Determination of the scissile bond in the hydrolysis of α-d-ribofuranose 1-phosphate by alkaline phosphatase, acid phosphatase and formic acid and in its conversion to d-ribose 5-phosphate

Biochimica et Biophysica Acta, 704 (1982) 427-436 427 Elsevier Biomedical Press

BBA31186

DETERMINATION OF THE SCISSILE BOND IN THE HYDROLYSIS OF a.D-RIBOFURANOSE 1.PHOSPHATE BY ALKALINE PHOSPHATASE, ACID PHOSPHATASE AND FORMIC ACID AND IN ITS CONVERSION TO D-RIBOSE 5-PHOSPHATE BY PHOSPHOGLUCOMUTASE

lSO SHIFT ON THE 31P-NMR AND MASS SPECTROSCOPIC EVIDENCE

FRANK JORDAN, DONALD J. KUO, SALVATORE J. SALAMONE and ALICE L. WANG

Carl A. Olson Chemical Laboratories, Rutgers, State University of New Jersey, Newark, NJ 07102 (U.S.A.)

(Received December 29th, 1981)

Key words: Ribose phosphate hydrolysis," Alkaline phosphatase; Acid phosphatase; Formic acid; Phosphoglucomutase

a-D-Ribofuranose 1-[1804]phosphate (made enzymatically employing purine nucleoside phosphorylase, inosine, and plSo4) was hydrolyzed in H2160 by alkaline phosphatase, acid phosphatase, formic and hydrochloric acids. The two enzymes cleaved the O-P bond, the acids the C-O bond. Phosphoglucomutase formed the O-P bond of D.ribose 5-phosphate when starting with a.D-ribofuranosyl 1-[ lSO4]phosphate. The two techniques employed in the analysis (lSO shift on the 31p.NMR and mass spectrometry) were in total agreement with the above conclusions. The mass spectroscopic approach was also employed to demonstrate O-P bond cleavage by alkaline phosphatase in 5'-AMP and in D-ribose 5-phosphate. These results were obtained by hydrolysis of 160-labeiled substrate in 85 atom% H21sO on a micro level (50 pl).

Alkyl phosphate monoesters (R-O-PO3 2- where R can be a variety of groups, including sugar, nucleosides, etc.) are substrates for many enzymatic reactions that involve transfer of phosphate either to water or to another acceptor. To study the details of the mechanism of such a reaction employing, for example, chiral phosphates, kinetic and equilibrium isotope effects and positional isotope exchange kinetics, conditions must be found under which exclusive R-O or O-P scission takes place. Such conditions are reported here for a-D-ribofuranose 1-phosphate. This mole- cule is of importance in the purine salvage pathway, the first reaction of which is catalyzed by purine nucleoside phosphorylase (EC 2.4.2.1):

guanosine + Pi ~ guanine (inosine) (hypoxanthine)

+ a-D-ribofuranose 1-phosphate (1)

0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

This enzyme can form very highly enriched ot-D- ribofuranose 1-[18 04 ]phosphate from pI804, since it proceeds by C-O cleavage [1,2]. The highly 180- labelled a-D-ribofuranose 1-[1804]phosphate was found to be cleaved exclusively at the C-O bond by acids, and at the O-P bond enzymaticaUy. Determination of the scissile bond was confirmed by two techniques: (a) the ~80 isotopic shift on the 31p-NMR [3,4] and (b) mass spectroscopy on the volatile triethyl phosphates. Both techniques are much simpler than the isotope-ratio mass spectroscopic method employed in the pioneering studies of this genre [5]. The two methods gave results in total agreement with each other. Also, in some cases a mass spectroscopic approach was employed subsequent to cleavage of unlabelled substrates in highly enriched H2~80 on as little as 50 /xl solution volume.

