Specificity of the phosphate donor in the hexokinase reaction

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 93, 508-513 (1961)

Specificity of the Phosphate Donor in the Hexokinase Reaction ~: R A F A E L J. M A R T I N E Z From the Department of Pediatrics and Biochemistry, University o] Buffalo School of Medicine, BufaIo, New Yorl¢ Received January 9, 1961 ATP and ITP have been demonstrated to transphosphorylate in the yeast hexokinase reaction. Other nueleoside triphosphates were ineffective as phosphate donors. The apparent K,~ for ITP in this reaction is 3.7 × 10.8 M when glucose is the acceptor sugar, and 2.9 × 10.8 M when mannose is the aeceptor. The relative rates of phospholTlation with ATP and ITP as donors for the various sugar acceptors were found to be significantly different. InhibLtion of sugar phosphorylation has been observed with ATP and ITP. The occurrence of inhibition at high triphosphate concentrations is not only a function of the donor but is also related to the phospha£e acceptor. INTRODUCTION

donors other t h a n A T P m a y be physiologically active in the hexokinase reaction, and t h a t the relative rates of m e t a b o l i s m of different sugars m a y be a function of t h e availability of various phosphate donors. The present s t u d y describes experiments on the ability of y e a s t hexokinase to t r a n s fer the terminal phosphate group of A T P , I T P , U T P , C T P , and G T P to hexoses. F o r this kinase, A T P and I T P a p p e a r to be t h e only donors capable of significant t r a n s phosphorylation.

A l t h o u g h the sugar kinases, p a r t i c u l a r l y hexokinase, have been studied extensively in recent years, few a t t e m p t s have been m a d e to determine the specificity of the p h o s p h a t e donor in the reaction. P a r k s et al. (1) reported t h a t I T P 2 and U T P could n o t replace A T P in the fructokinase reaction; whereas Ling and L a r d y (2) demons t r a t e d the p h o s p h o r y l a t i o n of fructose 6phosphate by the e n z y m e phosphohexokinase with I T P and U T P as phosphate donors. Glycerolkinase has been shown to t r a n s p h o s p h o r y l a t e wi~h U T P ; I T P being inactive in this reaction (3). T h e ubiquitous occurrence of the 5'-tr;ohosphates of uridine, inosine, cytosine, ~ nd guanosine makes it interesting to eonslJer t h a t phosphate

MATERIALS AND METHODS ENZYME PREPARATION

1This investigation was supported in part by Research Grants No. G-9618 from the National Science Foundation and No. B-1960 and B-9092 of the National Institutes of Health, U. S. Public Health Service. ~The following abbreviations are employed: ATP, ITP, UTP, GTP, and CTP; the 5'-triphosphates of adenosine, inosine, uridine, guanosine, and cytosine; AMP and ADP, the 5'-mono- and diphosphates of adenosine; Tris, tris(hydroxymethyl)aminomethane; P~, inorganic orthophosphate ; G-6-P, glucose-6-phosphate.

The yeast hexokinase used for these experiments was obtained from the Sigma Chemical Company (Type IV). Assays were made to determine if this preparation contained contaminating enzymes which might react with componer, ts: of the incubation mixture. The enzymes analyzed for and the assay methods used were as follows: glucose-6-phosphatase, liberation of Pi from G-6-P (4); adenosinetriphosphatase, liberation of Pi from ATP; phosphohexoisomerase,'conversion of G-6-P to ketose phosphate (5); ITP-ADP transphosphorylase, determination of ATP chromatographically after incubation of ITP and ADP in the reaction mixture; myokinase, determination of ATP chromatographically after incubation of ADP in the reaction mixture. In all cases, these

