[43c] Shikimate dehydrogenase1–2 (peas)

354

[43c]

AROMATIC AMINO ACIDS

Reproducibility The average optical density for 0.03 micromole of shikimic acid in 12 assays at 3 different times was 0.296 with a standard deviation of + 0.013. Specificity The method is relatively nonspecific, since strong reactions are given by such compounds as deoxyribose, guanosine, guanylic acid, and adenosine. About 20% of the color is given by N-acetylneuraminic acid. Some preliminary purification is necessary before mixtures containing these materials and shikimic acid can be assayed. 5-Dehydroshikimic acid reacts in the assay but no color is obtained with shikimic acid 5-phosphate, unless it has been pretreated with intestinal phosphatase or hydrolyzed in 0.4 N HCI for 4 hours at 120 °.

[43c] S h i k i m a t e D e h y d r o g e n a s e

1-2 ( P e a s )

By DORIS BALINSKY and ALAN W. DENNIS

coo-

coo.H

0

OH

Dehydroshikimate

+ NADPH + H+

H ~ H HO

+

NADP +

OH

8hikimate

Assay Method The enzyme is assayed in the direction of shikimate oxidation by following the production of NADPH spectrophotometrically at 340 m/~.a Coupling the reaction with GSSG reductase, as previously described, 2 is not necessary, as NADPH production is linear for at least 5 minutes. 1Alternative names: EC 1.1.1.25 Shikimate: NADP oxidoreductase; 5-dehydroshikimic acid reductase. The preparation of this enzyme from E. coli was described in Vol. II [39]. The preparation of 5-dehydroshikimic acid was described in Vol. VI [74]. laA recent paper on Moraxella calcoacetica [J. L. Canovas, M. L. Wheelis, and R. Y. Stanier, EuropeanJ. Biochem. 3, 293 (1968)] has described an inducible shikimate dehydrogenase which is not pyridine nucleotide-linked but can be coupled to 2,6-dichlorophenolindophenol. Its Michaelis constant for shikimate is 20 times greater than is that of the NADP-linked enzyme in the same organism. ZB. D. Davis, C. Gilvarg, and S. Mitsuhashi, Vol. II [39]. SD. Balinsky and D. D. Davies, Biochem.J. 80,292 (1961).

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SHIKIMATE DEHYDROGENASE

355

Reagents G l y c i n e - N a O H buffer, 0.1 M, pH 9.0 NADP, 0.005 M Shikimic acid, 0.03 M (5.22 mg/ml) Enzyme. Dilute the solution with 0.05 M sodium phosphate buffer, pH 7.4, containing 0.001 M cysteine, so that the sample being assayed contains ca. 0.03 unit. Procedure? Into a quartz cuvette of 1-cm light path are pipetted 2.7 ml of buffer, 0.1 ml of the NADP solution, 0.1 ml of diluted enzyme, and 0.1 ml of the shikimate solution (in an "adder-mixer") 4 to start the reaction. The increase in optical density at 340 m/z is followed spectrophotometrically. Definition of Unit and Specific Activity. A unit of activity is defined as the amount of enzyme catalyzing the production of 1 micromole of NADPH per minute, assuming a molar extinction coefficient for NADPH of 6.22 × 103.5 Specific activity is the activity per milligram protein. Protein is determined by measuring the optical density at 280 mtz, assuming a protein concentration of 1 mg/ml to give an optical density reading of 1. Application of Assay Method to Crude Preparations. The assay procedure can be used with crude preparations, provided a blank cuvette containing all reagents except shikimate is used as control. Purification P r o c e d u r e °

This procedure gives an enzyme preparation of higher purity and greater stability than that published earlier by Balinsky and Davies? Materials

Peas (Pisum sativum, var. "Greenfeast") are surface sterilized by soaking in 1% sodium hypochlorite solution at room temperature for 30 minutes. They are rinsed and allowed to swell in a jar (covered with a piece of cheesecloth) through which running tapwater is passed. After 2 days they are planted in wooden trays containing vermiculite, watered, and allowed to grow in the dark, preferably at 25-30 ° to permit a reasonable rate of growth. This treatment produces etiolated epicotyls with only a few tiny leaves. When the epicotyls are 2-3 inches in height (approximately 5 days after planting) the seedlings are harvested, and any in4p. D. Boyer and H. L. Segal in "The Mechanism of Enzyme Action" (W. D. McElroy and B. Glass, eds.), p. 520. Johns Hopkins Press, Baltimore, Maryland, 1954. 5B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). 8A. W, Dennis, M.Sc. thesis, University of the Witwatersrand, Johannesburg, South Africa, 1967.

