate Fractionation. To the supernatant solutior, from step 3 add 1/20 volume of 1 M potassium phosphate buffer, pH 7.4. Add solid ammonium sulfate, 176 mg per milliliter of enzyme solution, to bring the salt concentration to 30% of saturation. Add the salt slowly while stirring, and continue stirring for 15 minutes after the addition is complete. Centrifuge the material at 27,000 g for 15 minutes, and discard the precipitate. Add 162 mg ammonium sulfate per milliliter to the supernatant solution to bring the salt concentration to 55% of saturation. Centrifuge the suspension and discard the supernatant solution. The precipitate can be kept frozen for a year without appreciable loss of activity. Step 5. Dialysis. Dissolve the frozen ammonium sulfate precipitate from step 4 in a minimum volume of 10 mM potassium phosphate buffer, pH 7.0, containing 1 mM mercaptoethanol and 10 ~M MgS04, and dialyze against 400 ml of the same buffer until the dialyzate gives only a faint color with Nessler's reagent. Change the dialyzate every 30 minutes; about 6 liters of buffer are required. The enzyme may be frozen at this stage; however, 50% loss of activity is observed after 2 weeks of storage. Step 6. Column Chromatography. Either DEAE- or ECTEOLAcellulose may be used for the final purification step. DEAE- and ECTEOLA-celluloses are prepared as follows: The material is suspended in distilled water and allowed to settle for 20 minutes. The fines are decanted. The process is repeated until the liquid above the bulk of the material is clear after a 20-minute settling interval. The material is washed twice with saturated KC1 and collected using a Biichner funnel. It is then washed with distilled water until the washings are free of chloride. The material is suspended in 10 mM potassium phosphate buffer, pH 7.0, filtered, resuspended in the same buffer, and stored. Regeneration is done the same way. The cellulose exchanger is packed in a jacketed column to give a bed size either 1 or 2 cm in diameter and 20 cm in length. Column temperature is maintained at 4 °. Packing is achieved by pouring the DEAE or ECTEOLA suspension into the column and allowing the material to settle. Apply nitrogen at 5 psi to the top of DEAE-cellulose columns several times to hasten preparation and pack tlle cohmm tightly. Th(, 1 em columns are washed with 150 ml of cquilibr'tting buffer; the 2 cm

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columns, with 500 ml. Flow rates are maintained at 1 ml per 3-5 minutes by adjusting the stopcock at the bottom. The procedure employed with the larger DEAE column is as follows: Apply 5 ml of the enzyme solution (about 100 mg protein) to the column, followed by 95 ml of 10 mM potassium phosphate buffer, pH 7.0. Then wash the column with 150 ml of the same buffer containing 0.2 M KC1. Elute the enzyme with 100 ml of 10 mM potassium phosphate buffer, pH 7.0, containing 0.7 M KCI. Collec~ 10 ml fractions in test tubes containing 0.1 ml of 0.1 M mercaptoethanol. Under these conditions, the enzyme elutes sharply with the 0.7 M KCI front. Only a 1- to 2-fold dilution of the enzyme results. If the 1 X 20-cm column is used, all volumes are decreased to one-fourth of those reported above. Elute the ECTEOLA column with a linear gradient from zero to 0.5M KC1 at a constant level of 10 mM phosphate buffer, pH 7.0. Use a refrigerated fraction collector to collect 10 ml fractions. The enzyme is eluted at about 0.25 M KC1. Usually only one er two active fractions are obtained; the total dilution is about 2-fold. The eluted enzyme is unstable and should be used immediately o1" precipitated with ammonium sulfate, which is added to saturation. Tris-HC1 buffers of the same molarity and pH may be substituted for the phosphate buffers with minor changes in the recoveries of the enzyme. Data for a typical preparation are shown in the table? ° Specific activities are determined at pH 7.0, since aspartase becomes increasingly unstable as the pH is elevated. However, maximum rates are obtained at pH 8.5 (in the presence of saturating levels of substratO °) and the rate of fumarate production is 5-fold greater than that at pH 7.0. The specific activity of the final fraction shown in the table would be approximately 175 instead of 35 if measurements had been made at pH 8.5. Properties General Protein Characteristics

Aspartase is also discussed in an earlier volume. 11 Aspartase is an acidic protein with an isoelectric point of 4.8, determined by starch electrophoresis? 2 It attaches quite tightly to DEAE-cellulose in the neutral pI-I range. Sucrose density gradient centrifugation using eatalase as reference shows that the enzyme has a molecular weight of approximately 180,000.1° It can be dissociated into four subunits of equal weight by t°V. R. Williams and D. J. Lartigue, d. 3iol. Chem. 2242, 2973 (1967). 11A. I. Virtanen and N. Ellfolk, Vol. II, p. 386. '~J. 8. Wilkson and V. R. Williams, Arch. Biochem. Biophys. 93, 80 (1961).

