Vol. 176, No. 17JOURNAL OF BACrERIOLOGY, Sept. 1994, p. 5505-55120021-9193/94/$04.00+ 0Copyright (C 1994, American Society for Microbiology

Ketohexokinase (ATP:D-Fructose 1-Phosphotransferase) from aHalophilic Archaebacterium, Haloarcula vallismortis:

Purification and PropertiesVIDHYA RANGASWAMYt AND WIJAYA ALTEKAR*

Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre,Trombay, Bombay-400 085, India

Received 4 March 1994/Accepted 19 June 1994

Ketohexokinase (ATP:D-fructose 1-phosphotransferase [EC 2.7.1.3]), detected for the first time in aprokaryote, i.e., the extreme halophile Haloarcula vaUlismortis, was isolated and characterized from the samearchaebacterium. This enzyme was characterized with respect to its molecular mass, amino acid composition,salt dependency, immunological cross-reactivity, and kinetic properties. Gel filtration and sucrose densitygradient centrifugation revealed a native molecular mass of 100 kDa for halobacterial ketohexokinase, whichis larger than its mammalian counterpart. The enzyme could be labeled by UV irradiation in the presence of[_y-32P]ATP, suggesting the involvement of a phosphoenzyme intermediate. Other catalytic features of theenzyme were similar to those of its mammalian counterparts. No antigenic cross-reactivity could be detectedbetween the H. vallismortis ketohexokinase and the ketohexokinases from different rat tissues.

Studies of fructose metabolism in halophilic archaea (la-3)from our laboratory revealed the operation of a modified EMPpathway for the utilization of fructose. The pathway differedfrom the conventional EMP route in employing fructose1-phosphate (F1P) as the intermediate between fructose andfructose 1,6-bisphosphate (FBP). The formation of FlP fromfructose was mediated by an ATP-dependent reaction (2). FlPis not an unusual intermediate in fructose metabolism. How-ever, its formation, which is to an ATP-mediated phosphory-lation of fructose in prokaryotes, is certainly unusual. Incontrast, in eubacterial fructose metabolism, the formation ofFlP from fructose is mediated by the phosphoenolpyruvate(PEP)-dependent fructose phosphotransferase system. Thusdemonstration of such a reaction in the halobacterial strainsHaloarcula vallismortis and Haloferax mediterranei was indeed asalient feature. PEP could not replace ATP in this reaction.This ATP-dependent phosphorylation of fructose to FlP inhalobacteria is attributed to the enzyme ketohexokinase (EC2.7.1.3) (2), which has thus far been reported only for mam-malian systems and has never been reported for a prokaryote(10, 12). Therefore, the presence of this activity in halobacteriacame as a surprise. On the one hand, the halophilic archaeathat are thought to have originated early in evolution lack thePEP-F-PTS, which is considered the primordial ancestor of allPEP-PTS systems (30). On the other hand, they possessketohexokinase, an enzyme that is so far found only in highlyevolved tissues, e.g., mammalian liver. This finding adds to thelist of similarities with eukaryotes that are encountered inmembers of the archaeal group.The study of ketohexokinase from halophilic archaea, the

third domain of life, and comparison with its eukaryoticcounterpart will therefore be an important study to undertakefor understanding evolutionary relationships. This paper de-scribes the purification and characterization of ketohexokinasefrom H. vallismortis.

* Corresponding author. Phone: 91-22-5563060, ext. 2252. Fax:91-22-5560750.

t Present address: Department of Microbiology, 203 Morrill ScienceCenter IV-N, University of Massachusetts, Amherst, MA 01003-5720.

MATERLILS AND METHODS

Materials. Fructose, ATP, reduced glutathione (GSH), rab-bit muscle aldolase, iodoacetic acid, 5,5'-dithiobis-(2-nitroben-zoic acid) (DTNB), N-ethylmaleimide, phenylmethylsulfonylfluoride (PMSF), DNase, acrylamide, N,N,N',N'-tetramethyl-ethylenediamine, bisacrylamide, sodium dodecyl sulfate (SDS),DEAE-cellulose, carboxymethyl cellulose (CM-cellulose), stan-dard protein markers, 1,4-bis(5-phenyloxazolyl)benzene (POPOP)and 2,5-diphenyloxazole (POP) were from Sigma Chemical Co.Sepharose 4B, Sepharose CL-6B, phenyl-Sepharose, and cali-brated G-25 columns or PD-10 columns (5 by 1.5 cm) wereproducts of Pharmacia, Uppsala, Sweden. Hydroxylapatite andammonium persulfate were from Sisco Research Laboratories,Bombay, India. [Tr-32P]ATP (specific activity, 3,000 Ci/mmol)was obtained from Isotope Division, BARC, Bombay, India.All salts used were analytical grade.Organism and growth conditions. H. vallismortis (ATCC

34679) was grown in a synthetic medium, as described byRodriguez-Valera et al. (29), containing 20% NaCl, 0.1%CaCl2, 0.4% KCl, 0.6% Tris, 3.6% MgSO4 * 7H20, and 0.5%fructose. To this were added the following filter-sterilizedsolutions: 2 ml of FeSO4 - 7H20 (0.25% in 0.001 N HCl), 2 mlof K2HPO4 (5% in distilled water) and 5 ml of NH4Cl (20% indistilled water) per liter. The bacteria were grown as shakencultures at 150 rpm in Fernbach flasks at 37°C, pH 7.5, for72 h. The conditions for growth and preparation of cell extractby sonication were as described earlier (la, 2).Enzyme assay. Ketohexokinase catalyzes the phosphoryla-

tion of fructose to FlP in an ATP-dependent reaction asfollows:

ketohexokinasefructose FlP

ATP * Mg

In the assay, the product FIP, which was phosphorylated in thepresence of 1-PFK to FBP, is subsequently cleaved by aldolaseinto triose phosphates, which are trapped and estimatedcolorimetrically. The reaction mixture for the assay of keto-hexokinase consisted of 5 mM fructose, 5 mM ATP, 2 mMMgCl2 - 6H20, 2 mM GSH, 30 pLg of purified halobacterial

5505

on May 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

5506 RANGASWAMY AND ALTEKAR

x

EFCD

gwUIm.4

80 100 120 140FRACTION NO.

