238

BBA 32217

Biochimica et Biophysics Acta 829 (1985) 238-243

Elsevier

Fucokinase, its anomeric specificity and mechanism of phosphate group

transfer *

William Butler and George S. Serif **

The Department of Biochemistry, The Ohio Slate University, 484 West 12th Avenue, Columbus, OH 43210 (U.S.A.)

(Received September 18th. 1984)

(Revised manuscript received March 4th, 1985)

Key words: Fucokinase; Anomeric specificity; Phosphate transfer

Fucokinase phosphorylates L-fucose at the anomeric position and, as such, might use either the a or /I anomer as its substrate. Examination of the utilization of radiolabelled a and a,/3 mixtures established p-L-fucose as the required substrate. Phosphorylation at the anomeric center might involve either the loss or retention of the anomeric oxygen. The mechanism has been shown to involve anomeric oxygen retention through mass spectrometric analysis of the product phosphate derived from ‘*O-labelled L-fucose.

Introduction

Kinases capable of phosphorylating sugars at an anomeric carbon are relatively rare. In the hexose series, in addition to L-fucokinase [l-4], only D-galactokinase, N-acetyl-D-galactosamine kinase and D-glucuronic acid kinase have been identified [5-91. D-Galactokinase alone has been examined with respect to anomeric specificity and mechanism of enzyme action. Thus, Howard and Heinrich [lo] have determined that yeast D-galac- tokinase utilizes a-D-galactose as its substrate, and Gulbinsky and Cleland [ll] reported that Escherichia coli D-galactokinase proceeds through a random sequential mechanism. The anomeric specificity for fucokinase has not been determined. The mechanism for kinase action at an anomeric center has not been examined for fucokinase or

* This work was taken from a dissertation submitted by

William Butler to the Graduate School of the Ohio State

University in partial fulfillment of the requirements for the

M.Sc. degree.

** To whom reprint requests should be addressed.

Abbreviation: Mops, 4-morpholinepropanesulfonic acid.

any other of the above kinases to date. In the case of kinase action at a non-anomeric hydroxyl group, the mechanism appears to involve an SN, attack of the negatively charged oxygen on the y-phos- phate of ATP as depicted in mechanism A of Fig. 1 [12]. An alternate, unconventional possibility exists for phosphate group transfer to an anomeric

MECHANISM A

MECHANISM B

Fig. 1. Alternate mechanisms for fucokinase action in phos

phoryl group transfer (R = adenosine).

0167-4838/85/$03.30 Q 1985 Elsevier Science Publishers B.V. (Biomedical Division)

center. Thus, a water molecule or an hydroxide ion might attack the y-phosphorus of ATP, while the "t-phosphorus oxygen simultaneously attacks the anomeric center (Fig. 1 mechanism B). This un- usual mechanism might be effected by virtue of the capability of the anomeric center to lose its hydroxyl group with creation of a carbonium ion as demonstrated to occur during acid and en- zymatic hydrolysis of various glycosides and poly- s accharides [ 13-15 ].

In the first of these two mechanisms, as applied to fucokinase, the substrate for the enzyme would have to be the fl anomer, since the product of the reaction has been shown to be the fl-l-phosphate derivative [3] and anomeric inversion does not occur by that mechanism. Additionally, mecha- nism A would require retention of the anomeric oxygen, whereas mechanism B would require its loss. This report presents the results of studies to define the mechanism of fucokinase action through identification of the required anomer and through ~80-labelling studies.

Methods

Fucokinase assay. The determinat ion of anomeric specificity was accomplished by studying the rate of enzymatic phosphorylation of L-fucose anomers using an assay in which the production of ADP from fucokinase action was coupled through pyruvate kinase to pyruvate formation and its reduction to lactic acid by lactate dehydrogenase and NADH. The resulting NAD ÷ was measured fluorometrically.

