The purification and properties of l -rhamnulokinase

BIOCHIMICA ET BIOPHYSICAACTA

489

BI3A 65111 THE

PURIFICATION

AND PROPERTIES

OF L-RHAMNULOKINASE

T. H. CHIU AND DAVID SIDNEY FEINGOLD Microbiology Section, Department of Biology, University of Pittsburgh, Pittsburgh, Pa. (U.S.A.)

(Received June 26th, 1964)

SUMMARY L-Rhamnulokinase (ATP:L-rhamnulose i-phosphotransferase, EC 2.7.1.5) has been purified 34-fold from extracts of Escherichia coli K4o. The purified enzym~ catalyzes the phosphorylation of L-rhamnulose, but not of L-rhamnose, in the presence of Mg2+ and adenosine 5'-triphosphate. Uridine triphosphate, cytidine 5'-triphosphate, guanosine 5'-triphosphate, and thymidine triphosphate also can act as phcsphoryl donors, but less well than adenosine 5'-triphosphate. The product of the phosphorylation of L-rhamnulose by the enzyme has been isolated and characterized by periodic acid oxidation studies as L-rhamnulose I-phosphate. INTRODUCTION Many different organisms can utilize L-rhamnose as sole source of carbon and energy1. L-Rhamnose metabolism by Escherichia coli has been studied with crude extracts *,3 and with partially purified enzymes 4. Oil the basis of these studies it has been proposed that the initial steps of L-rhamnose metabolism in E. coli are as follows : L-rhamnose--> L-rhamnulose-~L-rhamnulose 1-phosphate. However, L-rhamnulokinase free from contaminating isomerase activity has not been reported, nor has the structure of the rhamnulose phosphate formed been determined. In this communication we describe the partial purification of L-rhamnulokinase (ATP:L-rhamnulose I-phosphotransferase, EC 2.7.1.5) from E. coli K4o and the characterization of the reaction product as L-rhamnulose 1-phosphate. MATERIALS AND METHODS Phosphoenolpyruvic acid, NADH v D-fructose 1-phosphate, calcium phosphate gel, alumina gel, ATP, GTP, UTP, CTP, TTP, and lactate dehydrogenase (containing pyruvate kinase) were obtained from Sigma Chemical Company. L-Rhamnose was obtained from the Pfanstiehl Laboratories, Inc. It was epimerized to L-rhamnulose with pyridine 5 and the L-rhamnulose was isolated from the pyridine-free reaction mixture by chromatography on a column of powdered cellulose using a mixture of Biochim. Biophys. Acta, 92 (1964) 489-497

49 °

T. H. CHIU, D. S. FEINGOLD

butanone-acetic acid-H20 (75:25:1o, v/v) as developing solvent (Solvent I). The isolated L-rhamnulose was homogeneous when examined by paper chromatography in Solvent I as well as in the following solvent systems : (2) n-propanol-conc. N H 4 O H H20 (60:30 :IO, v/v); (3) u-butanol-acetic acid-H20 (52:13:35, v/v); (4) 80°/) aqueous phenol. Paper electrophoresis was carried out in o.I M ammonium acetate (pH 5.8) ~. The following spray reagents were used to detect compounds on paper electrophoretograms and chromatograms: ammoniacal AgNO.~ (ref. 7) for free sugars and L-rhamnulose phosphate, inolybdic acid s for Pi and organic phosphate, urea phosphate 9 for L-rhamnulose phosphate (deep purple spot), aniline-xylose 1° for glycolic acid and phosphoglyeolic acid. Authentic pho~phoglycolic acid was a generous gift from I)r. C. E. BALLOU University of California, Berkeley. In addition, phosphoglycolic acid was prepared from D-fructose 1-phosphate. To b-fructose 1-phosphate (34 #moles) were added 2 ml o f o . I M HIOa and 0.5 ml of 7. 5 N H2SO 4, and the reaction mixture was kept at 25 ° for I h in the dark. Excess H I O 4 was destroyed b y addition of 1.2 ml of 2 M N a A s Q and 3 ml of i N NaOH. The mixture was then applied to a column (15 cm ;~ I cm 2) containing Dowex-i X - i o (C1- form, 200-400 mesh) and washed with IOO ml of I-I90. The column was eluted with an increasing gradient of HC1, obtained with 500 ml of 0.05 N HCI in a constant-volume mixing vessel and I 1 of 0.4 N HC1 in the reservoir. Fractions (3.3 ml) were collected; an organic phosphate emerged in Fractions 18-27. This compound had a paper-electrophoretic mobility at p H 5.8 slightly greater than that of glycolic acid; it gave a positive reaction for organic phosphate with molybdic acid spray. Hydrolysis with acid phosphatase yielded electrophoret i c a l l y - d e m o n s t r a b l e P l and a compound identical in mobility to glycolic acid. Analysis of the hydrolysate for P i and for glycolic acid (chromotropic acid method) gave a ratio of o~95, demonstrating that the compound obtained from the periodate oxidation of D-fructose 1-phosphate was phosphoglycolic acid. Acid phosphatase (EC 3.1.3.2) was purified from seminal fluid 11. Reducing sugar was determined by the method of PARK AND JOHNSON12; L-rhamnulose b y the cysteine-carbazole method of DISCHE AND BORENFREUND 13, with 36.2 Klett units -o.oi /~mole14; glycolic acid by the chromotropic acid method 1~. Protein was determined by the method of WADDEL16 and P i by the technique of ROCKSTEIN AND HERRON17. Light absorption was measured with a Beckman DU spectrophotometer equipped with a device to keep the temperature at 37 °.

