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The significance of ribokinase for ribose utilization by Escherichia coli
I~3
SHO~R.T COMMUNICATIONS
in a metabolic pathway. Mass action ratios could not be calculated for those reactions where either the substrate or product was not measured or where the reaction includes pyridine nucleotides, which are compartmented in the cell so that total amounts are not proportional to levels at a particular enzyme. Mass action tatios that could be calculated are compared with equilibrium constants in Table II. Mass action ratios are far displaced from equilibrium constants only for the reactions of phosphofructokinase and pyruvate kinase. This is typical of other tissues and is consistent with the ~cole of these enzymes as major control points of glycolysis. The reactions of aconitase and fumarase are also displaced from equilibrium but not to an extent that would suggest a control point. Levels of metabolic intermediates v a r y with the metabolic state of the tissue. Although data here were obtained from platelets with a reduced rate of metabolism due to low temperatures, they probably approximate the levels in metabolically active pla}elets. This research was supported b y grant H E 10099 from the National Heart Institute., U.S. Public Health Service. I am grateful for the technical assistance of Mr. Michael De Jesus, Jr.
Department of Biochemistry, State University of New York, Downstate Medical Center, Brooklyn, N.Y., 112o3 IUoS.A.)
THOMAS C. DETWILEI~
I T. ~BI~cHER AND W . RI~SSMANN, Angew. Chem. Intern. Ed. Engl., 3 (1964) 426. 2 B. H E s s AND IK. BRAND, Clin. Chem., i i (1965) 223. 3 0 . ~ . LowRY, J. V. PASSONN/~AU, F. X. HASS/~LBERGER AND D. W. SCHULZ, ./. Biol. Chem., 239 (1964) 18. 4 N. D. C~OLDBERG,J. ~'. PASSONNEAIJ AND O. I-I. LOWRY, J. Biol. Chem., 241 (1966) 39975 J. 1~. WILLIAMSON, ./. Biol. Chem., 240 (1965) 2308. 6 C'r. PFLI!:IDERER, in K . U. BERGMEYER, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965, p. 597 ~ . KLINGENB•RG, in H. U. BERGMEYER, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965, p. 531. 8 H. U. BERGMEYI~R, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965. 9 P. K. MAITRA AND R. W . ESTABROOK, Anal. Biochem., 7 (1964) 472. IO ~ . W. ]~STABROOK AND P. K. MAITRA, Anal. Biochem., 3 (1962) 369.
Reoeived September 26th, 1968 Biochim. Biophys. Acta, 177 (1969) 161-163
BBA 23483
The: significance of ribokinase for ribose utilization by Escherichia coli It has been establishedZ, ~ that Escherichia colt take up a variety of sugars from their media via a phosphoenolpyruvate-dependent phosphotransferase system ~ through which the sugars are converted to sugar phosphates. This implies that ATPdependent hexokinases are not necessarily involved in sugar utilization, as also shown b y the observation that mutants lacking glucokinase grow normally on glucose ~, Biochim. Biophys. Acta, 177 (1969) 163-165
Ib 4
SHORT COMMUNICATION S
whereas m u t a n t s lacking a c o m p o n e n t of the phosphotransferase system, b u t possessing glucokinase, grow very slowly~ I t is the purpose of this c o m m u n i c a t i o n to show t h a t in c o n t r a s t to results o b t a i n e d with a v a r i e t y of hexoses, the n~itization of ribose requires the a c t i v i t y of a n A T P - d e p e n d e n t ribokinase. M u t a n t s of E. colt K~2. strain P A 309, u n a b l e to grow on ribose as sole source of c a r b o n a n d energy were o b t a i n e d b y chemical mutagenesis with e t h y l m e t h a n e s u l p h o n a t e ~ a n d s u b s e q u e n t penicillin selectionL One such mutan{ R~, was unabIe ~o grow on ribose b u t grew n o r m a l l y on glucose, s n c c m a t e or n u t r i e n t broth. Like its p a r e n t organism, R~ did n o t grow on ~)-xytose or uridine as sole carbon source' however, f u r t h e r m u t a n t s of R_~ which grew on these s u b s t a n c e s still failed zo grow on ribose. This suggests t h a t Rz a n d the n m t a n t s derived from it