!0_11

.6 E wI _

_.6 <IC ~;Iz

-2 E.s'

9

Z D

W

)- x

FIG. 1. Separation of H. vallismortis 1-phosphofructokinase and ketohexokinase activities on a Sepharose 4B column. A 185-mg amount ofprotein was purified on this column. The column was developed with a decreasing linear gradient of (NH4)2SO4 from 2.5 to 0.5 M in 50 mMTris-HCl-5 mM MgCl2-0.1 mM PMSF (pH 9.0) buffer. Details are given in Materials and Methods.

1-PFK from H. vallismortis (33), 1.3 U of rabbit musclealdolase, 35 mM Tris-HCl (pH 9.0), 1.2 M KCl, 56 mMhydrazine sulfate, and 0.05 ml of enzyme in a total volume of0.5 ml. After incubation at 37°C for 30 min, the reaction wasterminated with 0.5 ml of 10% trichloroacetic acid (TCA). Thetrapped triose phosphates in the supernatant were assayedcolorimetrically by the method described by Sibley and Leh-ninger (32). The activity was linear under the conditions of theassay over a period of 45 min. One unit of ketohexokinaseactivity was expressed as the amount of enzyme which gave 2,umol of triose phosphates per min at 37°C.For testing inhibition by FlP, the ketohexokinase reaction

mixture was coupled to an ADP indicator system whichconsisted of 5 mM PEP, 9.27 U of pyruvate kinase, 0.2 U oflactate dehydrogenase, 0.4 mM NADH, and 80 mM Tris-HCl(pH 7.5). Oxidation of NADH to NAD was monitored spec-trophotometrically at 340 nm. The coupling enzymes were inexcess and were active at the salt concentration of 1.2 M KClthat was used in the assay of ketohexokinase.

Unless otherwise mentioned, all data presented were ob-tained from two or four experiments. The kinetic data wereanalyzed with the least-squares regression program in a pro-grammable calculator.

Protein. The amounts of protein were determined by themethod described by Bradford (7), with bovine serum albuminas the standard.

Purification of ketohexokinase. A typical procedure devel-oped for the purification of ketohexokinase from H. vallismor-tis is described below. Preparation of cell extract and(NH4)2SO4 precipitation were carried out at 4°C, while theremaining steps were performed at 25°C.

Cell extract. H. vallismortis cells (40 g) which were sus-pended in 80 ml of 1 M KCl-1 M (NH4)2SO4-50 mM Tris-HCl(pH 9.0) were sonicated in a Vibracell Sonicator at 50% dutycycle for about 5 min at 4°C. A 200-,ug amount of DNase and0.1 mM PMSF were added to the sonicate, and the sonicatewas stirred for 30 min and centrifuge,d at 11,000 x g for 15 minat 4°C. The supernatant was used as the crude enzymepreparation.

(NH4)2SO4 precipitation. To the cell extract were added

simultaneously, with stirring, 1 volume of 100 mM Tris-HCl(pH 9.0) and 71.2 g of (NH4)2SO4 to obtain 80% saturation ofthe total suspension. The suspension was allowed to stand for18 h at 4°C and was centrifuged at 13,000 x g for 30 min. Thesupernatant containing ketohexokinase activity was collected.

(NH4)2SO4-mediated chromatography on Sepharose 4B.The supernatant was adsorbed on a column of Sepharose 4B(20 by 4 cm) that was equilibrated with 2.5 M (NH4)2SO4-0.1mM PMSF-5 mM MgCl2-50 mM Tris-HCl (pH 9.0) (buffer 1).The column was washed with 2 liters of buffer 1 and wasdeveloped with a linear gradient consisting of 600 ml of buffer1 and 600 ml of 0.5 M (NH4)2504-0.1 mM PMSF-5 mMMgCI2-50 mM Tris-HCl (pH 9.0). Fractions (5 ml) werecollected at a flow rate of 60 mi/h. Separation of ketohexoki-nase activity from the 1-PFK present in the crude extract waspossible at this step (Fig. 1). Active fractions were pooled.

CM-cellulose chromatography. The (NH4)2SO4 concentra-tion in the pooled ketohexokinase fraction was brought to 2.5M, and the fraction was loaded on a CM-cellulose column (8by 2 cm) equilibrated with buffer 1. The column was washedwith the same buffer, and protein was eluted batchwise withdecreasing concentrations of (NH4)2SO4. Ketohexokinase ac-tivity eluted in 2 M (NH4)2SO4.

IEAE-cellulose chromatography. The CM-cellulose frac-tion containing ketohexokinase was applied on a DEAE-cellulose column (8 by 2 cm) that was equilibrated with buffer1. The ketohexokinase activity was eluted with 2 M KCl-0.1mM PMSF-5 mM MgCl2-50 mM Tris-HCl (pH 9.0). Anexchange of KCl for (NH4)2SO4, as well as concentration ofthe enzyme, was achieved by this step.