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ASPARTASE

359

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360

REACTIONS LEADING TO AND FROM TItE CYCLE

[54]

treating native aspartase with p-hydroxymercuribenzoate. Active tetramer is regenerated by treating the mercury derivative with mercaptoethanol. Aspartase has not been crystallized, and its amino acid composition is unknown. Characteristics as a Catalyst a. Specificity toward Substrate. The enzyme has long been regarded as displaying absolute specificity toward L-aspartate, fumarate, and NH~. However, Emery5 has reported that hydroxylamine can replace NH3 in the addition reaction, forming N-hydroxyaspartic acid. b. K',~. At pH 7.0 in Tris-HCl buffer, K',~ for aspartate is 1.5 raM; at pH 7.0 in potassium phosphate buffer, K ' , is 20 raM. An analysis of the pH dependence of K'~ and Vm~ suggests that imidazole and sulfhydryl groups are present at the active site. is c. pH Optimum. In Tris-HC1 buffer the pH optimum is near 8.5.~° Most earlier reports cite a pH optimum of 7.0-7.5, attributable to the inhibition of aspartase by secondary phosphate anion and the choice of low substrate concentration, or both. The observed pH optimum is influenced greatly by the concentration of substrate in the assay system. The optimum of 8.5 was determined from a plot of extrapolated Vma. as a function of pH. d. Thermodyna.mic Constants. At pH 7.2, K',q for the elimination reaction is approximately 20 mM at 39° and 10 mM at 20°. 12,14 e. Stability. Aspartase shows maximal stability between pH 6.0 and 7.5; pH should be maintained in this range during purification. Aspartase is inactivated rapidly on cold storage unless it is frozen as the ammonium sulfate precipitate from step 4. Storage deterioration is irreversible. ]. Organic Cofactors. Aspartase appears to have no organic cofactors essential to catalysis. However, Scott~5 obtained significant reactivation of extensively dialyzed preparations with either inosine monophosphate, adenosine monophosphate, or guanosine diphosphate. Guanosine triphosphate was highly inhibitory. These results suggest heterotropic interactions between nucleotide and enzyme. We have obtained confirmation of these findings. ~° g. Metal Ion Activators. Aspartase possesses a divalent cation requirement of low specificity. 12,~6 The best activators are Mg** and Mn +*. lSD. J. Lartigue, Ph.D. Dissertation, Louisiana State University, 1965; University of Michigan Microfilm No. 65-11, 394. "V. R. Williams and R. T. McIntyre, J. Biol. Chem. 217, 467 (1955). i~R. M. Scott, Ph.D. Dissertation, University of Illinois, 1959; University of Michigan Microfilm No. 59-4, 566. ~R. It. Depue and A. G. Moat, Y. Bacteriol. 82, 383 (1961).

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ASPARTASE

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Methods employed commonly to convert metalloenzymes into their apoenzymes are not highly effective with aspartase. The metal ion is either tightly bound or well shielded from solvent. h. Cooperative El~ects o/ Substrate. Aspartase exhibits typical Miehaelis kinetics at pH 6.0, but substrate concentration-activity curves deviate increasingly from the hyperbolic shape as the pH is increased. Above pH 7.5 they are markedly sigmoidal, suggesting that the effect of substrate is cooperative, as is the Bohr effect observed with hemoglobin. The molecular weight of aspartase is the same at pH 6.0 and 8.0. ~° i. Enzyme Mechanism. Although the stereochemistry of the aspartase reaction is known, the mechanism of the elimination is uncertain. Englard 17 and Krasna TM showed that NH~ is removed from aspartate and added to fumarate in a stereospecific manner. These workers proposed independently that the reaction involves c/s-elimination; however, it was shown to be trans by Gawron and Fondy29 Neither deuterium exchange nor a deuterium isotope effect has been observed in the elimination reaction2 ~

Distribution Although aspartase was thought to occur only in bacteria and a few species of higher plants, 11 Kurata 2o has reported its presence during the ontogeny of the frog Rhacophorus schlegelii var. arborea. Salvatore et al., 21,~ report wide distribution of aspartase activity in animal tissue, particularly in sharks and bony fishes. The properties of bacterial aspartase indicate it may be a regulatory enzyme: its synthesis is glucoserepressed, 2~ it possesses quaternary structure, and it exhibits cooperative effects of substrate and heterotropic interactions with various nucleotide activators. 1° Its role in animal metabolism is unknown. Acknowledgment The methods described in this report were developed with the research support of Grant GM-11016 from the United States Public Health Service and Grant GB-5017 from the National Science Foundation.

"S. Englard, J. Biol. Chem. 233, 1003 (1958). ~SA. I. Krasna, J. Biol. Chem. 233, 1010 (1058). 1'0. Gawron and T. P. Fondy, J. Am. Chem. Soe. 81, 6333 (1959). ~oy. Kurata, Exptl. Cell Res. 28, 424 (1962). '~ F. SaIvatore, V. Zappia, and C. Costa, Comp. Biochem. Physiol. 16, 303 (1965). :*V. Zappia, C. Pietropaolo, C. Costa, and F. Salvatore, Ab~.tr. 150th Meeting Am. Chem. Soc., Atlantic City, Sept., 1965, p. 37e. Spaulding-Moss, Boston, Massachusetts. ~3M. A. Farley and H. C. Lichstein, Can. J. Microbiol. 9, 835 (1963).