tacked the abi!itv to convert externa! ribose ~o i n t e r n a l ribose p h o s p h a t e due to a defect in either r~bose permease s or ribokinase. To i n v e s t i g a t e this possibility cells were grown on various carbon sources anc~ the presence of ribokinase m ceil-free extracts o b t a i n e d therefrom was determined, The extracts were prepared b y u l t r a s o n i c a t i o n of cells suspended in 50 mM Tris-HC! buffer, p H 7.4, followed b y c e n t r i f u g a t i o n at ~ o o o o ~ g for z~o rain ~o remove N A D H - o x i d a s e activity. The ribokinase a c t i v i t y was measured as the ribose-dependent f o r m a t i o n of A D P in a coupled spectrophotometric assay i n v o l v i n g pyruva~e kinase a n d lactate dehydrogenase. Table I shows t h a t for the p a r e n t s t r a i n r i b o k i n a s e coutd be detected i n succinate-, uridine- a n d n u t r i e n t - g r o w n cells a n d to a lesser extent in glucose-grown cel!s. The a c t i v i t y in ribose-grown cells was appreciably higher. ~n contrast, ~o ribokinase a c t i v i t y couId be detected in a n y of the extracts prepared ~rom the m u t a n t celis. Mixtures of m u t a n t a n d wiid-tv~e extracts showed t h a t a m o u n t of ribokinase expected from the c o n t r i b u t i o n of the wild-type extract alone suggesting t h a t there was no i n h i b i t o r of ribokinase present in the extract of the m u t a n t . R i b o k i n a s e was also assaved b y following the A T P - d e p e n d e n t r e m o v a l of ribose in a d i s c o n t i n u o u s assay ". I d e n t i c a l results to those g~ven in T a b i e I were found b v this m e t h o d confirming the absence of r i b o k i n a s e in extracts of the m u t a n t . A q u a l i t a t i v e confirmation t h a t the p r o d u c t was ribose 5-phosphate was pro~ vided b y utilizing the ability of crude extracts to t r a n s f o r m ribose 5-phosphate to fructose 6-phosphate t h r o u g h the reactions of t e e n o n - o x i d a t i v e b r a n c h of the pentose p h o s p h a t e pathway. Whilst m u z a n t a n d wild-type extract formed fructose 6-phosp h a t e at the same rate in such a system o n l y the wild-type extract was able ro catalyse the reaction when ribose plus A T P replaced the ribose 5-phosphate. T h e results presented above suggest t h a t the m u t a n t fails to grow- on ribose because it lacks ribokinase. This essential role of ribokinase was confirmed b y the selection of five clones of R I which had regained the a b i l i t y to grow on ribose. Such r e v e r t a n t s grew on ribose a~ the n o r m a l rate with m e a n g e n e r a t i o n times of _r2o-z36 rain at 37 ° a n d all five c o n t a i n e d the same a m o u n t of r i b o k i n a s e as did the originat strain. Since ribokinase was presen~ to an appreciable e x t e n t in succinate- a n d n u t r i e n t grown cells of the p a r e n t s t r a i n it is clear t h a t the synthesis of the e n z y m e is not d e p e n d e n t on e x t e r n a l ribose as an inducer. Hence the failure of the m u t a n t "co form. ribokinase c a n n o t be due to an i n a b i i i t y to a c c u m u l a t e ribose. Little is k n o w n a b o u t ~
-
~iochim. B~ophys. dcta, ~77 (~969) ~63-x65
SHORT COMiVrUNICATION S
165
TABILE I S P E C I F I C A C T I V I T Y OF R I B O K I N A S E D U R I N G GROWTH ON VARIOUS SUBSTRATES
R i b o k i n a s e a c t i v i t y w a s m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y (see text). T h e r e a c t i o n m i x t u r e at 30o c o n t a i n e d in i ml : I o o / ~ m o l e s of Tris-HC1 (pH 8.o), 5o/~moles of KC1, 5 / , m o l e s of 3/[gC12, 2/~moles of p h o s p h o e n o l p y r u v a t e , o.15 /,mole of N A D H , 2/*g of crystalline p y r u v a t e kinase, 5 # g of c r y s t a l l i n e l a c t a t e d e h y d r o g e n a s e , 2o-5o ~ g of E. colt protein, 2 / * m o l e s of A T P a n d 5 /~moles of ~)-ribose. T h e ribose w a s a d d e d last after t h e r a t e d u e to A T P a s e h a d been obtained.
