[52] Cytidine deaminases (from Escherichia coli and human liver)

[52]

CYTIDINE DEAM1NASES

401

Sensitivity to p-mercuribenzoate. Cytidine deaminase from yeast is highly sensitive to p-mercuribenzoate. When added at 0.2 ~ concentration simultaneously with substrate, this agent causes a 50% inactivation, while the reaction rate is immediately reduced to zero at 2/aM pmercuribenzoate concentration. 1 Heat Sensitivity. When the enzyme preparation is held for 5 min at temperatures between 40--62 °, no reduction is observed in the reaction rate tested at 27 ° in the presence of 0.3 mM cytidine; heat treatment for 5 min at 50-60 °, however, completely abolishes CMP and substrate inhibition. 1

[52] C y t i d i n e D e a m i n a s e s (from Escherichia Human Liver)

coli and

B y D A V I D F . W E N T W O R T H a n d RICHARD W O L F E N D E N

Cytidine + H20 m uridine + NHz

The equilibrium for the hydrolytic deamination of cytidine, catalyzed by cytidine deaminase, lies far in the direction of hydrolysis, with Keq = [uridine][NHa]/[cytidine][HzO] = 78, expressed in terms of the molar concentration of uncharged reactants and products, with water activity taken as unity.1 A. Cytidine Deaminase from E. coil. 2 Cytidine deaminase, originally detected in extracts of E. coli by Wang et al., 3,4 has been purified extensively but not to homogeneity. Assay Method Principle. Enzyme activity can be determined by a direct spectrophotometric assay based on the loss of absorbance when cytidine is converted to uridine, at 282 nm where Ae = -3600 for a 1-cm light path at pH 7.5. When high levels of extraneous protein or the presence of certain nucleoside inhibitors cause the background absorbance to be too 1 R. z R. a T. 4 T.

M. Cohen and R. Wolfenden, J. Biol. Chem. 246, 7566 (1971). M. Cohen and R. Wolfenden, J. Biol. Chem. 246, 7561 (1971). P. Wang, H. Z. Sable, and J. O. Lamprn, J. Biol, Chem. 184, 17 (1950). P. Wang, this series, Vol. 2, p. 478.

METHODS

IN

ENZYMOLOGY, VOL. L1

Copyright ~) 1978 by Academic Press, lnc, All rights of reproduction in any form reserved. ISBN 0-12-181951-5

402

PYRIMIDINE METABOLIZING ENZYMES

[52]

high at 282 nm, the reaction may be followed at 290 nm where Ae for cytidine transformation is -2100 at pH 8.0. When using cytidine at concentrations much above Km, the reaction may be followed at 290 nm or at 282 nm by using cuvettes of shorter path length (0.1-0.5 cm). Reagents 0.1 M Tris'HC1, pH 7.5 0.00167 M cytidine Procedure. To 0.5 ml of buffer and 0.1 ml of cytidine, add that amount of water that will result in a final volume of 1 ml after the addition of enzyme. Start the reaction by adding enzyme and record the early linear portion of the reaction. Definition of Unit and Specific Activity. One unit of activity is the amount of enzyme required to deaminate 1/zmole of cytidine per minute in the above standard reaction mixture at 25 °. Specific activity is units per milligram of protein as determined by the method of Lowry et al. 5 Purification Procedure Step 1. Preparation of Crude Extract. Frozen E. coli B cells (mid-log phase, obtained from General Biochemicals, Inc.) are thawed overnight. The resulting paste (150 g) is mixed for 10 min in a Waring Blendor with Superbrite glass beads (450 g) (obtained from 3M Co.) in Tris'HC1 buffer (210 ml, 0.01 M, pH 7.4, containing 0.01 M magnesium acetate). The cell extract, after low-speed centrifugation, is centrifuged for 3 hr at 105,000 g in a Spinco model L ultracentrifuge. To the resulting supernatant solution is added, with stirring, 0.2 volume of streptomycin B sulfate (5% solution in water), over a period of 30 min. After 20 min of additional stirring, the supernatant fluid is removed by centrifugation, divided into small portions (10 ml), placed in a water bath at 60 ° for 1 min, and then rapidly cooled. The precipitate is removed by centrifugation and discarded. Step 2. Ammonium Sulfate Fractionation. To the supernatant solution is added, in a ratio of 31.5 g/100 ml, a solid mixture containing ammonium sulfate and potassium bicarbonate in a weight ratio of 99: 1, respectively. The mixture is added slowly over a period of 50 min, and the solution is stirred for an additional 30 rain. The resulting precipitate is removed by centrifugation and discarded, and the supernatant fluid is 50. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