The assay solutions contained the following fi- nal concentrations of components in a total volume of 2.2 ml: 6 mM KF, 6 mM MgSO 4, 5 mM ATP, 0.5 mM phosphoenolpyruvate, 0.5 mM NADH, 0.182 M glycerol, 0.02 M Mops buffer to adjust the pH to 6.6, 0.35 units of pyruvate kinase, 5 units of lactate dehydrogenase and 2 .2 .10-3 units of fucokinase prepared as previously described [16]. At zero time, 10 #1 of an aqueous L-fucose solution, held for a period greater than 1 h to reach equilibrium between its a- and fl-pyranose forms, was added to the 2.2 ml assay solution such that the final L-fucose concentration was 100 /~M. The reaction in a second reaction vessel containing 2.2 ml of the assay solution was started 1 min later

239

by adding the same concentration of L-fucose but from an aqueous L-fucose solution prepared im- mediately before use by dissolving crystalline a-L- fucose in water. Aliquots (90 #1) from both reac- tion vessels were taken at zero time and at various time intervals to be assayed for NAD ÷ content. Each aliquot was added to a small reaction vial containing 30 /~1 of ice-cold 20% trichloroacetic acid. After a 4 rain interval, 120/tl of 12 N NaOH were added and the vial was placed in a boiling- water bath for 5 min. After cooling, the samples were centrifuged to remove precipitated protein and aliquots of the supernatant solutions were removed and diluted 1 : 5 with water. The fluores- cence was then measured against standards using an excitation wavelength of 360 nm and an emis- sion wavelength of 460 nm [17,18].

Preparation of L-[lSO]fucose. Labelled t-fucose was prepared by oxygen exchange between the anomeric oxygen of the sugar and [180]H20. 10 mg of L-fucose and 100 #1 of [180]H20 (55 atom%) were sealed in a polypropylene tube for several days to reach isotopic equilibrium. Excess [180]H20 was removed under reduced tempera- tures and pressure.

Phosphorylation of L-[ l SO]fucose. Phosphoryla- tion of 180-labelled L-fucose was accomplished in a reaction medium containing the following con- centration of components: 400 #M L-[~80]fucose, 6 mM MgSO4, 5 mM ATP, 8.3 mM NaF, 20 mM Mops buffer (pH 6.6), 80 mM glycerol and 2.0. 10 -2 units of fucokinase in a total volume of 25 ml. Approx. 40 000 dpm of marker 1-L-[laC]fucose were incorporated with the substrate t-fucose to permit detection of product L-fucose 1-phosphate at various stages in its isolation. The enzymatic reaction mixtures were incubated at 35°C for 5 h. These conditions should provide, theoretically, 6 /xmol of product from each reaction.

Isolation of L-fucose 1-phosphate. 5 ml of 0.06 M Na2B407 were added to each reaction mixture and a given mixture applied to a Bio-Rad AG 1 × 4 column (15 ml bed volume) previously equilibrated with 0.01 M Na2B407. The column was washed with 150 ml of 0.01 M Na2B407. The L-fucose 1-phosphate was subsequently eluted with 60 ml of a solution with 0.1 M NHnCI and 0.005 M Na2B407. ATP, ADP, AMP and inorganic phos- phate are not eluted from the column under these

240

conditions. 5 ml of Bio-Rad AG 50 × 4 resin in the hydrogen form were added to the eluted solu- tion of L-fucose 1-phosphate to remove cations, and it was subsequently removed by filtration. Several batches of methanol (100 ml each) were added to the filtrate followed by evaporation in vacuo after each addition to remove borate ion. The residual L-fucose 1-phosphate was taken up in 2 ml of water adjusted to a pH of 3.0 with HC1 and heated at 70°C for 15 min. The hydrolysis mixture was treated with 2.0 ml of 0.03 M triethyl- ammonium bicarbonate buffer (pH 7.8) and the solution was applied to a DEAE-Sephadex A-25 column (5 ml bed volume) equilibrated with 0.15 M triethylammonium bicarbonate buffer at a pH of 7.8. The column was washed with 30 ml of the buffer and the phosphate subsequently eluted with 20 ml of 0.4 M buffer at the same pH. The triethylammonium bicarbonate was removed by repeated evaporation with water in vacuo.

Derivatization of L-fucose and phosphate. Tri- methylsilylated ethers of L-fucose were prepared by introducing the sugar (1 mg), N-bis(trimethyl- silyl)trifluoroacetamide (100 /~l) and trimethyl- chlorosilane (20/~l) into a small glass vial contain- ing 40 #l of dry pyridine. The vial was capped and placed in a 37°C oven for 15 min. The samples were subsequently examined for completeness of reaction by gas chromatography on a capillary column (SE 54) prior to mass spectral analysis.