Growth substrate
Ribose phosphorylated (#moles/rag protein per rain) Strain : YPA 309
R z
Ribose
o. 133
--
Nutrient broth
0.047
o
Succinate
0.040
o
IJridine
0.050
o
Glucose
o.oi 7
o
the inducer of ribokinase and in these experiments growth on uridine which gives rise to ribose I-phosphate and ribose 5-phosphate 1°, fails to increase the amount of ribokinase above that of nutrient-grown ceils. Thus it seems unlikely that ribose phosphate is the inducer of ribokinase in contrast to the glycerol system where a-glycerophosphate is the inducer of glycerol kinase 11. Our thanks are due to Professor H. L. Kornberg, F.R.S. for his interest and advice. This work was performed during the tenure by A.A. of a studentship from the Science Research Council.
Department of Biochemistry, University of Leicester, Leicester (Great Britain) I 2 3 4 5 6 7 8 9 io II
A N N E ANDERSON
R. A. CooPeR
"W. IKUNDIG, F. D. IKUNDIG, ]3. ANDERSON AND S. ROSEMAN, dr. Biol. Chem., 241 (1966) 3243 . IH. IR. I52ABACK,~/. Biol. Chem., 243 (1968) 3711. W . KUNDIG, S. G~IOSH AND S. ROS~MAN, -Proc. Natl. dead. Sci. U.S., 52 (1964) to67. ~D. G-. ~FRAENKEL, ~F. FALCOZ-KELLY AND ]3. L. t7IORECKER, i°roe. Natl. Acad. Sci. U.S., 52 ,(1964) 12o 7 . '.S.TANAKA, ]). G. FRAENKEL AND ]~. C. C. LIN, Biochem. Biophys. Res. Commun., 27 (1967) 63. E. C. C. LIN, S. A. LERIVER AND S. E. JORGENSEN, Biochim. Biophys. ~cta, 60 (1962) 442. L. GORINI AND S. IKAIJFMAN, Science, 131 (196o) 604. L . V . EGGLESTON AND H. A. KREBS, Biochem. J., 73 (1959) 264. B. L. I-IoRECKtgR, in S. P. COLOWICK AND N. O. tS~.APLAN, Methods in Enzymology, Vol. 2, Acadelnic Press, N e w York, 1957, p. 188. FI. O. KAM/~EN, -Pacific Slope Biochemical Conference, drune, z967. IN. t~. COZZARtgLLI, W . ~B. • R E E D B E R G A N D ]~. C. C. LIN, J. Mol. Biol., 31 (I968) 371.
Received November 8th, I968 Biochim. Biophys. Acta, 177 (I969) 163-165
SHO~R.T COMMUNICATIONS
in a metabolic pathway. Mass action ratios could not be calculated for those reactions where either the substrate or product was not measured or where the reaction includes pyridine nucleotides, which are compartmented in the cell so that total amounts are not proportional to levels at a particular enzyme. Mass action tatios that could be calculated are compared with equilibrium constants in Table II. Mass action ratios are far displaced from equilibrium constants only for the reactions of phosphofructokinase and pyruvate kinase. This is typical of other tissues and is consistent with the ~cole of these enzymes as major control points of glycolysis. The reactions of aconitase and fumarase are also displaced from equilibrium but not to an extent that would suggest a control point. Levels of metabolic intermediates v a r y with the metabolic state of the tissue. Although data here were obtained from platelets with a reduced rate of metabolism due to low temperatures, they probably approximate the levels in metabolically active pla}elets. This research was supported b y grant H E 10099 from the National Heart Institute., U.S. Public Health Service. I am grateful for the technical assistance of Mr. Michael De Jesus, Jr.
Department of Biochemistry, State University of New York, Downstate Medical Center, Brooklyn, N.Y., 112o3 IUoS.A.)
THOMAS C. DETWILEI~
I T. ~BI~cHER AND W . RI~SSMANN, Angew. Chem. Intern. Ed. Engl., 3 (1964) 426. 2 B. H E s s AND IK. BRAND, Clin. Chem., i i (1965) 223. 3 0 . ~ . LowRY, J. V. PASSONN/~AU, F. X. HASS/~LBERGER AND D. W. SCHULZ, ./. Biol. Chem., 239 (1964) 18. 4 N. D. C~OLDBERG,J. ~'. PASSONNEAIJ AND O. I-I. LOWRY, J. Biol. Chem., 241 (1966) 39975 J. 1~. WILLIAMSON, ./. Biol. Chem., 240 (1965) 2308. 6 C'r. PFLI!:IDERER, in K . U. BERGMEYER, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965, p. 597 ~ . KLINGENB•RG, in H. U. BERGMEYER, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965, p. 531. 8 H. U. BERGMEYI~R, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965. 9 P. K. MAITRA AND R. W . ESTABROOK, Anal. Biochem., 7 (1964) 472. IO ~ . W. ]~STABROOK AND P. K. MAITRA, Anal. Biochem., 3 (1962) 369.