Enzyme assays L-Rhamnulokinase activity was measured by determining the rate of ADP formation from ATP at 37 ° in the presence of L-rhamnulose by either a one-step or two-step method as follows. (I) One-step method : The rate of release of ADP during the phosphorylation of L-rhamnulose was measured with phosphoenolpyruvate kinase and lactate dehydrogenase is. The reaction mixture (in a I-ml cuvette) contained the following (in btmoles) : L-rhamnulose, 2.0; N A D H 2, 0.2; GSH, o.67; EDTA, o.033; MgC12, 3.3; phosphoenolpyruvic acid, 1.7; Tris-HC1 buffer (pH 8.5), 30; ATP, 0.67; and 0.08 mg lactate Biochim. Biophys. ,4cta, 92 (1964) 489-497

L-RHAMNULOKINASEOF E. coli

491

dehydrogenase in a total volume of I ml. L Rhamnulokinase was added to start the reaction and the rate of dehydrogenation of N A D H 2 was ascertained b y following the decrease of absorbancy at 34 ° m/, for 3 rain. A control was run without sugar. A unit of activity is defined as the amount of enzyme required to produce I #mole of A D P per min, and specific activity as the numbe~ of units per mg protein. (2) Two-step method: The reaction mixture (0. 5 ml) contained the following (in #moles): t-rhamnulose, 2.0; ATP, 5.0; MgC1,2, 5.0; GSH, 5.0; KF, 5.0; Tris-HC1 buffer (pH 8.5), 5.0; and L-rhamnulokinase. After incubation at 37 ° for IO rain, the mixture was inactivated in a boiling water bath for I rain,cooled to room temperature, and centrifuged. ADP in the supernatant fluid was assayed by adding an aliquot (0.05 ml) to a reaction mixture containing the following (in/,moles) : NADH2, 0.2 ; potassium phosphoenolpyruvate, 0.5; sodium and potassium phosphate buffer (pH 7.o), IO; MgC12, 5; lactate dehydrogenase (containing pyruvate kinase), o.I rag; and H~O to make a total vol. of I.O ml. The decrease in absorbancy at 340 m# was read alter it had reached a constant value. ATPase activity was determined in incubation mixtures without L-rhamnulose. Enzyme activity is defined above. RESULTS AND DISCUSSION