Phenyl-Sepharose chromatography. The concentrated keto-hexokinase fraction from the step described above was broughtto a concentration of 4 M KCl by adding solid KCl and wasloaded on a phenyl-Sepharose column (9 by 1.5 cm) that wasequilibrated with 4 M KCl-5 mM MgCl2-50mM Tris-HCl (pH9.0) (buffer 2). The column was washed with 3 volumes ofbuffer 2 and then subsequently with 3 volumes of 3 M KCl-5mM MgCl2-50 mM Tris-HCl (pH 9.0). Ketohexokinase activ-ity eluted in the last buffer.

Hydroxylapatite chromatography. The ketohexokinase frac-

J. BACrERIOL.

on May 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

KETOHEXOKINASE FROM H. VALLISMORTIS 5507

TABLE 1. Purification chart of ketohexokinase from H. vallismortisa

Atotaf pc Total Reoey Purifi-Fraction total Sp act activity Re(cov) cationprotein (U) U) M (fold)(mg)

Crude extract 1,076 0.73 786 100 1.0(NH4)2SO4 super- 780 1.0 780 99 2.0

natantSepharose 4B 185 3.0 555 71 4.0CM-cellulose 113 4.3 486 62 6.0DEAE-cellulose 103 11.0 1,092 139 15.0Phenyl-Sepharose 12 29.0 348 44 40.0Hydroxylapatite 1.1 172.0 190 24 236.0

a Details of purification, starting with 40 g of cells, are given in Materials andMethods.

tion from the step described above was loaded on a hydroxyl-apatite column (2.5 by 1 cm) that equilibrated with 2.5 MKCl-5 mM MgCl2-50 mM Tris-HCl (pH 7.5). Sequentialbatchwise elution with 3 column volumes each of 50, 100, 150,and 200 mM K-phosphate in 2.5 M KCl-5 mM MgCl2 (pH 7.5)was carried out. Ketohexokinase activity eluted at a concen-tration of 50 mM phosphate.

Gel electrophoresis. Polyacrylamide gel electrophoresis(PAGE) using 10% gels in the presence of SDS was performedaccording to the method described by Laemmli (20) afterdesalting of the protein. Electrophoresis was also carried out inthe presence of the cationic detergent cetyltrimethylammo-nium bromide (CTAB) (15) such that the gels and the runningbuffer contained 0.1% CTAB; the electrophoresis was con-ducted by reversing the electrodes. CTAB gels were stainedand destained by the method used for SDS gels. The markerproteins used were bovine serum albumin (66 kDa), ovalbumin(45 kDa), rabbit muscle glyceraldehyde 3-phosphate dehydro-genase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen(24 kDa), trypsin inhibitor (20 kDa), and ot-lactalbumin (14kDa).

Determination of apparent molecular weight by sucrosedensity gradient ultracentrifugation. The molecular mass ofthe native halophilic ketohexokinase was estimated by sucrosedensity gradient ultracentrifugation (21). Sucrose gradients of5 to 20% (wt/vol) were prepared in a solution containing 2.5 MKCl-50 mM Tris-HCl (pH 7.5). Ketohexokinase (1 mg) dis-solved in the same buffer was layered on the gradient (11 ml)and centrifuged for 17 h at 35,000 x g in a Beckman SW41rotor at 4°C in a Beckman L7 ultracentrifuge. Fractions (0.5ml) were collected and assayed for enzymatic activity understandard conditions. To estimate the size of the native enzyme,0.5 mg each of following standards was run under similarconditions: bovine serum albumin (Mr, 66,000; 4.4S), rabbitmuscle aldolase (Mr, 160,000; 7.8S), bovine liver catalase (Mr,240,000; 11.2S), and bovine thyroglobulin (Mr, 690,000; 19.2S).Amino acid analysis. Halobacterial ketohexokinase (h-keto-

hexokinase) was desalted on a Sephadex G-25 column (5 by 1.5cm) and freeze-dried. The sample was then hydrolyzed in 6 NJICl and 0.5% phenol under a vacuum in a sealed ampoule for24 and 48 h at 110°C. Amino acid analysis was performed witha Beckman 119 CL automatic amino acid analyzer.Immunological cross-reactivity. Antiserum against h-keto-

hexokinase was raised in a rabbit by administering a subcuta-neous injection of 80 ,ug of desalted ketohexokinase inFreund's complete adjuvant. Boosters were given twice atfortnightly intervals. Blood samples were collected 10 daysafter the last booster injection, and antibodies that were

kDa

97-

66-

45-

FIG. 2. SDS-PAGE of h-ketohexokinase desalted by four differentmethods. The protein concentration in each lane was 33 ,ug. Lanes: 1,Centricon 30 microconcentrator; 2, gel filtration on a PD-10 column; 3,TCA precipitation; 4, dialysis against Tris-HCl (pH 9.0) buffer. Fordetails, see text.

obtained were used for checking immunological cross-reactiv-ity by Ouchterlony's double-diffusion tests (23).

Phosphorylation of ketohexokinase with [_y-32P]ATP (34).The reaction mixture consisted of 1.2 M KCl, 50 mM Tris-HCl(pH 9.0), 2 mM GSH, 5 mM MgCl2 6H20, 0.05 mM PEP, 20,ug of pyruvate kinase, 5 mM ATP, 200 jig of enzyme, and 50jiCi of [_y-32P]ATP in a total volume of 500 pl. After incuba-tion at 26°C for 25 min, the solution was passed over a column(1 by 30 cm) of Sephadex G-25 that had been previouslyequilibrated with 2.5 M KCl-5 mM MgCl2-50 mM Tris-HCl(pH 9.0) buffer. Elution was conducted at 26°C with the samebuffer. Fractions (0.5 ml) were collected. An aliquot (10 p,l)was assayed for 32p in an LKB-Rackbeta scintillation counter.For ketohexokinase activity, 25-pl aliquots were assayed by theusual colorimetric method.