[52]

CYTIDINE DEAMINASES

403

similarly treated with an additional quantity of solid ammonium sulfate mixture (17.5 g/100 ml). The resulting precipitate, containing most of the deaminase activity, is recovered by centrifugation, redissolved in a minimal volume of distilled water, and dialyzed overnight against a large volume (1000 ml/10 ml of enzyme solution) of potassium phosphate buffer (0.01 M, pH 8.8). Step 3. DEAE Column Chromatography. After dialysis, the enzyme solution is applied to a column of DEAE-cellulose (bed volume 230 ml) equilibrated with potassium phosphate buffer (0.01 M, pH 8.8) containing 0.01 M KCI. Elution is performed with a linear gradient, the first reservoir containing 1200 ml of the buffer used for equilibration, and the second reservoir containing 1200 ml of potassium phosphate buffer (0.01 M, pH 7.5) containing 0.5 M KCI. Step 4. To the pooled fractions containing maximal activity (eluted in the neighborhood of 0.25 M KC1) is added the solid ammonium sulfate potassium bicarbonate mixture described above (49 g of mixture per 100 ml of enzyme solution). The resulting precipitate is dissolved in a minimal volume of distilled water, dialyzed exhaustively against Tris.HC1 buffer (0.01 M, pH 7.5), and applied to a column of carboxymethylcellulose (75-ml bed volume) equilibrated with the same buffer. Deaminase activity is not retained appreciably on this column, which removes some inactive protein. The eluted enzyme solution is applied to a column of hydroxylapatite (100-ml bed volume) and is eluted with a linear gradient, the first reservoir containing Tris.HCl buffer (0.05 M, pH 7.5), and the second reservoir containing Tris.HC1 buffer (0.5 M, pH 7.5). Active fractions are pooled, dialyzed against potassium phosphate buffer (0.01 M, pH 8.8), and concentrated on a column of DEAEcellulose (5-ml bed volume) equilibrated with the same buffer. The enzyme, in its final state of purification, is eluted with potassium phosphate buffer (I M, pH 6.5). Overall purification, summarized in Table I, is approximately 175-fold. SUMMARY OF PURIFICATION OF CYTIDINE DEAM1NASE FROM E. coli

Total units Extract 75% (NH4)zSO4 preparation DEAE-cellulose, peak concentration Hydroxylapatite, peak concentration

209 142 57 6.7

Specific activity (units/mg)

Recovery (%)

0.051 0.12 0.69 8.9

(100) 68 27 3.2

404

PYRIMIDINE METABOLIZING ENZYMES

[52]

Properties

Stability. Cytidine deaminase, purified as described, is reasonably stable, retaining 55% of its activity after 3 weeks at 4 °. Specificity. The enzyme is specific for cytosine nucleoside derivatives. At pH 7.5 cytosine deoxyriboside a (Kin = 8.9 x 10-5 M) is converted 3-4 times more rapidly than cytidine (Kin = 2.1 x 10-4 M), whereas 5,6-dihydrocytidine 6 (Km = 1.1 x 10-4 M) is transformed 0.1 as rapidly as cytidine. 4-N-Methylcytidine is a poor substrate with Kin: Vmax at least three orders of magnitude higher than that of cytidine. No significant change in activity is encountered when uracil, adenine, hypoxanthine, guanine, or their respective nucleosides and 5'-nucleotides are included in the standard assay at concentrations equivalent to that of cytidine (2 × 10-4 M). The enzyme can add methylamine to uridine to form 4-N-methylcytidine, whereas dimethylamine, trimethylamine, tris(hydroxymethyl)aminomethane, hydroxylamine, or sodium sulfide do not serve as cosubstrates in the backward reaction. 1 Metal Involvement. No loss of activity is encountered when the enzyme is exhaustively dialyzed against EDTA (10 -2 M) nor does EDTA at this concentration affect activity when included in the assay. Borohydride Reduction. The enzyme is not inactivated by incubation with sodium borohydride (10 -3 M) for short periods in the presence or absence of substrate. Approximate Molecular Weight. The elution behavior of the enzyme on a calibrated column of Sephadex G-100, determined according to the procedure of Siegel and Monty, 7 indicates an apparent Stokes' radius of the enzyme of approximately 40 .~. Sucrose gradient centrifugation of the enzyme, according to the procedure of Martin and Ames, s yields a sedimentation coefficient of 4.4 S. Together, these results suggest an approximate molecular weight of 73,000, assuming a partial specific volume of 0.725 cm3/g. Effects ofpH and D20. There is little variation in Vmaxand Km in the range from pH 6.5-10.7. Below pH 6.5 there is a gradual reduction in Vmax. An irreversible loss of activity occurs at pH values below 4. The substitution of deuterium oxide (90 atom-% excess) for water as solvent at pH 7.1, 7.5, and 8.4 results in a slight increase (-10%) in Vm~xand no significant change in g m . The complete hydrolysis of cytidine in D20 in 6 B. E. Evans, G. N. Mitchell, and R. Wolfenden, Biochemistry 14, 621 (1975). r L. M. Siegel and K. J. Monty, Biochim. Biophys. Acta 112, 346 (1966). s R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961).