Phosphate samples to be esterified were dis- solved in I ml of methanol, and an ether solution of diazoethane in diethyl ether (prepared in a generator) was gradually added until a cloudiness was retained. The solvent was removed in vacuo and the process was repeated to insure complete ethylation. The samples were dissolved in chloro- form for mass spectral analysis.

Anomeric 150 loss. Loss of 180 from the anomeric position was studied by using 180- labelled t-fucose prepared as previously described. 10 mg of the labelled L-fucose was incubated in 150/L1 of distilled water at 37°C and aliquots were removed at appropriate intervals. These aliquots were frozen immediately and water was removed at freezing temperatures by lyophilization. The dried samples were then converted to their tri- methylsilyl derivatives as previously described and analyzed for 180 content by mass spectrometry.

Zero time samples showed no loss of 180 as a function of the handling procedure.

Mass spectral analysis was conducted at the Ohio State University Instrument Center with the kind assistance of Mr. David Chang, Ms. Mary Ann Seifert and Mr. Clemens Weisenberger. A Finnigan 4000 Gas Chromatography-Mass Spec- trometer and a Beckman M.S. 9 Mass Spectrome- ter were used in these analyses.

Results and Discussion

The fluorometric, coupled enzyme assay out- lined in the Methods section permitted the mea- surement of L-fucose phosphory la t ion by fucokinase as a function of the anomeric con- figuration of the sugar. Since fl-L-fucose is not available in pure form, the study was conducted with both the a-anomer and the t~,fl-equilibrium mixture (~ = 32%, fl = 68%). The concentration of L-fucose in both circumstances was set at 100 ~M, since that concentration of equilibrium mixture is known to be saturating with respect to the ap- propriate anomer. Fig. 2 presents the data ob- tained from this study. The ~,fl-mixture is seen to exhibit a linear increase in the production of NAD ÷ with time, coupled to ADP and fucose 1-phos- phate formation. The pure ct solution, on the other hand, exhibits a time lag with respect to an in- crease in NAD ÷ production, and it is only after approx. 10 min that the reaction rate approaches that observed with the t~,fl-mixture. This lag phase clearly identifies the fl-L anomer as the required

lOO

80

o 60 E Z- 4 0

"~ 20

a t B

J -o

4 s 12 16 0 24 28 2

M I N U T E S

Fig. 2. Separate effects of solutions o f the a anomer and the a +/3 equilibrium mixture of L-fucose on the course of the fucokinase reaction. The reaction is coupled to the production of NAD + measured fluorometrically.

substrate. Mutarotation of L-fucose tO an equi- librium mixture is relatively rapid (90% of equi- librium in 25 min). Thus, after approx. 10 min, sufficient a form has been converted to the accep- table substrate to permit saturation of the enzyme. Since the product of phosphorylation is fl-L-fucose 1-phosphate, as shown in an earlier report [3], it is apparent that no inversion of the anomeric carbon has occurred. Had inversion occurred, then mecha- nism A (Fig. 1A) would have been eliminated from consideration. Mechanism B, however, is also still feasible if there is a stereospecific attack of the ATP oxygen on the carbonium ion intermediate.

Labelling of the anomeric oxygen of L-fucose with 180 in this reaction should unequivocally establish the mechanism. Thus, 180 in the anomeric oxygen should be retained in the product in mech- anism A and lost in mechanism B.

L-Fucose was labelled with 180 through ex- change with [180]H20 as described in the Meth- ods section. A mass spectral analysis requires either the presence of a parent peak or a prior de- termination of which fragments possess the anomeric oxygen. No such prior determination has been conducted for L-fucose. The mass spectral patterns we obtained for unlabelled and labelled L-fucose in its trimethylsilylated forms are shown in Fig. 3. A parent ion was not observed. However, a comparative examination of the peaks shows that ion species represented by fragments 347, 393 and 437 in the control spectrum retain their 180 (peaks 349, 395 and 439) in the labelled fucose spectrum (Table I). Thus, these peaks were used to determine 180 content of the labelled L-fucose. This calculation yielded a figure of 53 atom% excess of 180 in the oxygen at the anomeric carbon of L-fucose.