428

Experimental procedures

Purine nucleoside phosphorylase from calf spleen was purchased from Boehringer Mannheim Biochemicals, Indianapolis, IN, as a crystalline suspension in 3.2 M (NH4)2SO 4 and had a specific activity of 26.4 units/mg protein. Xanthine oxidase isolated from buttermilk was purchased from Sigma, St. Louis, MO, as a suspension in 2.3 M (NH4)2SO 4 and had a specific activity of 1.68 units/mg protein.

Purine nucleoside phosphorylase activity was measured spectrophotometrically by the coupled xanthine oxidase method according to the method of Kalckar [6].

KH2P[180]O4 was synthesized from PC15 and HI280 (99 atom% 180, Norsk Hydro, New York) following the procedure of Risley and Van Etten [7]. According to 31p-NMR [3,4] it had at least 95% pI804 and the remainder p16OI803, ot-D- Ribofuranose 1-[18 04 ]phosphate was synthesized enzymatically. Typically, 81 mg [ISo4]P i (prepared as above, 555 #mol) was mixed with 210 mg (783 #mol) inosine in 20 ml 0.1 M (pH 7.4) Tris-HCl buffer. 10 units of xanthine oxidase and 8 units of purine nucleoside phosphorylase were added for each of three consecutive days at 4°C. The reaction progress was monitored by ultraviolet light at 249 nm (interconversion of inosine = hypoxanthine) and 293 nm (formation of uric acid from hypoxanthine). The reaction towards a- D-ribofuranose 1-phosphate synthesis was

inosine + Pi = hypoxanthine

xanthine + ribose- 1-P ~ uric acid (2)

oxidase

made irreversible by conversion of hypoxanthine to uric acid. The reaction could be made more efficient (80% or better yields of uric acid) by continuously bubbling 02 through the system. After the incubation the mixture was gravity- filtered and the filtrate absorbed on Norit A until the absorbances at 249 and 293 nm approached zero (nucleic bases and nucleoside were absorbed on the charcoal). Next, the filtrate was chromatographed on Dowex-l-HCO 3- and eluted with 0.15 M NH4HCO 3. The elution was monitored for both phosphate content and ribose content by the

Ames [8] and orcinol [9] tests, respectively allow- ing identification of the ribose 1-phosphate and Pi peaks. The fractions corresponding to a-D-ribofuranose 1-phosphate were pooled and lyophilized. 31p-NMR demonstrated that [1804] enrichment was nearly quantitative by addition of authentic a-D-ribofuranose 1-[1604]phosphate and measure- ment of the chemical shift difference between the two species [1,2].

Alkaline phosphatase (EC 3.1.3.1) type VII from calf intestine, acid phosphatase (EC 3.1.3.2) type III from potato, and phosphoglucomutase (EC 2.7. 5.1) from rabbit muscle were all obtained from Sigma, St. Louis, MO.

31p-NMR spectra were recorded at 145.7 MHz on a Bruker WH 360 spectrometer equipped with a deuterium lock and operating at 22°C in the Fourier transform mode. Samples were run in 10-mm sample tubes, sometimes with cylindrical inserts (Wilmad, N J) to reduce the sample volume. All experiments were performed in 80/20 (v/v) H20/2H2 O (the latter needed for lock). When sugar phosphates were examined the 31p spectrum was run under inverse gated decoupling conditions [101.

Preparation of samples for 31P-NMR experiments Alkaline phosphatase and formic acid hydrolysis.

A 4.5 ml 80/20 (v/v) H20/2H20 solution was made up to contain 16.5 mM a-D-ribofuranose 1-phosphate dicyclohexylammonium salt, 22 mM plSo 4, 10 mM EDTA, 4 mg of a hypoxanthine/inosine mixture (0.2 mM hypoxanthine/10.6 mM inosine) at pH 6.81--+0.1. 4 units of purine nucleoside phosphorylase from calf spleen were added, the solution was equilibrated at 37°C for 60 rain and the 31p-NMR spectrum was recorded. The pH of one 1.5-ml portion was raised to 10.5 and 20 units of alkaline phosphatase were added to it. The mixture was then incubated for 30 min at 37°C, the pH was readjusted to 8.1 and the 3~P-NMR spectrum was recorded. To another 1.5- ml portion an equal volume of 90% formic acid was added and the mixture was incubated for 20 min at 37°C. Next the solvents were evaporated to dryness in vacuo. The residue was redissolved in 1.5 ml 80/20 H20/2H20 and the pH was ad-

justed to 8.1 prior to recording of the 31p-NMR spectrum.