Reoeived September 26th, 1968 Biochim. Biophys. Acta, 177 (1969) 161-163
BBA 23483
The: significance of ribokinase for ribose utilization by Escherichia coli It has been establishedZ, ~ that Escherichia colt take up a variety of sugars from their media via a phosphoenolpyruvate-dependent phosphotransferase system ~ through which the sugars are converted to sugar phosphates. This implies that ATPdependent hexokinases are not necessarily involved in sugar utilization, as also shown b y the observation that mutants lacking glucokinase grow normally on glucose ~, Biochim. Biophys. Acta, 177 (1969) 163-165
Ib 4
SHORT COMMUNICATION S
whereas m u t a n t s lacking a c o m p o n e n t of the phosphotransferase system, b u t possessing glucokinase, grow very slowly~ I t is the purpose of this c o m m u n i c a t i o n to show t h a t in c o n t r a s t to results o b t a i n e d with a v a r i e t y of hexoses, the n~itization of ribose requires the a c t i v i t y of a n A T P - d e p e n d e n t ribokinase. M u t a n t s of E. colt K~2. strain P A 309, u n a b l e to grow on ribose as sole source of c a r b o n a n d energy were o b t a i n e d b y chemical mutagenesis with e t h y l m e t h a n e s u l p h o n a t e ~ a n d s u b s e q u e n t penicillin selectionL One such mutan{ R~, was unabIe ~o grow on ribose b u t grew n o r m a l l y on glucose, s n c c m a t e or n u t r i e n t broth. Like its p a r e n t organism, R~ did n o t grow on ~)-xytose or uridine as sole carbon source' however, f u r t h e r m u t a n t s of R_~ which grew on these s u b s t a n c e s still failed zo grow on ribose. This suggests t h a t Rz a n d the n m t a n t s derived from it tacked the abi!itv to convert externa! ribose ~o i n t e r n a l ribose p h o s p h a t e due to a defect in either r~bose permease s or ribokinase. To i n v e s t i g a t e this possibility cells were grown on various carbon sources anc~ the presence of ribokinase m ceil-free extracts o b t a i n e d therefrom was determined, The extracts were prepared b y u l t r a s o n i c a t i o n of cells suspended in 50 mM Tris-HC! buffer, p H 7.4, followed b y c e n t r i f u g a t i o n at ~ o o o o ~ g for z~o rain ~o remove N A D H - o x i d a s e activity. The ribokinase a c t i v i t y was measured as the ribose-dependent f o r m a t i o n of A D P in a coupled spectrophotometric assay i n v o l v i n g pyruva~e kinase a n d lactate dehydrogenase. Table I shows t h a t for the p a r e n t s t r a i n r i b o k i n a s e coutd be detected i n succinate-, uridine- a n d n u t r i e n t - g r o w n cells a n d to a lesser extent in glucose-grown cel!s. The a c t i v i t y in ribose-grown cells was appreciably higher. ~n contrast, ~o ribokinase a c t i v i t y couId be detected in a n y of the extracts prepared ~rom the m u t a n t celis. Mixtures of m u t a n t a n d wiid-tv~e extracts showed t h a t a m o u n t of ribokinase expected from the c o n t r i b u t i o n of the wild-type extract alone suggesting t h a t there was no i n h i b i t o r of ribokinase present in the extract of the m u t a n t . R i b o k i n a s e was also assaved b y following the A T P - d e p e n d e n t r e m o v a l of ribose in a d i s c o n t i n u o u s assay ". I d e n t i c a l results to those g~ven in T a b i e I were found b v this m e t h o d confirming the absence of r i b o k i n a s e in extracts of the m u t a n t . A q u a l i t a t i v e confirmation t h a t the p r o d u c t was ribose 5-phosphate was pro~ vided b y utilizing the ability of crude extracts to t r a n s f o r m ribose 5-phosphate to fructose 6-phosphate t h r o u g h the reactions of t e e n o n - o x i d a t i v e b r a n c h of the pentose p h o s p h a t e pathway. Whilst m u z a n t a n d wild-type extract formed