- Email: [email protected]
The phosphorylation of Tris by alkaline phosphatase
620
PRELIMINARY NOTES
ceae that contain agaritine 2. It has not been found in extracts of a variety of other mushrooms, bacteria, or higher plant and animal tissues. Thus it appears probable that the reversible enzymatic transfer of the glutamyl group between agaritine and y-glutamyl-p-hydroxyaniline plays a role in the metabolism, and perhaps also in the final stages of biosynthesis, of these two unusual substances. It m a y be anticipated that other ?~-glutamyltransferases similarly possessing unique donor and acceptor specificity will reveal their presence in other organisms from which compounds containing the y-glutamyl function in linkage with nitrogenous residues of a nona-amino acid character have recently been isolated ~7. Details of these findings will be published elsewhere in the near future. This investigation was supported by Grants E-2966 and GM-Io359 from the National Institutes of Health, U.S. Public Health Service.
Department of Biological Chemistry, The University of Michigan, Ann Arbor, Mich. (U.S.A.)
HELEN
J.
GIGLIOTTI*
BRUCE LEVENBERG**
1 j . JADOT, J. CASIMIR AND M. RENARD, Biochim. Biophys. Acta, 43 (196o) 322. 2 B. LEVENBERG, J. Am. Chem. Soc., 83 (1961) 503 . 8 R. B. KELLY, E. G. DANIELS AND J. W. HINMAN, J. Org. Chem., 27 (1962) 3229. 4 p. K. STUMPF, W. D. LOOMIS AND C. MICHELSON, Arch. Biochem. Biophys., 3° (1951) 126. 5 H. WAt~LSCH, in F. F. NORD, Advan. Enzymol., 13 (1952) 237, 8 A. LAJTHA, P. MELA AND H. WAELSCH, .]. Biol. Chem., 205 (1953) 553. C. G. HANES, V. J. R. HIRD AND F. A. ISHERWOOD, Nature, 166 (195 o) 288. s F. J. R. HIRD AND P. H. SPRINGELL, Biochem. J., 56 (1954) 417 • 9 F. BINKLEY, J. Biol. Chem., 236 (1961) lO75. 10 W. J, WILLIAMS AND C. B. THORNI~, J . Biol. Chem., 21o (1954) 203. 11 j . F. THOMPSON, D. H. TURNER AND R. K. GERING, Federation Proc., 21 (1962) 5. 12 H. WAELSCH, in S. P. COLOWICK AND ~T. O. KAPLAN, Methods in Enzymology, Vol. 2, Academic Press, New York, 1955, p. 267. 18 A. JEANES, C. S. \VISE AND R. J. DIMLER, Anal. Chem., 23 (1951) 415 . 14 M. GOLDENBERG, M. FABER, E. J. ALSTON AND E. C. CHARGAFF, Science, lO9 (1949) 534. 1~ ~V. H. ELLIOTT, Biochem. J., 49 (1951) lO6. 18 H. WAELSCH, in W. D. MCELROY AND B. GLASS, Phosphorus Metabolism, Vol. 2, J o h n s H o p k i n s Press, Baltimore, 1952, p. lO9. 1T j . F. TI~OMPSON, C. J. MORRIS, W, N. ARNOLD AND D. H. TURNER, in J. T. HOLDEN, Amino Acid Pools, Distribution, Formation and Function of Free Amino Acids, Elsevier, A m s t e r d a m , 1962, P. 54.
Received December I6th, 1963 P r e s e n t address: Scripps Clinic and Research F o u n d a t i o n , La Jolla, Calif. (U.S.A.) "* Research Career D e v e l o p m e n t Awardee (GM-3I I5-K-3), U.S. Public H e a l t h Service.