Acid phosphatase. A 3.0-ml 80/20 (v/v) H20 /2H20 mixture was made 6.6 mM in a- D-ribofuranose 1-phosphate dicyclohexylammonium salt, 7.85 mM in plSo 4, 5.0 mM in EDTA, 0.13 mM in hypoxanthine, 7.0 mM in inosine. This solution was incubated at 37°C for 90 min with 3 units of purine nucleoside phosphorylase from calf spleen at pH 6.91. Next, 1.5 ml of this was ad- justed to pH 4.77; 5.3 units of acid phosphatase were added and the mixture was incubated at 37°C for 3 h. The pH was then raised to 7.12 and the 31p-NMR spectrum was recorded.

Phosphoglucomutase. 200 #1 of a solution were made up to contain 35 mM a-D-ribofuranose 1- [16 04 ]phosphate dicyclohexylammonium salt, 17 mM a-D-ribofuranose 1-[1804]phosphate, 30 units phosphoglucomutase, 1.4 mM MgC12, 60 mM im- idazole, 3 mM Tris-HC1, approx. 0.1 mg glucose 1,6-diphosphate at pH 7.0 and equilibrated at about 25°C for 3 h. Before recording the spectrum 300 ~1 15 mM EDTA and 100 #1 2H20 were added and the pH was raised to 7.37.

Mass spectrometric determination of the position of bond cleavage

Chemical and enzymatic reactions producing Pi. Method 1. Cleavage of a-D-ribofuranose 1- [~804]phosphate in H~60.

(a) Cleavage in HC1 or formic acid. ct-D-RJbo- furanose 1-[ is 04 ]phosphate disodium salt (0.1 ml, 50 mM) was incubated at 25°C with 0.1 ml formic acid (97%) or 0.1 ml 12M HC1 for 1 h at approx. 25oc. Next, the reaction mixture was diluted to 2 ml with H20 and lyophilized. The residue was next taken up into 2 ml water and the solution was applied onto a Dowex-l-X-8 (C1- form) 0.5 5< 3 cm column that was washed previously with 10 ml water. The column was eluted with 20 mM HC1, the phosphate eluting in the first 12 ml. The eluate was lyophilized to remove the solvent and the residue was extracted with 2 × 1 ml methanol. The combined methanol extract was ethylated with diazoethane in diethyl ether. The diazoethane was generated in diethyl ether from N-ethyl-N'-nitro- N-nitrosoguanidine in a mixture of 1.5 (v/v) of 50% KOH/diethyl ether [11].

(b) Cleavage by phosphatases, Acid phosphatase cleavage was performed by incubation of a mixture of 500 #1 of 0.1 M Bistris (pH 6.11), 50

429

#1 acid phosphatase (0.6 units), 10/~10.1 M MgC12, and 260 #1 12 mM a-D-ribofuranose 1-[mSO4]phos - phate and 180 #1 water at 25°C for 1 h. Then the reaction mixture was diluted to 10 ml with water and applied to a Dowex-l-X (HCO~- form) 0.8 5< 37 cm column. The column was eluted with 0.15 M NH4HCO 3 and phosphate was collected between 107 and 120 ml of eluate. This 15-ml eluate was lyophilized to dryness and taken up in 5 ml water. The solution was next applied to a Dowex-l-X (C1- form) column (that was previously washed with 10 ml water) and was eluted with 0.1 M HCI. The first 5 ml of the eluate were lyophilized and ethylated as above.