Photoaffinity labelling (6). The reaction mixture in a totalvolume of 500 ,ul consisted of 1.2 M KCl, 2 mM GSH, 5 mMMgCl2- 6H20, 50 ,uCi of [,y-32P]ATP, 36 mM Tris-HCl (pH9.0), and 54 jig of enzyme. The mixture was illuminated at 25°Cin a thermostated quartz cuvette, which was placed 15 cm froma focused UV,lamp (long wave, 366 nm) and constantly stirred.After the irradiation, ATP was added to a final concentrationof 5 mM. The enzyme was precipitated with 0.5 ml of 20% TCAand kept on ice for 30 min. The precipitated protein was centrifuged,and the supernatant was discarded. The pellet was washed with20% TCA (four times). The final pellet was suspended in 25 ,ulof 0.5 M Tris-HCl (pH 7.5)-1% SDS and was electrophoresedon an SDS-10% polyacrylamide slab gel. X-ray film wasexposed to the stained and dried gel for 24 h, using intensifyingscreens.

RESULTS

Purification. Ketohexokinase from H. vallismortis was puri-fied 259-fold by the steps described above, and the finalpreparation had a specific activity of 172 U (Table 1). Consid-erable losses in activity were experienced in the Sepharose 4B,CM-cellulose, and phenyl-Sepharose steps. On SDS-PAGE,three major bands corresponding to 53, 47, and 25 kDa could

VOL. 176, 1994

on May 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

5508 RANGASWAMY AND ALTEKAR

20

161

121w-J4

U)

8

41

J. BACTERIOL.

20.. ThyroglobuI ins

CatalaseaeIkDa)

16

Lu 1 2

8 aRobbit muscle aldoluhyroglobul ~ I KTOHiEXOKINASE 110Thyroglobulin PFK(79kDo)

1j7 , ovine serum albumin

1\0I /2 3LOG M. wt.

\ Catalase

Rabbit muscle aldolase*KETOHEXOKINASE (6*9 5)IPFK (5-7 S)

ovine serum albumin

4 8 12 16 20 24FRACTION NO.

FIG. 3. Sedimentation proffle of h-ketohexokinase on sucrose density gradient ultracentrifugation. Other details are as given in Materials andMethods.

be detected in addition to minor impurities amounting to 5%(Fig. 2). This preparation was used for further characterizationof the enzyme.

Molecular mass. The apparent molecular mass of the activeketohexokinase determined by analytical gel-filtration wasfound to be 95.5 kDa (data not shown).

Sucrose density gradient sedimentation analysis of ketohex-okinase was carried out. From the standard plot of sedimen-tation coefficient (S) values against elution volume (Ve), avalue of 6.9S was obtained, which corresponds to a molecularmass of 100 kDa (Fig. 3).There were some ambiguities in preliminary runs of the

enzyme in denaturing PAGE. SDS-PAGE analysis of ketohex-okinase showed several bands when the enzyme was desaltedon a Centricon 30 microconcentrator. However, during TCAprecipitation of the enzyme, only three bands were detected,corresponding to 53, 47, and 25 kDa. To determine whetherthe mode and duration of desalting, i.e., the length of time forwhich the enzyme is left without salt, has any role in theappearance of numerous bands on SDS-PAGE, two additionalmethods of desalting, i.e., gel filtration on a PD-10 column anddialysis, were carried out, and the enzyme was subjected toSDS-PAGE. The protein concentration was kept constant inall four cases. Comparison of the number of bands revealedthat dialysis of the sample led to maximum denaturation of thepolypeptide chain, whereas TCA precipitation (since it is thequickest method of desalting) resulted in fewer bands when theprecipitate was subjected to SDS-PAGE (Fig. 2).CTAB-PAGE of the ketohexokinase yielded only two bands

corresponding to molecular masses of 74 and 46 kDa.Kinetic properties. (i) Effect of salt on ketohexokinase

activity. Ketohexokinase activity at various concentrations ofdifferent salts was tested, and the enzyme was found to behalophilic. The enzyme was maximally active in the presence of1.2 M KCl (Fig. 4). Activity in the presence of RbCl and CsClwas similar to that observed in the presence of KCl. NaCl was

comparatively less stimulatory, yielding only 50% of the activ-ity observed in equimolar KCl. NH4Cl and LiCl were inhibitoryfor ketohexokinase activity.

(ii) Efrect of salt on ketohexokinase stability. Ketohexoki-nase was fully active in 2.5 M KCl-50 mM Tris-HCl (pH 9.0)even at the end of 9 weeks, whereas the enzyme was totallyinactivated in 2 h at 25°C in 0.1 M KCl-50 mM Tris-HCl (pH9.0). KCl concentrations between 0.1 and 2.5 M were interme-diate in their effect on the stability of the enzyme, which

c

C>

00~E._a.