356

AROMATIC AMINO ACIDS

[43c]

fected parts are removed. The seedlings are then thoroughly washed and chilled. All subsequent operations are carried out in a cold room at 4 ° or in an ice-bath. Step 1. Extraction of Enzyme. The seedlings are homogenized in a Waring blendor in 0.1 M buffer 7 (1 ml of buffer is used per 2 g material). The homogenate is squeezed through cheesecloth to remove large cell debris. Step 2. First Ammonium Sulfate Fractionation. Solid ammonium sulfate is added to 35% saturation s (21.0 g/100 ml solution). The resulting precipitate and remaining cell debris are removed by centrifugation at 14,000 g for 20 minutes. T h e ammonium sulfate concentration is raised to 52% saturation (11.9 g/100 ml solution), and the precipitated protein is collected by centrifugation at 14,000 g for 20 minutes. The precipitate, redissolved in a minimal volume of 0.1 M buffer, is dialyzed for 4 hours against 12 volumes of the same buffer. Step 3. Second Ammonium Sulfate Fractionation. The enzyme solution is refractionated, a 95% saturated solution of ammonium sulfate being used. 9 Of this solution, 43 ml is added to each 100 ml of dialyzed protein solution, to 28% saturation. The resulting precipitate is removed by centrifugation as above. The supernatant is raised to 44% saturation by addition of 46 ml of 95% saturated ammonium sulfate per 100 ml of dialyzed protein solution. The precipitate is collected by centrifugation and redissolved in a minimal volume of 0.05 M buffer. The solution is dialyzed overnight against 15 volumes of 0.005 M buffer to remove most of the salt. It is adjusted to pH 6.3 by dialyzing for a further 3 hours against 25 volumes of 0,005 M buffer, pH 6.3. Step 4. Adsorption on Calcium Phosphate Gel. To the enzyme solution is added one-fifth of its volume of calcium phosphate gel. t° After 5 minutes the gel is centrifuged down at 800 g for 5 minutes. The supernatant solution is assayed for enzyme activity. The procedure is repeated until all enzyme activity has been adsorbed onto the gel. The gel portions which have adsorbed most of the activity are washed with ¢"Buffer" refers (unless otherwise stated), to sodium phosphate buffer, pH 7.4, conraining 0.001 M cysteine to protect the enzyme from oxidation. SAmmonium sulfate saturations are calculated according to the nomogram of M. Dixon, Biochem.J. 54, 457 (I953). ~Ninety-five percent saturated a m m o n i u m sulfate solution is prepared by dissolving 700 g of a m m o n i u m sulfate in 1 liter of 0.1 M buffer, and readjusting to pH 7.4 with a m m o n i u m hydroxide. l°D. Keilin and E. F. Hartree, quoted in Vol. I [11]. T h e gel was allowed to age for at least 3 weeks before being used. It was used as a thick suspension.

[43c]