Alkaline phosphatase cleavage was performed by incubation of a mixture of 50 #1 1.0 M Tris-HC1 (pH 8.3), 50 /~1 alkaline phosphatase (6.5 units), 260 #1 12 mM a-D-ribofuranose 1-[lSO4]phosphate, 10/~1 0.1 M MgC12 and 630 pl water for 1 h at 25°C. The incubation mixture was next diluted to l0 ml with water and applied to a Dowex-l-X (HCO; form) 0.8 5< 37 cm column. The column was eluted with 0.1 M NH4HCO 3 and phosphate was collected between 108 and 125 ml of eluate. This fraction was next taken through the procedure outlined under acid phosphatase.

Control reactions were run for the alkaline and acid phosphatase to rule out enzyme-catalyzed pI804 -F Ht960 ~ pI604 exchange taking place during the reaction by processing a mixture of 50/50 (w/w) plSo4/pl604 instead of ct-D-ribofuranose 1-[~804]phosphate through all the steps outlined above.

Method 2. Cleavage of 5'-AMP and of D-ribose 5-phosphate by alkaline phosphatase (the procedures for the two were identical: that for 5'-AMP will be described in detail). A mixture of 50 /al 0.2 M Y-AMP (pH 8.4, obtained by pH adjust- ment of a solution of the disodium salt), 3/~1 1 M Tris-HC1 (pH 8.3) and 2#1 of 0.3 M MgC12 was evaporated to dryness on a vacuum pump. To the residue were added 55 #1 85 atom% HI2sO and 5 #1 (13 units) of alkaline phosphatase and the resulting solution was incubatedat 25°C. The progress of the reaction was monitored by determining the amount of free phosphate released [8]. At 60 and 100 min the reaction was 80 and 90% completed, respectively. After 60 rain a 35-#1 portion was removed and pipetted into 1 ml of 0.1 M HC1

430

containing l0 mM EDTA. This solution was next applied to a 0.6 × 16 cm Dowex-l-X-8 formate column (100-200 mesh, formed by elution of the C1- form with 1 N ammonium formate) and eluted with I N formic acid. 5'-AMP was eluted first, followed by phosphate. The fractions containing inorganic phosphate were combined and lyophilized. Next the residue was chromatographed on Dowex-l-X-8 (C1- form) and converted to the triethyl phosphate as described under Method 1. The remainder of the sample after 100 min was treated in the same manner as the sample removed after 60 min. A method with GC, MS determination of tris(trimethylsilyl) phosphate was recently employed to study the position of bond cleavage in Y,5'-cyclic GMP by a phosphodiesterase [12].

Mass spectrometric measurements The solution that was employed in the ethyla-

tion procedure was evaporated to dryness in vacuo (1 Torr). The residue remaining was reconstituted in ethyl acetate and an aliquot of this solution was analyzed by GC-MS using a Finnigan Model 1015D mass spectrometer equipped for chemical ionization and a Finnigan model 9500 GC. The glass GC column 1.83 M long with a 1-mm inside diameter, was packed with 10% SP-1000 on 120- 140 mesh acid-washed chromasorb W. Methane was used as GC carrier gas (3.4 atm) and chemical ionization reagent gas (0.5 Torr). The column, injector, interface oven and transfer-line tempera- tures were 140, 200, 200 and 200°C, respectively. The retention time of triethyl phosphate under these conditions is approx. 90 s. A Finnigan model 6000 data system with revision I software was used to set the mass spectrometer to scan every second between m/e 150 and m/e 250 (10-ms dwell time) and to collect, store and display the resulting spectra. The preference for ethylation of Pi was suggested by a recent comprehensive review of the subject [13] that indicated less spillover and cleaner fragmentation pattern of the triethyl compared to the tris(trimethylsilyl) phosphate and trimethyl phosphate. It should also be emphasized that the analytical GC-MS method employed in these studies was able to de termine quanti ta t ively p180,,/PI604 ratios (by integration) to a few tenths of a percent accuracy. This novel method (to be published elsewhere) would be applicable to a

number of other experiments, e.g., determination of 180 isotope effects in chemical and enzymatic processes.