Purification of z-rhamnulokinase Growth of cells: E. coli K4o was grown in the following medium (g/l) : K~HP04, 7.0; KH2PO,, 3.0; (NH4)2SQ, I.O,; MgSO4.7H20 , o.I; I,-rhamnose, 2.0. The sugar was sterilized b y Seitz filtration and added aseptically to the sterile salts solution. 2-1 flasks containing i 1 of medium were inoculated with IOO ml of starter culture with a Klett reading of approx. 200 (Filter No. 42). After 12-16 h incubation on a rotary shaker at 37 ° , the cultures were in the stationary phase of growth. The cells were then harvested and washed twice with ice-cold 0.85 % NaCI solution. Approx. 2 g of packed cells per 1 culture medium were obtained. Preparation of cell-free extracts: Cell-free extracts were prepared by disrupting the cells in a Raytheon Io-kcycles sonic oscillator cooled by circulation of ice-water. Io-g batches (wet wt.) of cells were suspended in 25 ml of cold 0.02 M sodium and potassium phosphate buffer (pH 7.o), and sonicated for 15 rain. The suspension was centrifuged in the cold at 30 ooo × g for 20 min and the pellet was discarded. The supernatant fluid released ADP from A T P when either L-rhamnose or L-rhamnulose was used as substrate, indicating the presence of L-rhamnose isomerase as well as L-rhamnulokinase. These enzymes were inducible, since they were not present in the crude enzyme solution obtained from cells grown in a medium in which the L-rhamnose was replaced b y D-glucose. MnCl 2 treatment: (This and all subsequent operations were conducted at o-4°.) The supernatant fluid from the previous step was diluted with 0.02 M phosphate buffer (pH 7.o), to give a protein concentration of 30 mg/ml, and o.I volume of 0.5 M MnCI 2 was added. The mixture was allowed to stand for 30 min and then centrifuged at 30 ooo × g for io rain. The residue was washed with a small amount of 0.02 M phosphate buffer and the washings were combined with the supernatant liquid. (NH4)2SO 4fraetionation I: To the supernatant solution (9.2 mg protein per ml) saturated (NH4)2SO 4 solution (pH 7.0) was added to 33% saturation. (In all (NH4)2SO * Biochim. Biophys. Acta, 92 (I964) 489-497

492

T. H. CHIU, D. S. FEINGOLD

precipitations 5 rain were allowed to elapse between addition of (NH4)2SO 4 and centrifugation.) The precipitate was discarded and the supernatant solution was brought to 55 % saturation by addition of a saturated solution of (NHa)aSO 4 (pH 7.o). The precipitate, which contained most of the L-rhamnulokinase activity, was dissolved in a volume of o.oi M phosphate buffer (pH 7.o), o.oi M in respect to EDTA, equal to the volume of the crude enzyme solution. At this point the protein concentration was approx. 4 mg/ml. Calcium phosphate gel treatment: Calcium phosphate gel (12 mg dly wt. per mg protein) was added to the enzyme solution. The suspension was stirred occasionally for 15 min and the gel was then removed by centrifugation. (NH4)2SO 4 fractionation II: The above supernatant solution was flactionated as previously between 35 % and 5 ° % saturation with (NH4)2SO a. The precipitate was dissolved in sufficient o.o2 M phosphate buffer (pH 7), o.oi M in respect to EDTA, to make the protein concentration approx. 1.5 mg/ml. Alumina gel treatment: Alumina gel (7 mg dry wt. per mg protein) ~as added'to the above solution. After 5 min the gel was spun down and discarded. The supernatant was treated with a second portion of gel to adsorb the enzyme (9 mg dry wt. per mg protein). The mixture was stirred occasionally for 15 min and the gel was then spun down and washed once with a volume of cold water equal to one-half the w~lume of the supernatant fluid in the adsorption step. The enzyme was eluted from the gel with o.i M sodium and potassium phosphate buffer (pH 7.6). Usually two elutions, each with one-half the w)lume of the supernatant fluid in the adsorption step, sufficed to elute the bulk of the enzyme. TABI.E [ SUMMARY OF PURIFICATION OF L-RHAMNULOKINASF

Fraction

Volume (ml)

Protein (mg/ml)

Specific activity* (units)

Total units

Purification (-fold)

°i, recovery

Cru de e x t r a c t

15.o

30.00

0.20

90.0

1.o

ioo

MnCI~

I6.5

9.2o

0.54

82.0

2. 7

91

(NH4)2SO a f r a c t i o n a t i o n I Ca3(PO4) 2 gel (NH4)2SO 4 f r a c t i o n a t i o n 11 A1203 Gel

i5.o 15.2 5.o 2.5

3.90 i .28 J-44 o.63

0.92 1.96 4-o5 6.83

54.0 38.o 29.o io.8

4.6 9.8 2o.3 34.2

60 42 32 12

* # m o l e s of A D P released a t 37 ° per m i n per m g p r o t e i n (see t e x t ) .