0

-

0-5 1-0 15 2-0

M, Salt concentration2-5 3-0

FIG. 4. Salt-dependent activity of h-ketohexokinase. The standardassay mixture was used in the presence of different salts. 0, KCI; A,NaCl; 0, LiCl; O, NH4CI; X, RbCl; A, CsCl. The protein concentra-tion was 40 ,ug. Other details are as given in Materials and Methods.

b l\ \\ N

2 _ \\ \ XK

1_AX\N1\

LI

I I I I I I I . I

on May 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

KETOHEXOKINASE FROM H. VALLISMORTIS 5509

r--

>- .C

LUZIno 0; on

Yn E

0

200H

1001-

I I I6 7 8 9 10 l

pH

5 6 7'; 8 9 10 11

pH

FIG. 5. (A) Effect of pH on h-ketohexokinase activity. Enzymereaction was carried out at the pH indicated in the specified buffers. 0,

K-phosphate; X, Tris-HCl; A, glycine-NaOH. The protein concentra-tion was 10 ,ug. Other details are as given in Materials and Methods.(B) Plot of log of Vversus pH to determine the pKa of the amino acidbeing titrated. Log of Vvalues were taken from the experiment on theeffect of pH on ketohexokinase activity described for panel A.

increased (ranging from 30 to 98% of the total) with increasingKCl concentrations. Addition of 50 mM phosphate aided instabilization of ketohexokinase activity in 0.1 M KCI for about24 h. Although it was inhibitory for expression of ketohexoki-nase activity (also see Table 1), (NH4)2SO4 was found to bestabilizing.

(iii) pH optimum for ketohexokinase activity. Ketohexoki-nase exhibited activity over a wide range of pH values, steadilyincreasing about twofold from pH 7.5 to 10.5 (Fig. 5A).The straight lines produced from the plot of log of Vversus

pH intersect at a pH value equal to 7.15 (Fig. SB), the pK ofthe ionizing groups which either form part of the active centeror are closely associated with it.

(iv) Kinetic constants. Ketohexokinase exhibited a Km of 50.1 mM toward fructose, whereas the Km for ATP was 3.3

0.15 mM at pH 7.5.Ketohexokinase was most active at a Mg2+ concentration of

1 mM. Mg2+ could not be replaced by Mn2+. ATP * Mg in the

cE0

I 008

srdi 0-06U)0

- 0-04E

0-02_I>

A0.I-

0-04 K; * 0-1 mMl(ADP)

0_ 6 _ ADP

-'0-02 ;

a I I-0-2513 01-25 O-S

AD P, mM /

Z xO~~~~x-25mM/x

G r~~~OmMl Il-0 4 -0-2 0 0-2 0.4 -1 1;0

1 (mM)[ATPJ

0I04-0a 0-08 _-w 0 03 - / D008 0.0 ADP

-0E -0-02 K0.2 mM [ADP] 05mM

E 0-06 -0 01

xL 0-2 ,25mM

LI -i ADP. mM

-0-5 0 0-4 0.6 1-1

i [mM]AT,P

2

FIG. 6. (A) ADP inhibition of h-ketohexokinase at pH 9.0. Insetshows plot of slope versus ADP concentration. The protein concen-tration was 5.4 ,ug. Other details are as given in Materials andMethods. (B) ADP inhibition of h-ketohexokinase at pH 7.5. Insetshows plot of slope versus ADP concentration. The protein concen-tration was 5.4 ,ug. Other details are as given in Materials andMethods.

ratio of 1:2 forms the actual substrate for ketohexokinaseactivity.

Ketohexokinase activity was unaffected by its product FlP,whereas the other product of reaction, i.e., ADP, competitivelyinhibited ketohexokinase activity to various extents at differentpHs. Thus, Kis of 0.1 ± 0.05 and 0.2 ± 0.05 mM were obtainedat pH 9.0 (Fig. 6A) and at pH 7.5 (Fig. 6B), respectively.

Thermostability and temperature dependence of ketohex-okinase activity. The enzyme was incubated for variousamounts of time at different temperatures (without substrate),and the residual ketohexokinase activity was determined by thestandard assay. The enzyme was relatively thermostable, ex-hibiting 95% of its activity when exposed for 30 min at 75°C(Fig. 7). Arrhenius plotting of the data (data not shown)showed a linear increase with no breakpoint in the tempera-ture range of 27 to 45°C, with an apparent activation energy of68.2 kJ/mol.Amino acid composition. The amino acid composition of

h-ketohexokinase is shown in Table 2. Amide values were notdetermined. Tyrosine and tryptophan contents were deter-mined spectrophotometrically by the method described byBencze and Schmid (5).

Involvement of phosphoenzyme intermediate. To determinewhether ketohexokinase forms a phosphoenzyme intermedi-

A

/x

II#

I1Z

VOL. 176, 1994

A

on May 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

5510 RANGASWAMY AND ALTEKAR

00

X 20

W:3~ ~ ~ ~ I

WtaN40=-

C O0h 1h 2h 24hTIME

FIG. 7. Effect of temperature on h-ketohexokinase activity. Theenzyme was incubated for various amounts of time at the differenttemperatures indicated in standard buffer, and residual activities weremeasured. The protein concentration was 86 ,ug. 0, 27°C; X, 37°C; 0,45°C; OI1, 55°C; A, 75°C; *, 85°C.

ate, ketohexokinase was incubated with [y-32P]ATP andpassed through a Sephadex G-25 column (1 by 30 cm) toremove the unincorporated label. The enzyme isolated by gelfiltration was radioactive, suggesting the transfer of the termi-nal phosphoryl group of ATP to the enzyme (Fig. 8).

In another experiment, the enzyme was incubated with[_y-32P]ATP in the presence of UV light, which aids in co-valently linking the phosphoryl group of ATP to the enzymeand thereby forming a relatively stable phosphoenzyme com-plex. Incorporation of the label increased with the amount oftime of exposure to UV light. In comparison with the patternobtained by SDS-PAGE, the signals appearing on the autora-diogram corresponded to the 54- and 27-kDa subunits.