Results and Discuss ion

Hydrolysis of a-D-ribofuranose 1-phosphate in acids Fig. 1 shows the 31p-NMR spectrum of the

Fornic (~Jd*~O

Po 4

12.5 HZ

P~

Fig. 1. 31p-NMR spectrum (145.7 MHz) of the products of hydrolysis of a mixture of a-D-ribofuranose 1-[1804 ]phosphate and a-o-ribofuranose 1-[]604]phosphate in 50% formic acid. The initial mixture is shown in Fig. 4. Below the spectrum the peak separation is indicated in Hz. Proton decoupling was employed. The asterisked oxygen denotes lSo.

00 60

Fig. 2. Chemical-ionization mass spectrum of the triethyl phosphate obtained from ethylation of the product of hydrolysis of a-D-ribofuranose 1-[lsO4]phosphate in 50% formic acid.

100

.I . . . . . I

65

. . . . . , . . . . . . . . . , . . . . . . . . . , . . . . . . . . . . . . . . . . , . . . . . . . . . , . . . . . . . . . , . . . . . . . . . , = :

IBI ZOO ZDU m / e

Fig. 3. Chemical-ionization mass spectrum of the triethyl phosphate obtained from ethylation of the product of hydrolysis of a-D-ribofuranose 1-[1804]phosphate in 6 M HCI.

431

a-D-glucose 1-phosphate as well [5]. A likely mechanism involves an Sr~l (or A1) process leading to the formation of an oxocarbonium ion which is trapped by water to form ribose, although the results cannot distinguish this stepwise pathway from one involving concerted expulsion of phosphate by water.

Hydrolysis of phosphates by alkaline phosphatase Fig.4 shows the 31P-NMR spectrum of the

products of hydrolysis of a mixture of a-D-ribofuranose 1-[1804]phosphate and a-D-ribofuranose 1-[1604]phosphate by alkaline phosphatase in HI260. Scheme I indicates the products expected depending on whether C-O or O-P cleavage takes place. The fact that p16OI803 is observed suggests O-P cleavage. Under the conditions of the experi- ent exchange of oxygens between phosphate and water is very slow (such exchange would lead to a distribution of Pi s [14] including p1601802 and

products of hydrolysis of a mixture a-D-ribofuranose 1-[!SO4]phosphate and a-D-ribofuranose 1-[ 16 04 ]phosphate in 50% formic acid. The separation of the Pi resonances (12.5 Hz at 145.7 MHz) clearly indicates formation of Pl804 and pI604 [3,4] and C-O cleavage. Figs. 2 and 3 provide the mass spectra of the triethyl phosphates (role 183, 185, 187, 189 and 191 for 0, 1, 2, 3 and 4 180 atoms, respectively) formed in the hydrolysis of a-D-ribofuranose 1-[18 04 ]phosphate by 50% formic acid and 6 M HC1. The observation of role = 191 demonstrates that the phosphate contains no oxygen from H1260 and confirms C-O bond cleavage. It was demonstrated, employing a mass spectral method, that acids cleave the C-O bond of

HO / ~OH H2PO2

H O O +

HO / \OH

4- H 2 0 --, D-r ibose

(3)

R 4 - P

Fig. 4. 31p-NMR spectrum (145.7 MHz) of the products of hydrolysis of a mixture of a-D-ribofuranose 1-[ I s 04 ]phosphate and a-D-ribofuranose l-[1604]phosphat¢ catalyzed by alkaline phosphatase. The upper spectrum demonstrates the purine nucleoside phosphorylasc-catalyzed equilibrated mixture starting with a-D-ribofuranos¢ 1-[1604]phosphate and PIsO 4 (no proton decoupling). The lower spectrum is that of the hydrolysis mixture. The asterisked oxygens denote ISO. Below the spectra the peak separation is indicated in Hz.