508

PHOSPHATE DONOR SPECIFICITY assays were carried out in the buffer system used for the hexokinase determination after incubation of the reaction mixtures for 30 min. at 30° employing 10 ~g. hexokinase for the assays. Under the assay conditions employed, less than 0.05 ~moles P~ was liberated from 5 tLmoles G-6-P or 5 ~moles ATP. The enzyme preparation, when incubated with 5 ~moles ITP, and 5 tLmoles ADP under the above conditions, did not convert more than 0.08 t~mole of the ITP to ATP (0.08 t~mole ATP is readily detectable by the chromatographic procedure employed). Thus, the hexokinase preparation used was free from significant contamination by enzymes known to metabolize G-6-P or ATP. SVBSTRATES The nucleotides employed were products of Pabst Laboratories. Paper chromatography of the nueleotides and reaction mixtures was carried out using the following solvent system in % ( v / v ) : isobutyric acid-NH~Ott-water (60:10:30). Even when mieromolar amounts of the nucleotides were applied to the paper, there was no contamination of one type of nucleoside phosphate with another type, although the triphosphates showed trace contamination with the corresponding diphosphates. G-6-P was identified by paper chromatography using in % (v/v) methanol-formic acid-water (80:15:5) as solvent (6). The phosphate esters were detected on the paper using the molybdic acid spray of Hanes and Isherwood (7).

509

1.0

o 0.6

g =~ 0,2
as contro!, and net activity was determined by the difference in free sugar concentration between control and experimental tubes. No measurable change occurred in the free sugar concentration on incubation of the controls. The validity of the assay procedures employed was tested by free sugar recovery experiments using reaction mixtures devoid of phosphate donor or enzyme or mixtures containing heat-killed enzyme. The recoveries of free sugar under these eonditions ranged from 98 to 103%. RESULTS

HEXOKINASE ASSAY The reaction mixtures had the following composition except where noted in the legends of the tables or in the text: sugar, 1 ~mole; MgCI_~, 40 /mmles; Tris-HC1 buffer, pH 8.0, 10 ~moles; nucleotide triphosphate, 3 t~moles; and su~eient enzyme in a total volume of 1 ml. The reaction mixtures were prepared in an ice bath, and the reaction was started by the addition of the enzyme. Incubation was carried out at 30°C. The readtion was stopped by placing the tubes in boilir~g water for 1 rain. Hexokinase activity was follow~e}t by the disappearance of free sugar from the reaction mixture. When glucose was used as substrate, residual free glucose was measured with the Glueostat reagent (Worthington Biochemical Corporation) after adjusting the pH of the reaction mixtures to 7 with phosphate buffer. When other sugars were tested, the phosphate esters were precipitated with Ba(OtI)~-ZnS04 (8), and free sugar was determined using the cysteineI-I_,SO~ reagent of Dische (9). A reaction mixture without added nueleoside triphosphate was used

ENZYME CONCENTRATION AND TIME R e a c t i o n r a t e was n o t s t r i c t l y p r o p o r tional to e n z y m e c o n c e n t r a t i o n w h e n less t h a n 2 ~g. e n z y m e was e m p l o y e d , p r e s u m a bly due to e n z y m e d e h a t u r a t l o n . H o w e v e r , as seen in Fig. 1, e x c e l l e n t p r o p o r t i o n a l i t y was o b t a i n e d a t h @ i e r e n z y m e e o n e e n t r a tions. E s s e n t i a l l y a]:: of t h e a d d e d s u g a r d i s a p p e a r e d a t t h e l~igher e n z y m e e o n c e n t r at i o n s. T h e d i s a p p e a r a n c e of free s u g a r c o n t i n u e d a t a c o n s t a n t r a t e for 20 rain. with 2 ~g. h e x o k i n a s e d u r i n g w h i ch t i m e 4 5 - 5 0 % of t h e sugar in t h e i n c u b a t i o n m i x t u r e was p h o s p h o r y l a t e d (Fig. 2). O c c a s i o n a l l y i t Was n e c e s s a r y t o v a r y the i n c u b a t i o n t i m e an d t h e e n z y m e eoneentration, depending upon the sugar and the t r i p h o s p h a t e e m p l o y e d , in o r d e r t h a t the reaction would not proceed beyond 40% p h o s p h o r y l a t i o n so t h a t t h e m e a s u r e m e n t s

510

MAIZTINEZ

~ 0.6 o

/

o .J {.9 ,,I _J 0

0.4


I0

2'0

3'0

MINUTES

FIG. 2. Rate of glucose phosphorylation with ATP as phosphate donor and glucose as acceptor. ReaCtion mixtures as in Fig. 1 using 2 ~g. enzyme, in a total volume of 1 ml.