[52]

CYTIDINE DEAMINASES

405

the presence of cytidine deaminase leads to no isotope incorporation at position 5, as indicated by the nuclear magnetic resonance spectrum of the product uridine.

Inhibitors. The product uridine and its reduced derivatives, 5,6dihydrouridine and 3,4,5,6-tetrahydrouridine, serve as competitive inhibitors of cytidine deamination under the usual assay conditions. Tetrahydrouridine (Ki = 2.4 x 10-7 M) is more than four orders of magnitude more effective as an inhibitor than uridine (Ki = 2.5 × 10-3 M) or dihydrouridine (Ki = 3.4 × 10-3 M). Inhibition by tetrahydrouridine is instantaneous, purely competitive, and fully reversed by dilution within the time (approximately 5 sec) required to initiate the standard assay. The unusual affinity of cytidine deaminase for tetrahydrouridine may be due to its resemblance to a transition-state intermediate in direct water attack on cytidine. The following compounds, at a concentration (3 × 10-4 M) in excess of the K m value of the substrate, do not show significant inhibition in the standard assay with 1.67 × 10-4 M cytidine: 3-N-methylcytidine, 4-O-methyluridine, 4-thiouridine, 4-N,N-dimethylcytidine, and 1-(fl-D-ribofuranosyl)-4-sulfonyl-2-pyrimidone. The product ammonia shows no significant inhibition at quite high concentrations, an indication that its Ki value is well in excess of 1 M. Inhibition is not observed in ammonia-ammonium chloride buffers at concentrations as high as 1 M at pH 9.2, at which ammonia and its conjugate acid are present in approximately equal concentration. B. Cytidine

Deaminase

from Human

Liver 9

Camiener and Smith TM examined cytidine deaminase activity in various human tissues (liver, kidney, heart, and muscle) and in liver from various species (man, monkey, rabbit, rat, dog, guinea pig, mouse, frog, pigeon, cat, and pig) and found the highest activity in human liver. Assay Method

Principle. The deamination of cytidine can be followed by measuring the decrease in A290n m where the change in extinction coefficient corresponding to complete conversion to uridine is -2100 for a 1-cm light path at pH 8.0. Because the Km for 6-azacytidine (A¢ = -800 at 305 nm, pH 8.0) is high, observation of the reaction is facilitated by use of cuvettes of shorter path length (0.5 cm). The deamination of 5-azacyti9 D. F. Wentworth and R. Wolfenden, Biochemistry 14, 5099 (1975). lo G. W. Camiener and C. G. Smith, Biochem. Pharmacol. 14, 1405 (1965).

406

PYRIMIDINE METABOLIZING ENZYMES

[52]

dine (he = -1300 at pH 8.0) can be followed at 270 nm, but in the presence of Tris.HC1 or ethanolamine buffer a nonenzymic reaction takes place which requires correction. 8

Reagents 0.002 M cytidine 0.02 M Tris.HCl, pH 8.0

Procedure. To 0.5 ml of buffer and 0.1 ml of cytidine, add that volume of water that will give a final volume of 1 ml after addition of enzyme. Start the reaction by adding enzyme. Definition of Unit. One unit of activity is that amount of enzyme required to deaminate 1 /.~mole of cytidine per minute at 25 ° in the standard assay above. Purification Procedure