As can be seen from Table I, an overall purification of M-fold was achieved, with a recovery of about 12%. The purified L-rhamnulokinase was free of ATPase, NADH2-oxidase, and L-rhamnulose isomerase. L-Rhamnose was not phosphorylated b y the enzyme.

Properties of L-rhamnulokinase Optimum pH: The optimum p H for the reaction was determined by incubating the reaction mixture with o.I M sodimn and potassium phosphate buffer between p H 6 and 7, with o.I M Tris-HC1 buffer between p H 7.6 and 9, and with o.I M Biochim. Biophys. Acla, 92 (i964) 489-497

L-RHAMNULOKINASE OF

~

4.C

&.

,9o3.0 a. .,.,

/L

E. coli

493

5.0 oJ 4 . 0

~3.0

'~2.0

@

•

0

~ 2.0

~1.0

1.0 pH

5 10 15 20 MicrogrQms of protein

Fig. i. The effect of p H on the activity of L-rhamnulokinase.

25

Fig. 2. Reaction rate as a function of L-rhamnulokinase concentration.

glycine-NaOH buffer between pH 9 and io. As can beseen in Fig. i, L-rhamnulokinase has a rather sharp optimum at pH 8.5. Effect of enzyme concentration: A linear relation was obtained between activity and amount of enzyme (Fig. 2). Effect of substrate concentration: The effect of ATP and L-rhamnulose concentration respectively on reaction rate is shown in Fig. 3 and 4- The Km values at 37 °, determined by the method of LINEWEAVERAND BURK19, are 8.2.10 -5 M for Lrhamnulose and I . i . 1 0 -4 M for ATP.

0.5

~5.o 0.6

2

0.4 '

J_ V

0.4

2~ 14, 6J 8, 10 ' '12 ES]M x 10"3 M J

0.3

:vI 0.3 0.2 0.~ 0.1 0.1

8

12 16 ! x 1 0 -3 Is]

20

214

28

Fig. 3. The effect of L-rhamnulose concentration on the activity of L-rhamnulokinase.

1-- x 10 - 3

F.s.]

Fig. 4- The effect of A T P concentration on the activity of L-rhamnulokinase.

Biochim. Biophys. Ac/a, 92 (1964) 489-497

494

T . H . CHIU, D. S. FEINGOLD

Activation by metal ions: The kinase required certain divalent metal ions for m a x i m u m activity, Km for Mg 2+ at 37 ° being 2. 7 • lO -4 M (Fig. 5)- Purified preparations of the enzyme were stimulated 6-fold b y 2.5" lO -3 M Mg 2+. The relative effect of the same concentration of other metal ions was as follows: Mg 2+, I.O; Mn 2+, I.O; Co 2÷, 0.90; Fe 2+, o.85; Ca 2+, 0.77; Cu ~+, o. Nucleoside triphosphate requirement: The kinase could use other nucleotides t h a n A T P as phosphoryl donor. The ratio of activity with various nucleotides was :ATP,I ; UTP, 0.9; CTP, 0.7; GTP, 0.25; TTP, o.io.

12

0.18 0.16 0.14

1 V

b

j

; A & ~ ~ If IS .M ]×10-3i • 10

0.12 0.10 0.08 0.06 0,04 0.02 I

I

[~x10-3

Fig. 5. The effect of Mg 2÷ concentration on the activity of L-rhamnulokinase.

Substrate specificity: Purified L-rhamnulokinase could catalyze the phosphorylation of a n u m b e r of ketoses other t h a n L-rhamnulose, with relative activities as follows (many of the compounds tested were the generous gift of Dr. E. C. H E A T H , J o h n Hopkins University): L-rhamnulose, i.o; L-fuculose, 0.30; L-xylulose, O.II; D-xylulose, 0.02; L-ribulose o.oi; D-fructose o.oi; D-psicose, O; I)-tagatose, o; L-sorbose, o. No a t t e m p t was made to determine the structure of the phosphates formed from sugars other t h a n L-rhamnulose. Stoichiometry : I mole each of L-rhamnulose phosphate and A D P was formed for each mole of L-rhamnulose utilized in the reaction. Preparation and properties of L-rhamnulose i-phosphate A large-scale reaction was run with the following quantities of reactants (mmoles) : L-rhamnulose, 2.2 ; ATP, 3.0 ; MgC12, I ; Tris-HC1 (pH 8.5), 8.0 ; and 52.3 mg of e n z y m e protein in a total volume of 300 ml. After incubation at 37 ° for I h the reaction mix13iochim. Biophys..4cta, 9z (1964) 489 497