Effect of -SH compounds on ketohexokinase activity. Ke-tohexokinase activity was tested in the presence of SH com-

TABLE 2. Amino acid composition of ketohexokinasefrom H. vallismortis

Mol%Amino acida Mol/100 mol

h-ketohexokinase Liver

Aspartate 130.0 13.4 9.7Threonine 48.9 5.1 3.4Serine 37.0 3.8 7.0Glutamate 137.0 14.2 11.1Proline 11.0 1.1 3.0Glycine 84.0 8.7 8.7Alanine 107.0 11.0 8.7Valine 82.3 8.5 9.4Methionine 11.9 1.2 1.3Isoleucine 37.0 3.8 4.4Leucine 91.0 9.4 9.1Tyrosine 31.0 3.2 1.7Phenylalanine 32.3 3.3 4.7Histidine 26.0 2.7 1.7Lysine 25.2 2.6 5.4Arginine 46.0 4.7 6.0Tryptophan 30.0 3.1 1.3Cysteine 3.4

Total 967.6

a Amide content was not determined. Tryptophan content was estimatedspectrophotometrically (5).

5000

4000[- 3000)

x 2000

a, 1000

.- 100U,_ 20C,)

'---i 10

10 20 30 40 50 60

IJE v,

.g .

U^.03a 4

I _

w Yen o0-2 2 x.

CXO

01E x'Q,

0IFRACTION NO.

FIG. 8. Phosphorylation of h-ketohexokinase (200 ,ug) with[y-32P]ATP. Other details are as given in Materials and Methods.

pounds and reagents. Reduced glutathione was the mosteffective SH compound for expression of ketohexokinase ac-tivity. Cysteine and 2-mercaptoethanol could also attributebetween 87 and 94% of the activity observed with reducedglutathione. The absolute requirement of -SH groups duringcatalysis was indicated by the complete inhibition of activitydue to -SH-binding reagents such as DTNB and p-chloromer-curibenzoate.

Cofactor specificity. Except for ATP, other nucleotides(such as UTP, CTP, GTP, or ITP) could not act as phosphoryldonors in the ketohexokinase reaction.

Autofragmentation. To investigate whether any thermaldenaturation occurs during preparation of samples for electro-phoresis which eventually leads to the appearance of variousbands on SDS-PAGE, the enzyme was incubated with thesample buffer for various time intervals at different tempera-tures and was subjected to electrophoresis. No change in bandpattern was observed.

Immunological cross-reactivity. Antiserum against h-keto-hexokinase did not cross-react with ketohexokinase activity inrat liver.

DISCUSSION

The purification protocol for ketohexokinase is summarizedin Table 1. The final preparation had a specific activity of 172U. Since the enzyme is a kinase, MgCl2 at a concentration of 5mM was maintained throughout the purification. The concen-trations of salt had to be maintained at a high level (>1.5 M)throughout the purification. Since (NH4)2SO4 has an inhibi-tory effect on ketohexokinase activity, the estimated values forspecific activities at the (NH4)2SO4 supernatant, Sepharose4B, and CM-cellulose steps are much lower than the truevalues.The native h-ketohexokinase showed a molecular mass of

95.5 kDa by gel filtration. A sedimentation coefficient of 6.9S,which was obtained following sucrose density gradient centrif-ugation, corresponded to a molecular mass of 100 kDa, whichwas within the range. This value of 100 kDa is comparable withthe molecular masses of native yeast hexokinases and mam-malian hexokinase forms I, II, and III.

- S R~~~~~x

I %I v I7'

J. BACTERIOL.

----I

)k

on May 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

KETOHEXOKINASE FROM H. VALLISMORTIS 5511

TABLE 3. Comparison of molecular weights of ketohexokinasesfrom various sources

Source Native Mr No. of Subunit Mr(reference) (103) subunits (103)

H. vallismortis 100 2 74.45Rat liver (31) 28 1 28Bovine liver (27)a 56 2 29Human liver (4)a 75 2 39

a Homomeric dimer.

SDS-PAGE of ketohexokinase presented several anomalies,depending on the mode in which the enzyme was desalted.Halobacterial proteins lose their three-dimensional conforma-tion during exposure to low salt concentration, and subunitsget separated. When exposed to a milieu of no salt for an

extended period, these polypeptide chains can undergo ran-

dom association and dissociation processes, leading to numer-

ous bands on SDS-PAGE (1, 14, 19). Therefore, it is suspectedthat the difference in the numbers of bands in SDS and CTABprocedures observed here could be because the 74-kDa sub-unit dissociates further into 53- and 27-kDa moieties in SDS.Besides, in the photoaffinity labeling experiments, only bandscorresponding to 53 and 27 kDa showed incorporation of label,as is evident from the autoradiogram. This observation reiter-ates that the subunit of ketohexokinase involved in the transferof the phosphoryl group may be the 74-kDa subunit whichdissociates on SDS-PAGE. When the observations madeabove are taken into consideration, it is tempting to propose a

heterodimeric nature to ketohexokinase with subunits of 74and 45 kDa. This agrees well with the molecular mass of 100kDa obtained by gel filtration and sucrose density gradientcentrifugation experiments.

Similar confusion in ascertaining the subunit composition ofanother sugar kinase, i.e., yeast hexokinase, has been reportedelsewhere (9). SDS-PAGE gave erroneously low values, cor-

responding to 25 kDa. The source of error was the occurrence

of traces of yeast proteases in the preparation. Unless precau-tions are taken to destroy the protease (e.g., by boiling) (26), itdegrades the hexokinase peptide chain immediately when it isplaced in the denaturing solvent.