,,, 1.0

tive phosphate donor for glucose phosphorylation. The other nucleotides transphosphorylated to only a negligible extent (less than 0.02 t~mole), even when the incubation times were extended to 40 rain. and the enzyme concentration was elevated to 40 ~g. The product of the I T P - g l u c o s e reaction was identified as G - 6 - P by paper chromatography. Figure 3 demonstrates the effect of v a r y ing enzyme concentration on the hexokinase activity when I T P serves as phosphate donor for glucose phosphorylation. I t is obvious t h a t the rate of phosphorylation is much slower when I T P is employed and t h a t the rate of the reaction is proportional to enzyme concentration over a wide range. A Lineweaver-Burk (10) plot for I T P is represented in Fig. 4, from which an apparent Michaelis constant of 3.7 × 10 -3 M is obtained. The Vm,x for glucose phosphorylation under these conditions is 3.94 Fmoles glucose phosphorylated/min./mg, enzyme. Under the same conditions, the Vm,x for glucose phosphorylation with A T P as donor

0.6 W .J O

"~

4


3'0 50 #g.ENZYME I

t

I

FIG. 3. Effect of enzyme concentration on tion velocity with ITP as donor and glucose ceptor. Reaction mixtures as in Fig. 1 with 3 t~moles; and enzyme ih a total .volume of Incubation time, 20 rain. at 30°C ....

3 reacas acITP, I ml.

would all be made in the linear phase of the reaction. The product of the hexokinase reaction when A T P and glucose were the substrates was identified as G - 6 - P chromatographically by comparison with authentic G-6-P. PHOSPHORYLATION OF GLUCOSE WITH I T P AS PHOSPHATE DONOR Substitution of I T P , CTP, G T P , or A D P for A T P in the reaction mixtures revealed t h a t only I T P served as a suitable Mterna-

% 2

i

I

I

I

0.2

0.4

0.6

0.8

I / i T P x I0 -3 M

Fro. 4. Lineweaver-Burk plot for ITP with g]ucose as acceptor. Reaction mixtures as in Fig. 1 with 20 ~g. enzyme, and ITP in a total volume of 1 ml. Incubation time, 15 min. at 30°C.

PHOSPHATE DONOR SPECIFICITY is 11.8 /~moles/min./mg. enzyme. The respective Miehaelis constants for I T P and A T P did not v a r y with changes in the M g + + concentration. The reported Km for A T P in this reaction is 9.5 × 10 -5 M, and the K ~ for glucose (with A T P as donor) is 1.5 × 10 -5 M (11). Although the presence of an I T P - A D P transphosphorylation reaction could not be demonstrated in the y e a s t hexokinase preparation used, further experiments were run to determine if a preliminary transphosphorylation between I T P and A D P or A M P was required. Since the preincubation of 3 t~moles I T P with 1-9/~moles A D P or A M P and the enzyme prior to the addition of glucose did not increase the rate of glucose phosphorylation as would be anticipated if A T P were the actual phosphate donor, and since the I T P employed in these experiments was shown to be free of adenine nucleotides, it appears t h a t I T P donates its terminal phosphate group to glucose in the hexokinase reaction. PHOSPHORYLATION

OF OTHER SUGARS BY

I T P AND A T P

511 TABLE I

TRANSPttOSPHORYLATION OF SUGARS USING I T P AND ATP As PI~OSPHATE DONORS

Reaction mixtures contained 1 t,mole sugar; 3 t~moles nucleotide; 10 t,moles Tris-HC1 buffer pH 8; 40 t~moles NigCl: ; 2 ttg. hexokinase when ATP was donor and 10 ~g. enzyme with ITP as donor, in a total volume of 1 ml. The ATP-containing reaction mixtures were incubated for 20 rain. at 30°C.; the ITP-containing mixtures were incubated for 10 min. at 30°C. Nucleotlde

Sugar

Sugar phosphorylated #moles

umoles/ min./mg. enzyme

ATP

Glucose Fructose 2-Deoxyglucose Mannose

0.42 0.31 0.33 0.24

10.5 7.8 8.3 6.0

ITP

~V[annose

0.49 0.24 0.32 0

4.9 2.4 3.2 0

Glucose Fructose 2-Deoxyglueose

a Reaction mixture as above except that 4 ~g. enzyme was used and the incubation time was 15 rain.