This L-[180]fucose was used as a substrate with fucokinase as described in the Methods section. Product L-fucose 1-phosphate was isolated and the phosphate was hydrolytically cleaved from the sugar moiety at an acid pH reported to cleave the bond between the anomeric carbon and its oxygen [19]. Thus, inorganic phosphate is produced which should contain 180 if product a-L-fucose con- tained that label. The inorganic phosphate was converted to its triethylphosphate form and ex- amined by mass spectroscopy. Fragmentation pat- terns have already been established for triethyl-

100 - 319

241

>- I---

Z uJ I - z ~ LU _> I -

._1 ILl

347

I

3~9

395

349

M/E

Fig. 3. Mass spectral patterns for silylated 160- and 1sO-labelled L-fucose generated by chemical ionization (CH 4).

phosphate by McLafferty [20] who showed an initial double rearrangement leading to loss of a -C2 H 3 fragment and two successive -C2H 2 losses yielding prominent 155, 127 and 99 peaks. These prominent peaks, representing no loss in oxygen, and their single 180-substituted counterparts (157,

TABLE I

FRAGMENTATION PATTERN FOR TRIMETHYLSILYL- L-FUCOSE

M/E Loss Probable components lost

319 133 - O C H CH3(44), -OTMS a(89) 347 105 -HOTMS(90), - C H 3(15) 363 89 -OTMS a 393 59 -CH3, - O C H C H 3 437 15 - C H 3

a Anomeric -OTMS group, since 180 is lost from the resulting fragment.

242

129, 101) were used to calculate the a80 content of phosphate derived from L-fucose 1-phosphate. An 180 value of 16.3 atom% excess was obtained for the single 180-substituted phosphate species. This value is approx, one third the initial 180 content of the precursor fucose. The appearance of 180 in the derived phosphate unequivocally establishes mech- anism A as the appropriate enzymatic process, since release of 180 to water by any other mecha- nism would result in enormous dilutions of the tracer, such that it would be undetectable from natural abundance.

The extent of loss of label is puzzling and requires an explanation. Loss of label through cleavage of the wrong oxygen bond at the anomeric center or through exchange of orthophosphate oxygen seems unlikely under the conditions used [19,22]. An alternative possibility is that the mech- anism whereby L-fucose was labelled with 180 might operate sufficiently rapidly in reverse during the enzymatic incubation period to effect the loss. Thus, 180-L-fucose might mutarotate through the open chain aldehyde which, upon hydration-dehy- dration, loses its 180 to water, Studies with D-glu- cose [23] and D-galactose [24] set the mutarotation rate as approx. 1000-times faster than the rate for anomeric exchange with these sugars. We observed a half-time value of approx. 8 min for the mutaro- tation of L-fucose from the a, to the a, fl mixture at 25°C. Correlating this value for the examples D-glucose and D-galactose would suggest that a period of over 130 h might be required to effec- tively exchange one-half the 180 from anomeri- cally labelled L-fucose. In order to verify this value we studied 180 loss from the anomeric position of L-fucose as a function of time. Fig. 4 shows the data obtained from that study. Surprisingly, the half-time for anomeric exchange is seen to be approx. 1.8 h. Thus, the anomeric exchange rate for L-fucose is approx. 70-fold faster than that reported for D-glucose and D-galactose, based on the mutarotation relationship, This exchange rate accounts for the major portion of the 180 lost during the enzymatic incubation period. Why L- fucose exchanges more rapidly than D-glucose or D-galactose is not immediately clear, but the ex- planation must surely be concerned with the sta- bility of the cyclic ring structures [25].

The data of these studies clearly establish that

100

ao

.~ 60

40 t~

2C

HOURS

Fig. 4. Loss of anomeric 180 from L-fucose as a function of time.

the immediate precursor of fucokinase is fl-L-fucose and that the enzymatic mechanism operative in the formation of fl-L-fucose 1-phosphate involves a nucleophilic attack of the anomeric oxygen on the 7-phosphate of ATP as portrayed in mechanism A. Thus, the potential for carbonium ion formation, expressed in other systems, does not play a part in the enzymatic phosphorylation of L-fucose at its anomeric center. It is probable that similar cir- cumstances obtain with D-galactokinase, N-acetyl- D-galactosamine kinase and o-glucuronic acid kinase.

Acknowledgement

This work was supported by Grant No. PCM- 7911032 from the National Science Foundation.

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