Step 1. Preparation of Crude Extract. The enzyme can be prepared from frozen tissue which has been removed at autopsy. A chilled suspension of tissue (100 g) in Tris.HC1 buffer (300 ml, 0.013 M, pH 8.0) was homogenized for 2 rain in a Waring Blendor. The homogenate was rapidly frozen and thawed, and then cleared of cell debris by centrifugation at 0 ° for 20 min at 20,000 g in a Sorvall centrifuge. Step 2. Heat Treatment. In view of the observed heat stability of this enzyme, 11 the supernatant fluid was incubated in 10-ml portions at 75 ° for 10 rain, rapidly cooled, and then cleared by centrifugation at 0 ° for 60 rain at 20,000 g. This heat-treatment procedure was repeated once. Step 3. Ammonium Sulfate Fractionation. The supernatant fluid was then adjusted to 40% saturation by addition of solid ammonium sulfate (24 g/100 ml of supernatant fluid) with stirring at room temperature, and stirring was continued for 30 rain. After removal of the precipitate, the active supernatant fluid was adjusted to 70% saturation by further addition of ammonium sulfate (22.9 g/100 ml of supernatant fluid). The resulting precipitate, recovered by centrifugation, was dissolved in 5 ml of Tris.HCl buffer (0.01 M, pH 8.0) and dialyzed 3 times against 250 ml of the same buffer. This procedure was found to result in a 21-fold increase in the specific activity of the enzyme as compared with the crude extract so that the stock enzyme solution contained 0.2 unit of activity and 8 mg of protein/ml as determined by the procedure of Lowry et al. ~ n G. W. Camiener, Biochem. Pharmacol. 16, 1681 (1967).

[52]

CYTIDINE DEAMINASES

407

Properties

Stability. The partially purified e n z y m e may lose some activity upon storage, but activity may be restored by the addition of 2.5 mM dithiothreitol. Effect of pH.

The e n z y m e exhibits identical rates of deamination of

1 0 - 4 M cytidine at pH 5, 8, and 10.

Specificity. The e n z y m e is specific for cytidine derivatives and can deaminate several antineoplastic agents. At p H 8.0, 5-azacytidine (Kin = 5.8 × 10-5 M)is deaminated 0.17 as rapidly as cytidine (Kin -- 9.2 x 10-6 M), whereas 6-azacytidine (Kin -- 4.2 x 10-3 M) is converted 6.4 times more rapidly than cytidine. The e n z y m e can also deaminate cytosine arabinoside 11 (Km = 1.4 x l0 -4 M). Cyclocytidine (2,2'-anhydro-l-l]-Darabinofuranosylcytosine) is not a substrate. Camiener 12 has tested a number of cytidine analogs at 1 mM levels, pH 8.0, 37 °. Thus certain substituents at the 5-position are tolerated by the e n z y m e as the order of deamination rates is 5-chloro > 5-bromo > 5-H > 5-iodo > 5-methyl, whereas 3-methylcytidine is not a substrate. Cytosine 2'-deoxyriboside is a substrate, but epimerization at the 3'-position of cytosine riboside or cytosine arabinoside results in the loss o f substrate activity. Cytosine is also not a substrate. Time-dependent Inhibition. In contrast to the rapid inhibition of bacterial cytidine deaminase by 3,4,5,6-tetrahydrouridine, the onset of inhibition of the e n z y m e from human liver is relatively slow and can be observed with an ordinary recording spectrophotometer. Inhibition is reversible, and the ratio (k°n/k°n) of rate constants for binding (k°n = 2.4 X l04 M -1 Sec -1) and release (k°fr = 5.6 × 10-4 sec -1) is in reasonable agreement with a Ki value (2.9 x 10-8 M) measured separately under steady-state conditions. The e n z y m e from H e L a cells responds similarly to tetrahydrouridine with k°ff = 1.1 x 10a sec -~ and k°~ = 2.3 × 104 M -~ s e c - k Final steady-state rates indicate Ki = 4.0 x 10-8 M in accordance with the ratio (k°ff/k°"). Effect of D20. Substitution of deuterium oxide for water leads to no significant change in the rate o f deamination of 10-4 M cytidine or 10 -3 M 6-azacytidine at p H = pD = 8.0, and no isotope effect is observed on the rate of binding or release of tetrahydrouridine.

~2G. W. Camiener, Biochem. Pharmacol. 16, 1691 (1967).