Irrespective of its source, mammalian hexokinase exhibits a

size of about 100 kDa (8, 13, 24, 28) and is a monomer.

From the available information on ketohexokinase fromnonhalobacterial sources, it appears that the enzyme exists invarious sizes, ranging from 28 to 75 kDa, depending on thesource (Table 3). Thus, h-ketohexokinase appears to be largerthan its mammalian counterparts.No immunological cross-reactivity could be detected be-

tween the h-ketohexokinase antiserum and ketohexokinasefrom rat liver.

Ketohexokinase was a relatively thermostable enzyme,which is a property characteristic of several halophilic enzymes(16). Optimum temperature per se could not be determined,since the assay involved use of rabbit muscle aldolase, which isnot thermostable, as the coupling enzyme.

In concurrence with the known common feature of halobac-terial enzymes, ketohexokinase activity was also halophilic.Thus, activity was dependent on a high concentration of salt(>1.2 M). In a dilute salt solution (below 0.2 M) or in theabsence of salt, the enzyme activity was lost rapidly. However,salting-out types of salts, such as phosphate, stabilize ketohex-okinase activity for more than 24 h even at a KCl concentrationof as low as 0.1 M.

Ketohexokinase exhibited activity over a broad pH range, ashas been found in the case of ketohexokinases from othersources (25, 31). The pK. value of 7.15 obtained from the plotof log of Vversus pH does not match with the pKa values of anyamino acid, although histidine might be a likely candidate. It isknown that when it is present in a protein molecule, a givengroup may not have the same pKa value that it has when it ispresent in a free amino acid or other small molecule becauseof the influence of the environment of the group created byadjacent groups in the folded protein (11). SH reagentsinactivated h-ketohexokinase, suggesting the possible involve-ment of -SH groups at the active site.ATP-Mg in the ratio of 1:2 formed the actual substrate for

ketohexokinase reaction. In contrast, a ratio of 1:1 was foundfor mammalian ketohexokinase (25).

Like most kinases, ketohexokinase was inhibited competi-tively by ADP. However, FlP had no effect on ketohexokinasereaction. This rules out any apparent regulatory role forketohexokinase, unlike that observed in hexokinases which areinhibited by the product glucose 6-phosphate. As in the case ofmammalian hexokinase and ketohexokinase, no other nucleo-side triphosphate could substitute for ATP as a phosphoryldonor in the h-ketohexokinase reaction.

Several attempts to identify any phosphoenzyme complexformation during the hexokinase reaction were unsuccessful(22). However, in our experiments, incubation of ketohexoki-nase with [_y-32P]ATP resulted in incorporation of radioactivityin the enzyme. Given this lack of success in detecting aphosphoenzyme intermediate in any hexokinase reactions, thedemonstration of such a complex in a ketohexokinase reactionhas indeed become a salient finding. Further studies on thecharacterization of the phosphorylated enzyme intermediateare necessary to understand the nature of the site of phosphor-ylation and the stoichiometry and kinetics exhibited by it.

Since the presence of ketohexokinase in any archaeon or, forthat matter, in any prokaryote was first demonstrated by ourlaboratory, no comparison with other members of the archaeais possible.With the isolation of ketohexokinase, four consecutive en-

zymes in the EMP pathway, i.e., ketohexokinase, 1-phospho-fructokinase (33), FBP aldolase (18) and glyceraldehyde3-phosphate dehydrogenase (17), have been purified from H.vallimortis in our laboratory.

Recently, the primary structure of ketohexokinase from ratliver has been determined (12). The enzyme has been clonedand expressed in Saccharomyces cerevisiae, resulting in fructoseintolerance in this yeast. The primary structure did not showany significant homology with those of other eukaryotic hex-okinases; however, it revealed a highly conserved motif, i.e.,TX(A/G)AGDXL, which is present in three prokaryotic phos-photransferases that have furanose substrates, i.e., Escherichiacoli ribokinase (EC 2.7.1.15), E. coli minor phosphofructoki-nase (EC 2.7.1.11), and Vibrio alginolyticus fructokinase (EC2.7.1.4). The amino acids X are nearly always conservativeamino acid replacements. This consensus was also found in afifth protein, E. coli DNA topoisomerase I.

Thus, further studies on ketohexokinases from the halophilicarchaea would show whether this protein belongs within thegroup of furanose kinases discussed above. If so, it would alsolend support to the belief that this group of kinases representsan ancient family of proteins.

ACKNOWLEDGMENTThe award of studentship to V.R. during the course of this work by

the University Grants Commission, Delhi, India, is gratefully acknowl-edged.

VOL. 176, 1994

on May 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

5512 RANGASWAMY AND ALTEKAR

REFERENCES1. Altekar, W. Personal communication.la.Altekar, W., and R. Vidhya. 1990. Indication of a modified EMP

pathway for fructose breakdown in a halophilic archaebacterium.FEMS Microbiol. Lett. 69:139-144.

2. Altekar, W., and R. Vidhya. 1991. Ketohexokinase (ATP: D-fructose 1-phosphotransferase) initiates fructose breakdown viathe modified EMP pathway in a halophilic archaebacterium.FEMS Microbiol. Lett. 83:241-246.

3. Altekar, W., and R. Vidhya. 1992. Degradation of endogenousfructose during catabolism of sucrose and mannitol in halophilicarchaebacteria. Arch. Microbiol. 158:356-363.

4. Bais, R., H. M. James, A. M. Rofe, and R. A. J. Conyers. 1985. Thepurification and properties of human liver ketohexokinase. Bio-chem. J. 230:53-60.