SHIKIMATE DEHYDROGENASE

357

cold water and eluted with several portions of 0.05 M buffer, p H 7.4. Step 5. Concentration of the Enzyme. The eluate is concentrated by overnight dialysis against a 95% saturated solution of ammonium sulfate? The resulting precipitate is collected by centrifugation at 20,000 g for 20 minutes and redissolved in a minimal volume of 0.05 M buffer. At this stage the enzyme is stable for at least 6 weeks on storage at - 1 5 °. Several batches of enzyme can be prepared to this stage and combined for subsequent steps. Step 6. DEAE-Ceilulose Fractionation. The combined batches are dialyzed for 4 hours against 25 volumes of 0.005 M buffer, pH 6.3. DEAE-cellulose, washed as described by Peterson and Sober, 1~ is equilibrated with the same buffer and poured into a column. The enzyme is adsorbed onto the DEAE-cellulose. The column is washed with 0.005 M buffer, pH 7.4, to remove unadsorbed protein, and the enzyme is eluted with 0.05 M buffer, p H 7.4. Step 7. Concentration of the Enzyme. The enzyme is concentrated by dialysis against 95% saturated ammonium s u l f a t e , 9 the precipitate is redissolved in 0.05 M buffer and stOred a t - 1 5 ° until required. Properties Stability. 6 The presence of cysteine in the buffers protects the enzyme from inactivation. However, only after the calcium phosphate gel step is the enzyme sufficiently stable to be kept for weeks with little activity loss. The purest preparation of enzyme obtained was stable for at least 3 months? TM Specificity. The enzyme cannot utilize NAD as electron acceptor, nor can any of a large number of structural analogs of shikimate tested be utilized as substrates. 3 The enzyme from Escherichia coli similarly shows high specificity for NADP and shikimate. 2 Michaelis Constants. The limiting Michaelis constants for shikimate and NADP at pH 9.1 are 1.0 × 10 -4 M and 2.2 × 10-6 M , respectively. The constants have also been determined at other pH values. 6 In sodium phosphate buffer at pH 7.4 the Michaelis constants for shikimate and NADP are 6.0 × 10-4 M and 1.0 × 10 -5 M, respectively; and for dehydroshikimate and NADPH, 3.4 × 10-4 M and 4.3 × 10-e M, respectively. TM Effect ofpH. A pH of 10 is the optimum pH for the reaction when saturating levels of both substrates a r e u s e d . 2"6 The limiting Michaelis

11E. A. Peterson and H. A. Sober, Vol. V [1]. HaDilution of the purified enzyme prior to assay in buffer containing 0.001 M dithiothreitol instead of cysteine markedly increased the stability.

358

[43c]

AROMATIC AMINO ACIDS

¢ 0

Z

0

a ..=

eZ

..~

~

[43c]

SHIKIMATE DEHYDROGENASE

359

constants are minimal between pH 8.5 and 9.5. 6'13 Equilibrium Constant. The equilibrium constant [shikimate][NADP+] Keq [H ÷] = [dehydroshikimate][NADPH]

is 27.7 at pH 7.0 and 5.7 at pH 7.9. 2 At pH 7.6 a value of 10.3 was determined. 12 Activators. There appears to be no metal requirement since 0.006-0.01 M EDTA has no effect on enzyme activity, 2"3 and there is no activation by Mg 2+ or MnZ+; 0.02 M NaCI increases enzyme activity 20% at pH 9.0, but does not affect the Michaelis constant for shikimate. 13 Inhibitors. p-Hydroxymercuribenzoate inhibits the enzyme; this inhibition is reversible by cysteine. 3 Some aromatic structural analogs of shikimate inhibit the enzyme competitively with respect to its affinity for shikimate. Ki values at pH 9.0 for p-hydroxybenzoate, protocatechuate, guaiacol, and vanillate are 1.35 mM, 0.86 mM, 1.91 mM, and 0.57 mM, respectively; n and for gallate and vanillin, they are 0.38 mM and 0.093 mM, respectively? 4 Ki values at other pH values have been determined? 3 Distribution. The enzyme occurs in bacteria, yeast, and higher plants, but not in animals. 2 Its specific activity is higher in actively growing seedlings (3-5 milliunits/mg protein) than in dormant tissues (0.3-0.6 milliunit/mg protein)J 5 and is very high in tea-shoot tips (0.15-0.27 unit/mg protein), which actively synthesize flavonols? n The specific activity doubles during the first 8 days of growth of pea seedlings. 15

12D. Balinsky and W. W. Cleland, unpublished results. laA. W. Dennis and D. Balinsky, S. Afr.J. Med. Sci. 32, 96 (1967). 14D. Balinsky and D. D. Davies, Biochem.J. 80, 296 (1961). ~SD.Balinsky and D. D. Davies,J. Exptl. Botany 13, 414 (1962). 16G. W. Sanderson, Biochem.J. 95,248 (1966).