fructose 6-phosp h a t e at the same rate in such a system o n l y the wild-type extract was able ro catalyse the reaction when ribose plus A T P replaced the ribose 5-phosphate. T h e results presented above suggest t h a t the m u t a n t fails to grow- on ribose because it lacks ribokinase. This essential role of ribokinase was confirmed b y the selection of five clones of R I which had regained the a b i l i t y to grow on ribose. Such r e v e r t a n t s grew on ribose a~ the n o r m a l rate with m e a n g e n e r a t i o n times of _r2o-z36 rain at 37 ° a n d all five c o n t a i n e d the same a m o u n t of r i b o k i n a s e as did the originat strain. Since ribokinase was presen~ to an appreciable e x t e n t in succinate- a n d n u t r i e n t grown cells of the p a r e n t s t r a i n it is clear t h a t the synthesis of the e n z y m e is not d e p e n d e n t on e x t e r n a l ribose as an inducer. Hence the failure of the m u t a n t "co form. ribokinase c a n n o t be due to an i n a b i i i t y to a c c u m u l a t e ribose. Little is k n o w n a b o u t ~
-
~iochim. B~ophys. dcta, ~77 (~969) ~63-x65
SHORT COMiVrUNICATION S
165
TABILE I S P E C I F I C A C T I V I T Y OF R I B O K I N A S E D U R I N G GROWTH ON VARIOUS SUBSTRATES
R i b o k i n a s e a c t i v i t y w a s m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y (see text). T h e r e a c t i o n m i x t u r e at 30o c o n t a i n e d in i ml : I o o / ~ m o l e s of Tris-HC1 (pH 8.o), 5o/~moles of KC1, 5 / , m o l e s of 3/[gC12, 2/~moles of p h o s p h o e n o l p y r u v a t e , o.15 /,mole of N A D H , 2/*g of crystalline p y r u v a t e kinase, 5 # g of c r y s t a l l i n e l a c t a t e d e h y d r o g e n a s e , 2o-5o ~ g of E. colt protein, 2 / * m o l e s of A T P a n d 5 /~moles of ~)-ribose. T h e ribose w a s a d d e d last after t h e r a t e d u e to A T P a s e h a d been obtained.
Growth substrate
Ribose phosphorylated (#moles/rag protein per rain) Strain : YPA 309
R z
Ribose
o. 133
--
Nutrient broth
0.047
o
Succinate
0.040
o
IJridine
0.050
o
Glucose
o.oi 7
o
the inducer of ribokinase and in these experiments growth on uridine which gives rise to ribose I-phosphate and ribose 5-phosphate 1°, fails to increase the amount of ribokinase above that of nutrient-grown ceils. Thus it seems unlikely that ribose phosphate is the inducer of ribokinase in contrast to the glycerol system where a-glycerophosphate is the inducer of glycerol kinase 11. Our thanks are due to Professor H. L. Kornberg, F.R.S. for his interest and advice. This work was performed during the tenure by A.A. of a studentship from the Science Research Council.
Department of Biochemistry, University of Leicester, Leicester (Great Britain) I 2 3 4 5 6 7 8 9 io II
A N N E ANDERSON
R. A. CooPeR
"W. IKUNDIG, F. D. IKUNDIG, ]3. ANDERSON AND S. ROSEMAN, dr. Biol. Chem., 241 (1966) 3243 . IH. IR. I52ABACK,~/. Biol. Chem., 243 (1968) 3711. W . KUNDIG, S. G~IOSH AND S. ROS~MAN, -Proc. Natl. dead. Sci. U.S., 52 (1964) to67. ~D. G-. ~FRAENKEL, ~F. FALCOZ-KELLY AND ]3. L. t7IORECKER, i°roe. Natl. Acad. Sci. U.S., 52 ,(1964) 12o 7 . '.S.TANAKA, ]). G. FRAENKEL AND ]~. C. C. LIN, Biochem. Biophys. Res. Commun., 27 (1967) 63. E. C. C. LIN, S. A. LERIVER AND S. E. JORGENSEN, Biochim. Biophys. ~cta, 60 (1962) 442. L. GORINI AND S. IKAIJFMAN, Science, 131 (196o) 604. L . V . EGGLESTON AND H. A. KREBS, Biochem. J., 73 (1959) 264. B. L. I-IoRECKtgR, in S. P. COLOWICK AND N. O. tS~.APLAN, Methods in Enzymology, Vol. 2, Acadelnic Press, N e w York, 1957, p. 188. FI. O. KAM/~EN, -Pacific Slope Biochemical Conference, drune, z967. IN. t~. COZZARtgLLI, W . ~B. • R E E D B E R G A N D ]~. C. C. LIN, J. Mol. Biol., 31 (I968) 371.
Received November 8th, I968 Biochim. Biophys. Acta, 177 (I969) 163-165