5. Bencze, W. L., and K. Schmid. 1957. Determinatin of tyrosine andtryptophan in proteins. Anal. Chem. 29:1193-1196.

6. Bonet, M. L., and L. B. Schobert. 1992. The catalytic site is locatedon subunit I of the ATPase from Halobacterium saccharovorum.Eur. J. Biochem. 207:369-376.

7. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

8. Chou, A. C., and J. E. Wilson. 1972. Purification and properties ofrat brain hexokinase. Arch. Biochem. Biophys. 151:48-55.

9. Colowick, S. P. 1973. The hexokinases, p. 1-48. In P. D. Boyer(ed.), The enzymes, vol. VII. Academic Press, New York.

10. Crane, R. K. 1962. Hexokinases and pentokinases, p. 47-66. InP. D. Boyer, H. Lardy, and K. Myrback, (ed.), The enzymes, vol. 6.Academic Press, New York.

11. Dixon, M., and E. C. Webb. 1979. Enzyme kinetics, p. 138. In P.Boyer (ed.), The enzymes, 3rd ed. Academic Press Inc., New York.

12. Donaldson, I. A., T. C. Doyle, and N. Matas. 1993. Expression ofrat liver ketohexokinase in yeast results in fructose intolerance.Biochem. J. 291:179-186.

13. Easterby, J. S. 1971. The polypeptide chain molecular weight of amammalian hexokinase. FEBS Lett. 18:23-26.

14. Eisenberg, H., M. Mevarech, and G. Zaccai. 1992. Biochemical,structural and molecular genetic aspects of halophilism. Adv.Protein Chem. 43:1-62.

15. Eley, M. H., P. C. Burns, C. C. Kannapell, and P. S. Campbell.1979. Cetyltrimethylammonium bromide polyacrylamide gel elec-trophoresis: estimation of protein subunit molecular weights usingcationic detergents. Anal. Biochem. 92:411-419.

16. Keradjopoulos, D., and A. W. Holldorf. 1977. Thermophilic char-acter of enzymes from extreme halophilic bacteria. FEMS Micro-biol. Lett. 1:179-182.

17. Krishnan, G., and W. Altekar. 1990. Characterization of a halo-philic glyceraldehyde 3-phosphate dehydrogenase from the ar-chaebacterium Haloarcula vallisnortis. J. Gen. Appl. Microbiol.36:19-32.

18. Krishnan, G., and W. Altekar. 1991. An unusual class I (Schiffbase) fructose 1,6-bisphosphate aldolase from the halophilic ar-chaebacterium Haloarcula vallismortis. Eur. J. Biochem. 195:343-350.

19. Krishnan, G., and W. Altekar. 1993. Halobacterial class I aldolaseand glyceraldehyde 3-phosphate dehydrogenase: some salt depen-dent structural features. Biochemistry 32:791-798.

20. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

21. Martin, R. G., and B. N. Ames. 1961. A method for determiningthe sedimentation behaviour of enzymes; application to proteinmixtures. J. Biol. Chem. 236:1372-1379.

22. Najjar, V. A., and E. E. McCoy. 1958. Mechanism of action ofmuscle and yeast phosphoglucomutase and a suggested mecha-nism of yeast hexokinase. Fed. Proc. 17:1141-1144.

23. Ouchterlony, 0. 1967. In D. M. Weir (ed.), Handbook of experi-mental immunology, p. 655-706. Blackwell Scientific Publications,Oxford.

24. Paranjpe, S. V., and V. Jagannathan. 1971. Properties and kineticsof purified particulate ox heart hexokinase. Ind. J. Biochem.Biophys. 8:227-231.

25. Parks, R E., E. Ben-Gershom, and H. A. Lardy. 1957. Liverfructokinase. J. Biol. Chem. 227:231-242.

26. Pringle, J. R. 1970. The molecular weight of the undegradedpolypeptide chain of yeast hexokinase. Biochem. Biophys. Res.Commun. 39:46-52.

27. Raushel, F. M., and W. W. Cleland. 1977. Bovine liver fructoki-nase: purification and kinetic properties. Biochemistry 16:2169-2175.

28. Redkar, V. D., and U. W. Kenkare. 1972. Bovine brain mitochon-drial hexokinase. J. Biol. Chem. 247:7576-7584.

29. Rodriguez-Valera, F., F. Ruiz-Berraquerro, and A. Ramos Cor-menzana. 1980. Isolation of extremely halophilic bacteria able togrow on defined inorganic media with single carbon source. J.Gen. Microbiol. 199:535-538.

30. Saier, M. H., Jr., F. C. Grenier, C. A. Lee, and B. E. Waygood.1985. Evidence for the evolutionary relatedness of the proteins ofthe bacterial phosphoenolpyruvate: sugar phosphotransferase sys-tem. J. Cell. Biochem. 27:43-56.

31. Sanchez, J. J., N. S. Gonzalez, and H. G. Pontis. 1971. Fructoki-nase from rat liver. I. Purification and properties. Biochim.Biophys. Acta 227:67-78.

32. Sibley, J. A., and A. L. Lehninger. 1949. Determination of aldolasein animal tissues. J. Biol. Chem. 117:859-878.

33. Vidhya, R, and W. Altekar. Characterization of 1-phosphofruc-tokinase from halophilic archaebacterium Haloarcula vallismortis.Biochim. Biophys. Acta, in press.

34. Walsh, C. T., Jr., and L. B. Spector. 1971. A phosphoenzymeintermediary in phosphoglycerate kinase action. J. Biol. Chem.246:1255-1261.

J. BACTERIOL.

on May 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from