L-RHAMNULOKINASEOF E. coli

495

ture was chilled in ice; analysis of an aliquot indicated that all the L-rhamnulose had been phosphorylated. The reaction mixture was fractionated on a column of Dowex-i X-8 resin (formate form, 200-600 mesh) b y gradient elution with 2oo ml H20 in a constantvolume mixing chamber and I 1 of o. 4 N formic acid containing o.I M sodium formate in the reservoir. Fractions (15 ml each), containing ketose phosphate esters (Fractions 62-9 o) were pooled, concentrated at 37 ° to 6o ml and adjusted to p H 6. 4 with saturated Ba(OH)2 solution. Then 4 volumes of ethanol were added and the solution was left at o ° for I h. The precipitate was spun down and washed with cold 80% ethanol, washed with cold ether and dried in vacuum. Contaminating ultraviolet-absorbing material was removed b y treatment of an aqueous solution of the barium salts with charcoal (acid-washed Norit A) and subsequent reprecipitation of the barium salt as mentioned above. Analysis of the isolated phosphate ester for total phosphorus, reducing sugar, ketose, organic phosphate and Pl gave molar ratios of I . I :I.O:I.O :I. o :o.I. The phosphate ester was treated with seminal acid phosphatase and the saccharide released was examined by paper chromatography in Solvents 1- 4. A single reducing substance which corresponded in mobility to authentic L-rhamnulose was obtained in each case. The cystein-carbazole reaction-mixture of the hydrolyzate had an absorption spectrum identical to that obtained with authentic L-rhamnulose. These data indicate that the product is L-rhamnulose monophosphate. 100

8O O3 6C .~

.~ 4c

L.

20

n

Minutes Fig. 6. H y d r o l y s i s of L - r h a m n u l o s e 1 - p h o s p h a t e in i N HCI a t i o o ~.

The acid stability of the L-rhamnulose monophosphate was determined by hydrolysis in I N HC1 at IOO°. As shown in Fig. 6, approximately one-half the organic phosphate was liberated in 6. 5 min. Similar results have been obtained b y SAWADA4. The optical rotation of the barium salt of L-rhamnulose phosphate was determined; [a]~ + 8. 9 4- 0.03 ° in acetic acid, p H 4 (c, 5.2%). In order to confirm the position of the phosphate group, L-rhamnulose phosphate was subjected to periodate oxidation studies 2o. The reaction mixture contained 14.1 /~moles of L-rhamnulose phosphate, 300 jumoles of HIO4, and 6 mmoles of H2SO 4 in a total volume of IO ml. Control flasks contained no L-rhamnulose phosphate. The reaction was run in the dark at o °. Aliquots were removed at intervals and periodate consumption was determined b y titration .1. There was rapid initial periodate uptake, Biochim. Biophys. Acta, 92 (1964) 489-497

496

W.H. CHIU, D. S. FEINGOLD

44 #moles being consumed during the first hour. During the next 2 h, i. 5 additional /,moles were used. Total periodate consumption, 45-5 #moles, corresponded to 3.2 moles per mole of rhamnulose phosphate, in reasonable agreement with the theoretical value, 3 moles per mole of sugar monophosphate. These data show that the rhamnulose derivative is a sugar monophosphate but do not distinguish between the I- and 5-phosphates (Fig. 7). CH2OPO3 2-

I

C

CIt,.,()pOa 2-

I

O

COOH

---I---

HCOH - - - I- - HOCH ---

+ 3 HIO4 ......

2 HCOOH ~

_u

I---

ltOCIt

CHO

CH3

CH a

CH2OH

HCH() b CO2

C= O

--I--HCOH

HC()OH

3 HIO4 HOCH

I

2-OsPOCH

I

CH 3

4CH O

I

2-OsPOCH

1

CH 3

Fig. 7. Periodate cleavage of rhamnulose phosphate. Cleavage position expected is indicated b y dotted lines.