Yeast hexokinase is also known to phos- havior of the two sugars glucose and m a n phorylate mannose, fructose, and 2-deoxy- nose at high I T P concentrations. A very glucose (11). Since I T P was found to trans- dramatic inhibition of mannose phosphophosphorylate with glucose, it became of rylation at elevated I T P values is obvious interest to determine if this nucleotide was from Fig. 5 whereas high I T P concentraeffective as a phosphate donor when these tions do not strikingly affect the rate of other hexoses were substituted for glucose glucose phosphorylation (Fig. 4). An experiment was performed to test (Table I ) . A T P served as phosphate donor for all four sugars, while I T P reacted with whether A T P can also inhibit at high conglucose, mannose, and fructose, but curi- centrations. As shown in Table II, A T P ously not with 2-deoxyglucose. I t is inter- does inhibit, but not necessarily with the esting t h a t the relative rates of reaction same sugar aeeeptors. For example, the with A T P and I T P for the various sugars phosphorylation of glucose was not inhibwere different. Glucose was phosphorylated ited at high I T P concentrations and t h a t four times as rapidly by A T P whereas m a n - of mannose was, whereas the reverse was nose reacted at nearly equal rates with the true when A T P was the phosphate donor. two donors. Thus, it appears t h a t the inhibition obExperiments using mannose as acceptor served at high triphosphate concentrations and I T P as the phosphate donor revealed is a function not only of the phosphate donor, but is also apparently dependent on an apparent Michaelis constant of 2.9 x 10 -3 M (Fig. 5) for I T P at 10 -3 M m a n - the orientation of the hydroxyl g r o u p on nose, whereas the Km for A T P with either carbon 2 of the hexose molecule. mannose or glucose as acceptor is probably DISCUSSION of the same order (11). The natural occurrence of nucleoside 5'Of theoretical interest is the different be- triphosphates other than A T P r a i s e s the

512

MARTINEZ

07

0.5

0.3

0.1 I

0.2

I

I

0.6

I

I

1.0

__

I~T p x I0 -3 M

FIG. 5. Lincweaver-Burk plot for ITP with mannose as acceptor. Reaction mixtures as in Fig. 1 with 1 ~mole mannose, 10 ~g. enzyme, and ITP in a total volume of 1 ml. Incubation time, 10 rain. at 30°C. question of possible direct participation of these high-energy compounds in phosphorylation reactions, such as the hexokinase reaction. To date four such reactions have been investigated with respect to the specificity of the phosphate donors. Ling and L a r d y (2), using a purified preparation of 6-phosphofructokinase from muscle, were able to demonstrate fructose 1,6-diphosphage formation from fructose 6-phosphate with ATP, I T P and U T P as phosphate donors. The K ~ for the nueleotides was 3 × 10 -5 M for ATP, 7 × 10 .5 M for I T P , and 3.3 × 10 -5 M for U T P . The Vma~ was found to be only slightly higher with A T P than with the other nucleotides. Muntz (12) reported t h a t I T P was 52% as active as A T P in the 6-phosphohexokinase reaction using the brain enzyme. Bublitz and Kennedy (3) found t h a t the rate of phosphorylation of glycerol was ¾ as rapid with U T P as with ATP, and t h a t I T P was inactive in this reaction. Utter and co-workers (13) in an investigation of the oxalaeetie earboxylase enzyme found t h a t I T P was considerably more effective than A T P (from 3 to 7 times) in the conversion of oxalaeetate to