432

INITIAL MIX PRODUCTS

H O - - ~ ~, . OP03 . p o 4 c leave p o 4 p o 4

HO HO H20 P

HO-- ' y 'O" ,~ . . ~ . ' 0 PO 3 • p o 4 cleaveo_p pO 4 PC):

HO NO

Scheme I. Products of phosphate hydrolysis. O* = lSo.

p1603180--neither of which is observed). The ratio of the integrals corresponding a-D-ribofuranose 1-[1804]phosphate to pl804 compare well with the ratio of p I 6 o I 8 0 3 to p I804 . Th i s suggests that the resonance corresponding to p~60~803 arose from O-P cleavage of ct-o-ribofuranose 1-[I804]phos - phate absorbing one atom of ~60 from solvent. Fig. 5A illustrates the mass spectrum obtained for the triethyl phosphate resulting from alkaline phosphatase-catalyzed hydrolysis of a-D-ribofuranose 1-[1804]phosphate in H160. The role value of 189 confirms O-P cleavage. Fig. 5B demonstrates the result of the control experiment. A mixture of PI804 and p1604, when treated with the same amount of alkaline phosphatase under the same conditions, led to triethyl phosphates that did not indicate 'washout' of 180 from plSo 4 by n~60. These results are consistent with those of Cohn [5] obtained employing the isotope-ratio mass spectroscopic analysis on a-D-glucose 1-phosphate.

The next set of experiments was performed to exploit a novel and very simple technique, i.e., of performing enzymatic hydrolysis of 160-labelled substrates in highly enriched H~80. The high enrichment allows application of the same GC-MS technique to the derivatized triethyl phosphates for determination of the position of bond cleavage. This method is in contrast to the earlier work in which the availability of low-enrichment H~80 necessitated use of the much more cumbersome isotope-ratio mass spectral method [5,15]. In order to ensure that the observations are not com- plicated by exchange between solvent 180 and phosphate '60, readings of 5'-AMP (Fig. 6) and D-ribose 5-phosphate (Fig. 7) were taken at 60 and 100 min. The results at both times (different frac-

151

00

A

. . . . . . I . ,, .... I,,, q,,.,i.,, rlP,,,I,',, q .,., r ,. q ,;,T,,q,~, ;,1~ .,,1,,hp; ..p-,,T,,q;,,,,i.,, qv.. ~l

m / e zoo

50

00

B

rrv'e

25

Fig. 5. A. Chemical-ionization mass spectrum of the triethyl phosphate obtained from ethylation of the product of hydrolysis of a-D-ribofuranose 1-[ 1804 ]phosphate in H ~60 by alkaline phosphatase. B shows the mass spectrum of triethyl phosphates obtained from the ethylation of a mixture of pI604 and plSo 4 that was processed as was the ribose-l-phosphate in A.

tional reactions) emphasize an m/e value of 185, corresponding to O-P bond cleavage in both mole- cules. Stein and Koshland [15] confirmed O,-P cleavage for 5'-AMP employing the isotope-ratio mass spectroscopic method. The fact that one can

433

I00

A

1~ m/@

I

,55 iO0

A

1 m / e

55

00

B SS

i I,

m / e Fig. 6. Chemical-ionization mass spectrum of the triethyl phosphate obtained from ethylation of the product of hydrolysis of 5':[160,~]AMP in HI, so by alkaline phosphatase after 60 rain (A) and 100 rain (B).

tOO

i5

B

m/e

5S

Fig. 7. Chemical-ionization mass spectrum of the triethyl phosphate obtained from ethylation of the product of hydrolysis of D-ribose 5-[1604]phosphate in H[80 by alkaline phosphatase after 60 min (A) and 100 rain (B).

now perform such reactions on micro quantities, lequiring only 50 #1 highly enriched H~80, makes this approach a very economical one to employ.

A cid phosphatase Fig. 8 illustrates the 31p-NMR spectrum of the

products of hydrolysis of a mixture of a-D-ribo-

furanose 1-[18 04 ]phosphate and a-D-ribofuranose 1-[ 1604]phosphate by acid phosphatase. The pres- ence of pI6oISo3, along with that of pt604 and pISo 4 and the control (as outlined under alkaline phosphatase), indicating no 'washout' of ISo from phosphate by H 16 O during the course of the reaction, proves O-P bond cleavage. Fig. 9A provides

434

139 HZ 0.7 J'~

~ d ~,~.~,,~,o~- ~20

,o2

,

"~2 32 O13

Fig. 8. 31p-NMR spectrum (145.7 Mttz) of the products of hydrolysis of a mixture of a-D-ribofuranose 1-[ I804]phosphate and a-D-ribofuranose l-[1604]phosphate catalyzed by acid phosphatase. The upper spectrum demonstrates the purine nucleoside phosphorylase-catalyzed equilibrated mixture starting with a-D-ribofuranose l-[1604]phosphate and plSo 4 (proton decoupled). The lower spectrum is that of the hydrolysate. The asterisked oxygens denote lSo. Below the spectra the peak separation is indicated in Hz.

the mass spectrum obtained on the triethyl phosphate resulting from ethylation of the acid phosphatase-catalyzed hydrolysis product of a-D-ribofuranose 1-[1804]phosphate in H~60. The m/e value of 189 and the control (Fig. 9B), which indicates no exchange of oxygens between phosphate and water under the reaction conditions, demonstrate O-P bond cleavage. This was also demonstrated for a-D-glucose 1-phosphate [5] employing the isotope-ratio mass spectroscopic technique.

Both phosphatases, as has been reported earlier for other substrates, operate by cleavage of an O-P bond and ultimate attachment of a solvent oxygen to the phosphorus. It has been weU-estabhshed that there exists a phosphoryl enzyme intermediate that eventually does exchange the phosphate and solvent oxygens [ 14,16]. Clearly, conditions can be selected to allow nearly quantitative bond-break-

O0

A 50

i00

B

r77/@

30

I lll 11

Fig. 9. A. Chemical-ionization mass spectrum of the triethyl phosphate obtained from ethylation of the product of hydrolysis of a-D-ribofuranose 1-[ISO4]phosphate in H~60 by acid phosphatase. B shows the mass spectrum of triethyl phosphates obtained from the ethylation of a mixture of pI604 and pISo 4 that was processed as was the ribose l-phosphate in A.

ing prior to exchange taking place, i.e., the exchange pi601803 d- H~60 ~ p1601802 is much slower than the formation of phosphoryl enzyme complex from the sugar phosphate.

P hosphoglucomutase a-D-Ribofuranose 1-phosphate is a poor sub-

strate for rabbit muscle phosphoglucomutase [17]. In our hands it was virtually impossible to make rabbit muscle phosphoglucomutase work in the direction

D-ribose 5-phosphate

a-D-ribofuranose 1-phosphate (4)

even though the equilibrium constant glucose 6- phosphate = glucose 1-phosphate is greater than 0.05. Therefore we could only study the position of bond-formation on the D-ribose 5-phosphate starting with the 1-phosphate. Fig. 10 presents the 31p-NMR spectrum obtained when a mixture of a-D-ribofuranose 1-[1604]phosphate and a- D-ribofuranose 1-[18 04 ]phosphate is equilibrated with rabbit muscle phosphoglucomutase. The two new resonances formed correspond to D-ribose 5-[1604]phosphate and D - r i b o s e 5-[16O1803]phos - phate (the former identified by addition of authentic 1604 material and perfect overlap of the resonances). The 11-Hz separation corresponds to the upfield shift expected for three non-bridging

Pho..~nogiucomutase ÷

1 %POOH

RIBOSE-5-P

RIE~)SE-1 - P

PO4

Po;

.u I . 11 Hz 13.2 Hz

Fig. 10. 31p-NMR spectrum (145.7 MHz) of the mixture obtained by treatment of a mixture of a-D-ribofuranose I- [1604]phosphate and a-D-ribofuranose 1-[lSOa]phosphate (upfield) with phosphoglucomutase from rabbit muscle. The asterisked oxygens denote lSo. Below the peaks the peak separation is indicated in Hz.

435

lSO atoms in an alkyl phosphate, as demonstrated for a-D-ribose 1-phosphates [2]. This experiment demonstrates that the O-P bond of D-ribose is being formed by rabbit muscle phosphoglucomutase, but does not prove that there is an O-P cleavage in the a-D-ribose 1-phosphate. The latter question could only be answered if one could drive the reaction towards formation of a-D-ribose 1- phosphate.

Conclusions

It was demonstrated that the ~SO shift on the 31p-NMR and mass spectroscopic techniques are in total agreement concerning the position of bond-breaking in the phosphate transfers here examined. While the NMR technique is much the simpler of the two, the synthesis of PIsO4-1abelled sugar phosphate is essential. The reaction catalyzed by purine nucleoside phosphorylase is ideal for this purpose, since this enzyme transfers PO 4 intact, a rare case. For cases where synthesis of p~804 substrates is difficult, the direct method employing GC-MS is preferable. As was here demonstrated, one can employ pI604 substrates and hydrolyze in highly enriched H l2sO on a micro scale (50 #1), then convert to the volatile phosphate and perform the GC-MS determination.

Acknowledgements

We are grateful to the National Institutes of Health for grant No. GM 26682, the Charles and Johanna Busch Biomedical Grant (at Rutgers) and the Rutgers Research Council for financial sup- port. We also thank Drs. George McDonald and Dee Huang for help with the NMR spectrometer. The Middle Atlantic NMR facility at the Univer- sity of Pennsylvania is supported by the National Institutes of Health Grant RR 542. We are espe- cially grateful to Dr. William Garland of the Bio- chemistry and Drug Metabolism Section of Hoff- mann-La Roche, Inc., Nutley, N J, for performing the mass spectral determinations.

References

1 Jordan, F., Patrick, J. and Salamone, S.J. (1979) J. Biol. Chem. 254, 2384-2386

436

2 Jordan, F., Salamone, S.J. and Wang, A.L. (1981) ACS Symposium Series No. 171,585-589

3 Colin, M. and Hu, A. (1978) Proc. Natl. Acad. Sci. U.S.A., 200-203

4 Lowe, G. and Sproat, B.S. (1978) J. Chem. Soc. Perkin Trans. 1, 1622-1630

5 Cohn, M. (1949) J. Biol. Chem. 180, 771-781 6 Kalckar, H.M. (1947) J. Biol. Chem. 177, 477-486 7 Risely, J.M. and Van Etten, R.L. (1978) J. Labelled Compd.

Radiopharmacol. 15, 533-534 8 Ames, B.N. (1966) Methods Enzymol. 8, 115-118 9 Albaum, H.G. and Umbreit, W.W. (1947) J. Biol. Chem.

167, 369-376 10 Lerman, C.L. and Cohn, M. (1980) J. Biol. Chem. 255,

8756-8760

11 DeBoer, T.J. and Backer, H.J. (1954) Org. Synth. 34, 96 12 Goldberg, N.D., Walseth, T.F., Stephenson, J.H., Thomas,

P.K. and Graft, G. (1980) J. Biol. Chem. 255, 10344-10347 13 Hackney, D.D., Stempel, K.E. and Boyer, P.D. (1980)

Methods Enzymol. 64, 60-81 14 Black, J.L. and Cohn, M. (1978) J. Biol. Chem. 253, 4082-

4085 15 Stein, S.S. and Koshland, D.E., Jr. (1952) Arch. Biochem.

Biophys. 39, 229-230 16 Engstrom, L. and Argen, G. (1958) Acta Chem. Scand. 12,

357 17 Kammen, H.O. and Koo, R. (1969) J. Biol. Chem. 244,

488-4893

Documents

Determination of the scissile bond in the hydrolysis of α-d-ribofuranose 1-phosphate by alkaline phosphatase, acid phosphatase and formic acid and in its conversion to d-ribose 5-phosphate