The two alternatives can be distinguished, however, by identification of the products of periodate oxidation. The 1-phosphate will yield (moles/mole) : 1 phosphoglycolic acid, 2 formic acid, and i acetaldehyde; the 5-phosphate will yield (moles/ mole): I phospholactaldehyde, i formic acid, I formaldehyde and I C O 2. Therefore the L-rhamnulose monophosphate (49 /,moles) was oxidized with periodic acid as described under MATERIALSAND METHODSfor the oxidation of D-fructose I-phosphate and the oxidation products were separated and characterized. Acetaldehyde in 6o% yield was isolated from the reaction mixture and characterized as the dimedon derivative ~2. Phosphoglycolic acid was isolated as described. It was sharply eluted in Fractions 22-27. The isolated compound had the same paper chromatographic (n-propanol-conc.NH4OH-H20 ;6o :3o :IO,V/V) and electrophoretie mobility as authentic phosphoglycolic acid. Treatment with seminal acid phosphatase yielded Pi and a substance with the electrophoretic mobility of glycolic acid. Analysis of the phosphatase hydrolyzate for glycolic acid and Pl gave 38.o /,moles and 41.3 #moles respectively, indicating a minimum of 8o% recovery. These results indicate that L-rhamnulose i-phosphate is the product of L-rhamnulokinase action. Biocl~im. Biopkys. Acla,

92 0 9 6 4 )

489-497

L-RHAMNULOKINASE OF E. coli

497

ACKNOWLEDGE MENT

This investigation was supported by a grant, No. G 23 428, from the National Science Foundation. REFERENCES 1 R . G. EAGON, J. Bacteriol., 82 (1961) 548. 2 G. TECCE AND M. DIGIROLAMO, Giorn. Microbiol., I (1956) 286, 8 D. M. WILSON AND S. J. AjL, J. Bacteriol., 73 (1957) 4 lo, 415 • 4 H. SAWADA, Kanazawa Daigahu Kekkaku Kenkiyusho Nempo, Japan, 2o (1962) 195. s p. A. LEVENE AND D. W. HILL, J. Biol. Chem., lO2 (1933) 563 • 6 D. S. FEINGOLD, E . F. ~TEUFELD AND W . Z. HASSID, in H. F. LINSKENS, B. D. SANWAL AND M. V. TRACEY, Modern Methods oJ Plant Analysis, Vol. 7, Springer, Berlin, 1964, p. 747. 7 S. M. PARTRIDGE, Biochem. J., 42 (1948) 238. s R. S. BANDURSKI AND B. AXELROD, J. Biol. Chem., 193 (1951) 405 . 0 C. S. WISE, R. J. DIMLER, H. A. DAVIS AND C. E. RIST, Anal. Chem., 27 (1955) 33. 10 j . SAARNIO, E. NISKASAARI AND C. GUSTAFSSON, Suomen Kemistilehti, B, 25 (1952) 25. 11 G. SCI~MIDT, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 2, Academic Press, New York, 1955, p. 523 • 12 j . T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1959) 149. 18 Z. DlSCHE AND E. BORENEREUND, J. Biol. Chem., 192 (1951) 583 • 1, N. J. PALLERONI AND M. DOUDOROFF, J. Biol. Chem., 218 (1956) 535. 15 V. P. CALKINS, Anal. Chem., 15 (1943) 762. 16 J. J. WADDEL, J. Lab. Clin, Med., 48 (1956) 311. 17 M. ROCKSTEIN AND P. W. HERRON, Anal. Chem., 23 (1951) 15oo. is A. KORNBERG AND W. E. PRICER, JR., J. Biol. Chem., 193 (1951) 481. 1~ H . LINEWEAVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658. 20 F. S. H. HEAD AND G. HUGHES, J. Chem. Soc., (1952) 2046. 21 E. C. HEATH AND M. A. GHALAMBOUR, J. Biol. Chem., 237 (1962) 2423. ~ A. I. VOGEL, Practical Organic Chemistry, Longmans, Green, London, 1948, p. 33 r.

Biochim. Biophys. Acta, 92 (I964) 489-497