phosphoenolpyruvate. I D P was also found to be more effective than A D P in the reverse reaction. G T P has been implicated as an essential eofactor in the synthesis of protein, although its exact role is as y e t unknown. The hexokinase enzyme apparently has a requirement for either A T P or I T P as phosphate donors, the other triphosphates tested being inactive. I T P appears to react directly, i.e., without any prior transphosphorylation between I T P and ADP. Of some theoretical interest was the variation in the relative reactivity of a particular phosphate donor with the various hexoses tested. When A T P was the donor, m a n nose appeared to be phosphorylated at an appreciably lower rate than the other sugars tested. However, when I T P replaced A T P as the donor, mannose was by far the best phosphate aceeptor. I t is evident t h a t the relative reaction rates are a reflection of both the phosphate donor and aeeeptor, and t h a t the m a x i m u m velocity of transphosphorylation m a y v a r y over a wide range depending on the relative concentration of both the sugar and phosphate donor. Moreover, inhibition at high nucleoside triphosphate concentration is a function of the phosphate donor and phosphate acceptor in the reaction. I t was considered t h a t the inhibitions observed at high triphosphate levels might result from the formation of a ternary ES2 complex which was unable to b r e a k down TABLE I I INHIBITION OF SUGAR PHOSPHORYLATION BY Excess ATP Reaction mixtures contained: 1 t,mole sugar; 40 vmoles MgCI~ ; 10 ~moles Tris-HC1 buffer, pit 8; 4/,g. enzyme; and the designated amounts of ATP in a total volume of 1 ml. Incubation was for 15 min. at 30°C. ATP

Sugar

3 **moles

12 **moles

Inhibition

A **moles sugar

Glucose 2-Deoxyglucose Mannose

0.72 0.56 0.53

0.18 0.10 0.43

% 74. 83. 19.

PHOSPHATE DONOR SPECIFICITY to the products. The kinetics of this situation have been analyzed by H a l d a n e (14) who has found this to be a case of eompetit i r e inhibition by the substrate itself. The reaction rates are described by the equation y =

V K S 1 + g + K's

which predicts t h a t a plot of 1 / v against S is linear at high S values and extrapolates to a real value of 1 / v at S = 0. The data for inhibition of mannose phosphorylation by high I T P concentrations as well as t h a t for the inhibition of glucose and 2-deoxyglucose phosphorylation by high A T P concentrations plotted in the above manner produced nonlinear slopes which could not be extrapolated to real values of 1Iv. Theoretically, then, we cannot assume this inhibition to be one produced by a nondissoeiable t e r n a r y complex between enzyme and nucleotide. The data presented in this communication demonstrate t h a t I T P m a y serve as a transphosphorylating agent in the hexokinase reaction, although it is highly improbable t h a t this nueleotide plays a significant physiological role since the relatively high Km for I T P would indicate t h a t at the nucleotide concentrations prevailing in cells the reaction would be primarily with A T P .

513

ACKNOWLEDGMENTS The author wishes to thank Dr. Kenneth Paigen for his continued interest and encouragement of this work and for his valuable comments in the preparation of the manuscript, and to Miss Mary Anne Hayes for her assistance in some of the experiments. REFERENCES 1. PARKS, R. E., BEN-GERs~0:4, E., AND LARDY, H. A., J. Biol. Chem. 227, 231 (1957). 2. LING, K. H., AND LARDY,It. A., Y. Am. Chem. Soc. 76, 2842 (1954). 3. BVBLITZ,C., A~D KENNEDY,E. P., Y. Biol. Chem. 211, 951 (1954). 4. FISKE, C. H., A~'DSUBBARow,Y., J. Biol. Chem. 66, 375 (1925). 5. DISCHE, F., AND B0~ENSFREtrND, E., J. Biol. Chem. 192, 583 (1951). 6. BANDURSI,I, R. S., AND AXEI~ROD, B., J. Biol. Chem. 193, 405 (1951). 7. Ha?qES, C. S., a~-D ISHERW0OD, F. A., Nature

146, 1107 (1949). 8. SOMOGVI,M., J. Biol. Chem. 160, 69 (1954). 9. DISCHE, F., S:r=IETTLES,L. B., AND OSNOS, M., Arch. Biochem. 22, 169 (1949). 10. LINEWEAVER,H., AND BURK, D., J. Am. Chem. Soc. 56, 658 (1934). 11. McDoNALD, M. R., in "Methods in Enzymol-

ogy" (Colowiek, S. P. and Kaplan, N. 0., eds.), Vol. I, p. 269. Academic Press, New York, 1955. 12. MUNTZ,J. A., Arch. Bioehem. Biophys. 42, 435 (1953). 13. UTTER, M. F., ];~URAHASItI,K., AND ROSE, I. A., J. Biol. Chem. 207, 803 (1954). 14. HALDANE, J. B. S., "Enzymes." Longmans, Green and Co., Ltd., London, 1930.