Biochim. Biophys. Acts, 81 (1964) 618-62o
PN 10O93
The phosphorylation of Tris by alkaline phosphatase Solutions of Tris and its conjugate acid (pKa = 8.o) are frequently used as buffers in the study of enzyme-catalyzed reactions. In studies with alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) (Escherichia coli), it has been found that the enzyme activity increases with the concentration of Tris buffer much Biochim. Biophys. Acts, 81 (1964) 620-623
PRELIMINARY NOTES
62I
faster than it increases with the ionic strength of other salts such as NaC1 and Mg2SO4 (refs. I, 2). This unexpected behavior of Tris suggested to us that a transphosphorylation reaction may occur, especially since the substrate used in these experiments was p-nitrophenyl phosphate. Transphosphorylation has been shown with other acceptors using other phosphatases 3-6, but has not yet been demonstrated for Tris or for the enzyme from E. coli, nor has an increase in the rate of utilization of substrate been reported in these cases. If transphosphorylation should occur, we would be able in this case to reach theoretical conclusions concerning the mechanism and kinetics of the reaction, as well as to explain the enhanced activity in the presence of Tris. We show here that a lively transphosphorylation does occur. The amount of Tris phosphate that is formed is on the order of 25 times more than the amount of phosphate ester that would be in equilibrium with Tris and phosphate. According to the transfer theory of hydrolytic enzymes, we should expect a phosphoryl enzyme as an intermediate in the hydrolysis of phosphate esters, and in fact there is some evidence for such an intermediate ~ n. We will discuss the hydrolysis of p-nitrophenyl phosphate by phosphatase in the presence of Tris in terms of the transfer theory, although it will be clear that our demonstration that transphosphorylation occurs is independent of any theory. A reasonable scheme for the hydrolytic process, including transphosphorylation is: kl
ka
E + S ~ E S - - + E " + P~ k, \ Y/~5 Pa
I,,4X P4
where E is the enzyme, S is the substrate p-nitrophenyl phosphate, E' is the phosphoryl enzyme intermediate, P1 is p-nitrophenol, P2 is phosphoric acid, Pa is Tris phosphate, and ROH is Tris. This simple scheme is quantitatively appropriate within the theory if [Pll, [P2~ and [Pal are low, and if ROH (which is in high concentration) does not have an appreciable binding constant with either the enzyme or the phosphoryl enzyme. The relative amounts of p-nitrophenol and phosphate are determined by ksIROH 1 and h 4 (the concentration of water is included in k4). If transphosphoryla~ion occurs (ks[ROH 1 # 0), the amount of p-nitrophenol produced at any time will exceed the amount of phosphate. Our results indicate that k5 is slightly lower than k 4. Whether there will be an increase in the rate of utilization of substrate (increase in the rate of formation of p-nitrophenol) depends upon the relative values of k3 and k 4. If k.~ << ka, there will be no change in the rate of utilization of substrate in the presence of Tris, but since the substrate which reacts will be partitioned into two products, the rate of appearance of phosphate will be correspondingly decreased. I f on the other hand, ka is comparable to or greater than k4, i.e. if the dephosphorylation of the enzyme is a rate-controlling step, the rate of utilization of substrate will be increased in the presence of Tris. The relative values of k3 and k4 determine whether there will be a decrease in the rate of formation of phosphate with increasing concentration of Tris; if k3 >> k 4 there will be no decrease, but if k3 and k 4 are approximately equal there vdll be a decrease. Biochim. Biophys. Acta, 81 (1964) 620 623
622
PRELIMINARY
NOTES
W e have m e a s u r e d the r a t e of a p p e a r a n c e of p - n i t r o p h e n o l a n d p h o s p h a t e as a function of Tris concentration between o.I a n d 1.6 M at 25 °, p H 8.0 a n d I M ionic strength, held c o n s t a n t b y suitable a d d i t i o n s of NaC1. A t high ionic strength, the enzyme a c t i v i t y is insensitive to changes in ionic s t r e n g t h 1. I n a t y p i c a l experiment, the reaction was i n i t i a t e d b y the a d d i t i o n of o.I ml aqueous solution of alkaline p h o s p h a t a s e ( W o r t h i n g t o n Biochemical Co.) to I. I ml of a r e a c t i o n m i x t u r e containing Tris (Sigma) (pH 8.o), a n d p - n i t r o p h e n y l p h o s p h a t e (Sigma) at 25 °. F i n a l concent r a t i o n s in 1.2 ml of reaction m i x t u r e : I I M Tris, 7.86 mM p - n i t r o p h e n y l phosphate, I O . 4 # g enzyme per ml. Controls of (I) p - n i t r o p h e n y l p h o s p h a t e a n d Tris a n d (2) enzyme a n d Tris were run concurrently. The reaction was s t o p p e d after 3 min b y the a d d i t i o n of I ml of 5 N H2SO 4. I - m l aliquots of the reaction m i x t u r e were p i p e t t e d (I) into a 5-ml v o l u m e t r i c flask for the d e t e r m i n a t i o n of p h o s p h a t e b y the m e t h o d of DRYER et al. 1~, in which the final concentration of H2SO 4 was I N, a n d (2) into a I o - m l v o l u m e t r i c flask for the d e t e r m i n a t i o n of p-nitrophenol. P h o s p h a t e was 0.8C
0.7( o.6c
,/o/
p-Nitrophenof
-/
:x 0.5C
0.40
"°--°~°-~ 0.2
0.4
°~o~.Phosp o 0.6
0.8
1.0
h et e ~ 1.2 1.4 1.6 Tris (M)
Fig. i. Rate of liberation of p-nitrophenol and phosphate as a function of Tris concentration. The ionic strength was maintained at 1 M by suitable additions of NaC]. The basic assay mixture contained 7.9 mM p-nitrophenyl phosphate, lO.4,ug of enzyme per ml, and Tris buffer, pH = 8.0, 25 °. d e t e r m i n e d from the a b s o r b a n c y at 77 ° m/~, a n d p - n i t r o p h e n o l (diluted to volume w i t h I M Tris) from the a b s o r b a n c y at 41o m # (A = 1.62. lO 4 (ref. 8), p H 8.o, in I M Tris) w i t h a Zeiss s p e c t r o p h o t o m e t e r . Our results are shown in Figs. i a n d 2. F i r s t , we note t h a t there is a m a r k e d t r a n s p h o s p h o r y l a t i o n i n d i c a t e d b y the fact t h a t more n i t r o p h e n o l is formed t h a n p h o s p h a t e , a n d second, t h a t the r a t e of f o r m a t i o n of p h o s p h a t e decreases, b u t n o t v e r y r a p i d l y . F i n a l l y , we n o t e t h a t the r a t e of u t i l i z a t i o n of s u b s t r a t e increases in t h e presence of Tris; a result a l r e a d y found b y others 1. The r a t i o of n i t r o p h e n o l to phosp h a t e increases l i n e a r l y w i t h the c o n c e n t r a t i o n of Tris up to a b o u t I M. I n t e r m s of the scheme, these results i n d i c a t e t h a t ks, k4, a n d k 3 have c o m p a r a b l e values; ks/k4is o.93 a n d k3/k 4 is a b o u t 3, e v a l u a t e d from equations derived from the scheme. The first r a t i o can b y given with considerable confidence because a n y r e a c t i o n scheme is certain to contain t e r m s which are essentially the same as our k 5 a n d k 4. The second r a t i o depends more upon the scheme a n d even t h o u g h we t h i n k the scheme is essentiall y correct, we are not certain t h a t the r e l a t i v e l y small decrease in p h o s p h a t e f o r m a t i o n with high Tris c o n c e n t r a t i o n is not a consequence of t h e p a r t i c i p a t i o n of Tris in the r e a c t i o n in some m a n n e r n o t c o n t e m p l a t e d in t h e scheme. Tris in high c o n c e n t r a t o n m a y exert a non-specific i n h i b i t o r y action, or it m a y inhibit b y zinc chelation. F o r
Biochim. Biophvs. Acla, 8I (1964) 62o-623
PRELIMINARY NOTES
623
these reasons it is better to consider the value of 3 for ratio k3/k 4 as a minimum one
i.e. k3/k4 >- about 3.
/
2.5
2.0
o
./'/
1.5
/
/ 1.£ ./o
I 0.2
I 0.4
I 0.6
I 0.8
I 1.0
[ I I 1.2 1.4 1.6 Tris (hi)
Fig. 2. R a t i o of n i t r o p h e n o l to p h o s p h a t e as a f u n c t i o n of Tris c o n c e n t r a t i o n , a t ionic s t r e n g t h of i M. See l e g e n d to Fig. I.
The fact that Tris is not an inert component of the medium with respect to alkaline phosphatase reactions, will in some cases require a reevaluation of experimental results. The turnover number 13, 3200 molecules of p-nitrophenyl phosphate cleaved per molecule of enzyme at 25 °, i M Tris, p H 8.0, includes both hydrolysis and transphosphorylation, and should be reduced to about 19oo to correspond to hydrolysis in the absence of Tris. This work was supported by the Division of Research Grants and Fellowships of the National Institutes of Health, Grant B-573 (C I5), b y United States Public Health Service and Research Career Award GM-K3-I5, o12, and by National Science Foundation Grant 18926.
Departments of Biochemistry and Neurology, Columbia University College of Physicians and Surgeons, New York, N.Y. (U.S.A.)
JEAN DAYAN IRWIN B. WILSON
1 D. J. PLOCKE AND B. L. VALLEE, Biochemistry, i (1962) lO39. 2 A. GAREN AND C. LEVINTHAL, Biochim. Biophys. Acta, 38 (196o) 47 o. 3 j . APPLEYARD, Biochem. J., 42 (I948) 569. * B. AXELROD, J. Biol. Chem., 172 (1948) I. 5 0 . MEYERHOF AND H. GREEN, J. Biol. Chem., 183 (195 o) 377. 6 R. K. MORTON, Biochem. J., 7 ° (1958) 139. 7 S. S. STEIN AND D. E. KOSHLAND, Arch. Biochem., 39 (1952) 229-23 o. 8 L. ENGSTEOM AND G. ANGREN, Acta Chem. Scan&, 12 (1958) 357. 9 L. ENOSTEOM, Bioehim. Biophys. Acta, 52 (1961) 49. 10 H. GREENBERG AND D. NACHMANSOHN, Biochem. Biophys. Res. Commun., 7 (1962) 186. 11 J. H. SCHWARTZ AND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1996. 12 R. L. DRYER, A. R. TAMMES AND G. J. ROUTH, J. Biol. Chem., 225 (1957) 177. 13 D. J. PLOCKE. C. LEVINTHAL AND B. L. VALLEE, Biochemistry, i (1962) 373.
Received December ISth, 1963 Biochim. Biophys. Acta, 81 (i964) 62o-623
PRELIMINARY NOTES
ceae that contain agaritine 2. It has not been found in extracts of a variety of other mushrooms, bacteria, or higher plant and animal tissues. Thus it appears probable that the reversible enzymatic transfer of the glutamyl group between agaritine and y-glutamyl-p-hydroxyaniline plays a role in the metabolism, and perhaps also in the final stages of biosynthesis, of these two unusual substances. It m a y be anticipated that other ?~-glutamyltransferases similarly possessing unique donor and acceptor specificity will reveal their presence in other organisms from which compounds containing the y-glutamyl function in linkage with nitrogenous residues of a nona-amino acid character have recently been isolated ~7. Details of these findings will be published elsewhere in the near future. This investigation was supported by Grants E-2966 and GM-Io359 from the National Institutes of Health, U.S. Public Health Service.
Department of Biological Chemistry, The University of Michigan, Ann Arbor, Mich. (U.S.A.)
HELEN
J.
GIGLIOTTI*
BRUCE LEVENBERG**
1 j . JADOT, J. CASIMIR AND M. RENARD, Biochim. Biophys. Acta, 43 (196o) 322. 2 B. LEVENBERG, J. Am. Chem. Soc., 83 (1961) 503 . 8 R. B. KELLY, E. G. DANIELS AND J. W. HINMAN, J. Org. Chem., 27 (1962) 3229. 4 p. K. STUMPF, W. D. LOOMIS AND C. MICHELSON, Arch. Biochem. Biophys., 3° (1951) 126. 5 H. WAt~LSCH, in F. F. NORD, Advan. Enzymol., 13 (1952) 237, 8 A. LAJTHA, P. MELA AND H. WAELSCH, .]. Biol. Chem., 205 (1953) 553. C. G. HANES, V. J. R. HIRD AND F. A. ISHERWOOD, Nature, 166 (195 o) 288. s F. J. R. HIRD AND P. H. SPRINGELL, Biochem. J., 56 (1954) 417 • 9 F. BINKLEY, J. Biol. Chem., 236 (1961) lO75. 10 W. J, WILLIAMS AND C. B. THORNI~, J . