MALATE SYNTHETASE 233

a2 W. S. DENHAM AND H. WOODHOUSE, J. Chem. SOL, (1913) 1861. *8 R. R. GRUNERT AND P. H. PHILLIPS, Arch. Biochem., 30 (1951) 217. e4 M. CALVIN AND A. A. BENSON, Scielzce, Iog (1949) 140. as H. L. KORNBERG, Biochem. J., 68 (1958) 535. 26 J. P. HUMMEL, J. Biol. Chem., 180 (1949) 1225. 17 D. E. HUGHES, Brit. J. Exptl. Path., 32 (1951) 97. 28 E. R. STADTMAN, in S. P. COLOWICK AND N. 0. KAPLAN, Methods in Enzymology, Vol. I. Aca-

demic Press, Inc., New York, 1955, p. 596. 29 H. MICHL, Naturwissenschnften, 40 (1953) 390. e” P. W. WEST AND N. COLL, J. Am. Waterworks Assoc., 4g (‘957) 1485. 31 J. GERGELY, P. HELE AND C. V. RAMAKRISHNAN, J. Biol. Chem., 198 (1952) 323. 82 H. LINEWEAVER AND D. BURK, J. Am. Chem. Sot., 56 (1934) 658. 33 S. OCHOA, in S. P. COLOWICK AND N. 0. KAPLAN, Methods in Enzymology, Vol. I. Academic

Press, Inc., New York, 1955, p. 685. 34 K. BURTON, personal communication. s A. MEISTER, J. Biol. Chem., 197 (1952) 309. se T. WIELAND AND H. K~PPE, Ann. Chem. Liebigs, 581 (1953) I. 37 A. MARCUS AND B. VENNESLAND, 1. Biol. Chem., 233 (1958) 727. s8 J. Bovt, R. 0. MARTIN, L. L. INGRAHAM AND P. K. STUMPF, J. Biol. Chem., 234 (1959) ggg. 39 F. LYNEN, Federation Proc., 12 (1953) 683.

Biochim. Biophys. Acta, 41 (1960) 217-233

SUBSTR.4TE SPECIFICITY OF SACCHAROMYCES FRAGILE

GALACTOKINASE

FRANCISCO ALVARADO*

Department of Enzymology, Centro de Investigaciones Biologicas, C.S.I.C., Madrid (Spain)

(Received November zIst, 1959)

SUMMARY

The substrate specificity of Saccharomyces fragilis galactokinase has been investigated

using a series of compounds structurally related to galactose. Only those compounds,

which, having the a-pyranose configuration, differ from galactose at the level of C2

(z-deoxygalactose, galactosamine, talose) have been found to be phosphorylated.

Changes at C3, C4 or C6 (gulose, glucose, L-arabinose, D-fucose, D-glycero-D-galacto-

heptose) result in compounds towards which this enzyme is inactive. The effect of the

concentration of ATP on the activity of the enzyme has also been explored.

INTRODUCTION

Galactokinase was discovered independently and almost simultaneously by LELOIR

and collaborators1 and by WILLKINSON 2. Some general properties of the enzyme

from yeast and mammalian organs have been described (see ref. 3), but very little is

known about its substrate specificity. WILLKINSON has shown that tagatose is not

* Present address: Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, N.Y.

Biochim. Biophys. Actn, 41 (1960) 233-238

234 F. ALVARADO

phosphorylated by the yeast enzyme 2. CARDINI AND LELOIR described the phosphory- lation of galactose and galactosamine by yeast, brain and liver extracts4; however, the identity of the enzyme(s) catalyzing these phosphorylations was not conclusively established.

The characterization of the substrate specificity of the galactokinase of adapted Saccharomyces fragilis was of interest to us, not only from a general point of view, but also because of its relationship to studies undertaken in our laboratory on the mechanism of transport of galactose into the intact yeast cell 5.

In this work an experimental approach was used similar to that of SOLS AND CRANE in their fundamental s tudy of brain hexokinase 6. For a more detailed theo- retical discussion reference may be made to their paper. In order to establish which parts of the sugar molecule are essential for the enzyme-substrate complex formation, a series of compounds structurally related to galactose have been assayed. I t appears that galactokinase has a rather narrow spectrum of specificity. Both apparent affinity and maximal velocity decrease when modifications are introduced at the level of the second carbon atom of the galactose molecule (2-deoxygalactose, galactosamine, talose), but the resulting compound is still phosphorylated. Modifications of galactose at carbon atoms 3, 4 or 6 (gulose, glucose, L-arabinose, fucose, D-glycero-D-galacto- heptose) were associated with both a disappearance of measurable phosphorylative capacity by galactokinase and the absence of detectable inhibitory action on galacto- kinase. The effect of adenosinetriphosphate (ATP) concentration on the reaction rate has also been investigated.

MATERIALS AND METHODS

This work was carried out with a strain of Saccharomyces fragilis obtained from the Inst i tuto Jaime Ferr~n de Microbiologia at Madrid. The yeast was grown in Roux bottles containing a yeast extract-galactose medium* (2 % galactose).

Galactokinase was extracted** and purified as described by TRUCCO et al. 1. Further purification and elimination of contaminating hexokinase are described in the text.

Compounds used. Talose and D-glycero-D-galactoheptose were obtained from Dr. N. K. RICHTMYER; tagatose and 2-deoxygalactose from Light. Calcium gulonate was from General Biochemicals, Inc.; calcium was removed by means of potassium phosphate, pH 7- Galactose, galactosamine, dulcitol, L-arabinose, fructose and glucose were obtained from Pfanstiehl. Galactose was treated with baker 's yeast in order to remove contaminating glucose. L-arabinose was recrystallized from alcohol until its contaminating galactose content decreased below 0.2 %. Galactosamine hydrochloride was neutralized with KOH before use. All sugars were allowed to attain mutarotat ion equilibrium. Excepting L-arabinose, all the sugars belonged to the D-series.

ATP was obtained from Sigma. Ethylenediaminotetraacetate (Versene) was the disodium salt supplied by the Bersworth Chemical Co. Bentonite from Tidinit was washed with HC1 and neutralized before use.

* I wish to express m y grat i tude to Dr. R. BELTR/~ for her help in the prepara t ion of the cultures.

** Crude prepara t ions contained 4,o0o galactokinase and i o,ooo hexokinase units (as measured on glucose) per gram of dried yeast.

Biochim. Biophys. Acla, 41 (I96o) 233--238

SPECIFICITY OF YEAST GALACTOKINASE 235

Enzymic assays. Two methods were principally used; the colorimetric indicator method of SOLS AND CRANE 6 and the reducing power disappearance methodk In this latter case, baritum-zinc filtrates 7 of the incubation mixtures were analyzed for reducing power with the SOMOGYI method s for common sugars, or with the SHAFFER- SOMOGYI method for deoxysugars 9. In the case of galactokinase, since the reaction product is a non-reducing ester, Ba-Zn treatment can be omitted, as shown in Fig. I. The reaction may be stopped with Versene 1°, residual reducing power being directly estimated in aliquots. The ATP and protein at the concentrations used do not inter- fere. If a crude preparation of enzyme is used, protein must be eliminated.

Fig" I" P h ° s p h ° r y l a t i ° n ° f tal°se" 5 # m ° l e s °f ta - ~ lose, 7.5 /~moles of ATP, 3.75 # m o l e s MgSO4, io /~moles fluoride, 2 5 /~moles p H 7 m a l e a t e buffer, :~ a . a n d 25 un i t s of e n z y m e were i n c u b a t e d a t 3 °0 in a final v o l u m e of 0. 4 ml . Series C) was t r ea t ed ~, w i th B a - Z n I. Series • was t r ea t ed w i t hVer sene 1°. o~ Res idua l or free ta lose was e s t i m a t e d in a l iquots s.

/°

2 4 6 Time (hours)

An enzyme unit is defined as the amount of enzyme which can catalyze the phosphorylation of I ~mole of hexose in 15 min of incubation at 3 o°.

Protein was estimated according to LOWRY et al. n. If not stated otherwise, assays were carried out at pH 6.4-7.21 and in the presence of saturating concentrations of ATP and Mg (ratio ATP/Mg ---- 2). Fluoride was used in the incubation mixtures; no trace of interfering phosphatases was observed.

EXPERIMENTAL

In Table I, the Michaelis constantsl relative maximal rates and phosphorylation coefficients of the compounds tested are specified.

Evaluation of Michaelis constants. When possible (compounds I, 4, 5 and 6, Table I), Michaelis constants were measured directly by studying the effect of substrate concentration on reaction rate; LINEWEAVER AND B U R K plots 12 were used for the calculations. The colorimetric indicator method was employed whenever possible (all compounds except 4 in Table I) because of its sensitivity. The effect of varying concentrations of substrate on phosphorylation rate was studied at a constant volume. Galactosamine required a special approach, which is described later.

Minimal values for the inhibition constants of different compounds were calcu- lated from their inhibitory effect on galactose phosphorylation, using either the colorimetric indicator method (dulcitol, tagatose) or the reducing power disappearance method (dulcitol).

Relative maximal velocities. Vmax for galactose was measured directly at saturat- ing concentrations of substrate. For other substrates (compounds 4, 5 and 6, Table I), maximal velocities were calculated from the experimental data. A minimal value for the concentrations used was calculated in the case of the apparently inert com- pounds (those labeled with a dash, column 4, Table I).

Biochim. Biophys. Acta, 41 (196o) 233-238

236 F. ALVARADO

TABLE I

SUBSTRATE SPECIFICITY OF Saccharomyces [ragilis GALACTOKINASE

Compound Modified at Kin* Relative I'Phosphorylation carbon moles/l maximal rate** coe~cient***

I Galactose 3" IO~t i i 2 Dulcitol i - - - - - - 3 Tagatose i, 2 - - - - - - 4 Galactosamine 2 I • lO-2§ 0. 4 1.2. lO -2 5 2-deoxygalactose 2 2. lO -2 0. 4 6. lo -3 6Talose 2 2- io 1 o.i 1.5.to-4 7 Gulose 3 - - - - - - 8 Glucose 4 - - - - - - 9 Fucose§§ 6 - - - - - -

Io L-arabinose 6 - - - - - - 11 D-glycero-D-galacto- 6 - - - - - -

heptose

* The Km values are not corrected for mutarrotation equilibria. A dash in this column indicates a Km bigger than 5" lo-1 ~I.

** A dash in this column indicates an undetectable velocity (less than o.oI at a concentration of i . l o -2 M o r g r e a t e r ) .

*** A dash in this column indicates that the compound appeared to be inert both as a substrate and as a competitive inhibitor to the extent that it cannot have a phosphorylation coefficient greater than 5" lO-5.

§ At pH 8; see the text. §§ Unpublished observations of Dr. C. F. DE HEREDIA.

Phosphorylat ion coe~cients were c a l c u l a t e d f rom t h e Km a n d m a x i m a l r a t e

va lues n, or a m i n i m a l f igure was c a l c u l a t e d 1° in t h o s e cases w h e r e no a p p r e c i a b l e

p h o s p h o r y l a t i o n or i n h i b i t o r y effects cou ld be o b s e r v e d .

Test for fa lse positives. Since m o s t ava i l ab le sugar s c o n t a i n v a r i ab l e a m o u n t s

of o t h e r suga r s as impur i t i e s , e x p e r i m e n t s were ca r r i ed ou t to d e t e c t a n y p o s i t i v e

r e su l t s t h a t cou ld be e r roneous . A t an a v e r a g e c o n c e n t r a t i o n of I . IO ~ M , e v e r y

p r e s u m p t i v e s u b s t r a t e was i n c u b a t e d w i t h A T P - M g a n d an excess of e n z y m e long

e n o u g h to a s su re t h e close a p p r o a c h to r e a c t i o n c o m p l e t i o n . The fo l lowing p e r c e n t a g e s

of p h o s p h o r y l a t i o n were o b t a i n e d : 2 - d e o x y g a l a c t o s e , ove r 95; g a l a c t o s a m i n e , 90;

ta lose , 7 o. W i t h t h e o t h e r c o m p o u n d s t e s t ed , t he d i s a p p e a r a n c e of r e d u c i n g p o w e r

was less t h a n 5 %.

Glucose. To s t u d y g lucose as a p r e s u m p t i v e s u b s t r a t e , it was n e c e s s a r y to o b t a i n

g a l a c t o k i n a s e p r e p a r a t i o n s e s sen t i a l l y free of h e x o k i n a s e . Th is was a c c o m p l i s h e d

b y a d s o r b i n g the i n t e r f e r i n g e n z y m e on p H 6.4 b e n t o n i t e (IOO rag/ rag p ro te in ) .

T h e r e l a t i ve r a t e s of p h o s p h o r y l a t i o n of g lucose , f ruc tose a n d ga l ac to se in t h e c r u d e

e x t r a c t s were I : 1. 5 : 0.4, w h e r e a s in t he m o r e pur i f ied p r e p a r a t i o n s t h e y were I : 1.5 : 17.

T h e ra t io g l u c o s e : f r u c t o s e is s imi la r to t h a t f o u n d in a v a r i e t y of y e a s t s 13. T h e fac t

t h a t th i s r a t io d id n o t c h a n g e a f t e r t he r e l a t ive c o n c e n t r a t i o n of g a l a c t o k i n a s e ( abou t

4 ° t imes) sugges t s t h a t t he p h o s p h o r y l a t i o n of b o t h g lucose a n d f ru c t o s e is due on ly

to one e n z y m e , n a m e l y , h e x o k i n a s e . T h e negl ig ible a f f in i ty of g a l a c t o k i n a s e for

g lucose is also e v i d e n c e d b y t h e l ack of a p p r e c i a b l e i n h i b i t o r y effect of g lucose on

ga l ac to se p h o s p h o r y l a t i o n , e v e n a t t h e e n o r m o u s g lucose c o n c e n t r a t i o n u s e d (more

t h a n 400 t i m e s t h e c o n c e n t r a t i o n of ga l ac to se ; see Fig. 3)-

Galactosamine. The mod i f i ed r e d u c i n g p o w e r d i s a p p e a r a n c e m e t h o d h a s p r o v i d e d

Biochim. Biophys. Acla, 41 (196o) 233-238

SPECIFICITY OF YEAST GALACTOKINASE 237

an especially useful tool for the study of galactosamine phosphorylation. Neither the normal colorimetrie indicator method nor the reducing power disappearance method can be employed in this special case, since galactosamine is variably ionized at the pH range 6 to 8 and since free aminosugars are partially adsorbed on the barium

/

1 6 ~ - o o o o . . t -

L I . . t

2 ~ f J , I , I , I

0 1 3 [_1siX 10_ t S 7

Fig. 2. Effect of ga l ac t o sami ne concen t ra t ion on veloci ty. • : 5/~moles ga lac tosamine , 7.5 #,moles ATP, 3.75 /zmoles MgSO 4, 2oo/~moles t r i s (hy- d r o x y m e t h y l a m i n o m e t h a n e ) p H 8 and 6. 7 ga- l ac tok inase un i t s were i ncuba t ed for a n hou r a t 3 °0 . The final v o l u m e of the i ncuba t ion m i x t u r e s r anged f rom o.25 to 2.65 ml . Reac t ions were s topped wi th Versene 1° and v o l u m e s comple t ed to 5 ml . Res idua l ga l ac t o sami ne was de te rmined in I m l a l iquots 8. Veloci ty and concen t r a t ion are p lo t t ed as reciprocals 1~. A paral le l exper i - m e n t was pe r fo rmed wi th ga lac tose (©) in s t ead of ga l ac to samine (6. 7 ga lac tok inase uni ts , six

m i n u t e s of i ncuba t ion a t p H 7).

¢=

t -

I -

E

, . c D .

30C

20C

\ \

2~0 i 6~0 ~ l i l 0 80 100 120 140 Time (minutes)

Fig. 3- Glucose as a p r e s u m p t i v e subs t ra te . P h o t o m e t r i c ind ica tor me thod . Ga lac tok inase p repara t ion , descr ibed in the text . Jk, I M glucose; &, o.oo 5 M fructose; O, o.oo25 M galac tose; O , o.oo25 M galac tose p lus I M

glucose; + b l ank wi thou t sugar .

sulfate gel. A constant amount of galactosamine was incubated (conditions explained in Fig. 2) with the enzyme and an excess of ATP-Mg, the substrate concentration being varied by increasing with water the total volume of the incubation mixtures. That velocity changes are not due to the dilution of ATP, Mg or enzyme, is supported by the lack of unspecific dilution effects on the rate of phosphorylation of a substrate with high affinity, as galactose. The Km of galactosamine rises with pH from a minimum below pH 6.5 to a maximum above pH 8. Preliminary results indicate that the increase in apparent affinity describes an S-shaped curve which can be superimposed on the galactosamine hydrochloride titration curve. As it happens in the case of hexokinase and glucosamine 6 only the non-ionized form of the amino- sugar seems able to combine with the enzyme, the galactosammonium ion being inert.

Influence of A TP concentration. This has been studied by using a colorimetric mixture containing 3.7" lO-3 M Mg+÷ and galactose at a concentration more than ten times above Kin. Reactions were started by adding variable amounts of ATP. At a concentration of ATP of I . lO -4 M, velocity was about half maximal or less.

DISCUSSION

It seems clear that, since the galactokinase reaction product is the Kosterlitz ester, ~-D-galactopyranose-I-phosphateX, 2,14 phosphorylation takes place through the glycosidic hydroxyl of the a-pyranose ring. This is supported by the fact that com- pounds closely related to galactose, but lacking the adequate configuration, have undetectable affinity (dulcitol, tagatose). A compound such as the 1,5-anhydro- galactitol, which has the same configuration as galactopyranose, but lacks the

Biochim. Biophys. Acta, 41 (196o) 233-238

238 F. ALVARADO

hydroxyl group necessary for phosphorylation, might be expected to be a strong competitive inhibitor.

Galactokinase seems to have a rather narrow spectrum of specificity, since only compounds which differ from galactose by their configuration at carbon atom 2 have been found to be phosphorylated (2-deoxygalactose, galactosamine, talose). Even so, phosphorylation coefficients of these compounds are about or below I . lO -2. An inversion of the hydroxyl group at C3 (gulose) or C 4 (glucose); the absence of C6 (L-arabinose), the absence of the oxygen atom at C6 (D-fucose), or the presence of an extra C 7 carbon atom (D-glycero-n-galactoheptose) make the apparent affinity drop almost to zero. The list of compounds tested is small; other sugars seem worthy of study, particularly derivatives with slight modifications (deoxy, deoxy-amino, etc.) at the level of carbon atoms 3 and 4, where, together with C6, the enzyme-substrate complex may be formed. In analogy with hexokinase, it will probably be interesting to test N-acetylgalactosamine as a presumptive competitive inhibitor of the galacto- kinase. The high Km and low phosphorylation coefficient of galactosamine supports the view that this sugar is a marginal substrate of galactokinase, rather than the physiological substrate of a different enzyme.

The apparent Km of ATP is similar in the case of both galactokinase and hexose- kinase; similar value has been found for the hexokinase of S. cerevisiae 1°.

ACKNOWLEDGEMENTS

I wish to thank Dr. A. SOLS for his interest and advice in this investigation, and Dr. K. R. HANSON for his valuable criticism of the manuscript.

This work has been supported in part by a grant of the Comisaria de Proteccion Escolar, Ministerio de Educacion Nacional, Madrid, Spain.

R E F E R E N C E S

1 R. E. TRUCCO, R. CAPUTTO, L. F. LELOIR AND N. MITTELMAN, Arch. Biochem., 18 (1948) 137. 2 j . F. WILLKINSON, Biochem. J., 44 (1949) 460. 8 L. F. LELOIR AND R. E. TRUCCO, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology,

Vol. I, Academic Press, New York, 1955, p. 290. 4 C. E. CARDINI AND L. F. LELOIR, Arch. Biochem. Biophys., 45 (1953) 55. 5 A. SOLS, G. DE LA FUENTE AND F. ALVARADO, 4th Intern. Congr. Biochemistry, Abstr. commun.,

1958, p. 78. s A. SOLS AND R. K. CRANE, J. Biol. Chem., 21o (1954) 581. ? M. SOMOGYI, J. Biol. Chem., 16o (1945) 69. s M. SOMOGYI, J. Biol. Chem., 195 (1952) 19.

A. SOLS AND R. K. CRANE, J. Biol. Chem., 206 (1954) 925 . x0 A. SOLS, G. DE LA FUENTE, C. VILLAR-PALASI AND C. ASENSlO, Biochim. Biophys. Acts, 3 °

(1958) IOI. 11 O. FI. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 12 H. LINEWEAVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658. 18 A. SOLS, i . LOSADA AND M. ROSELL, unpubl ished observations. 14 H. W'. KOSTERLITZ, Biochem. J., 37 (1943) 318, 322.

Biochim. Biophys. Acts, 41 (196o) 233-238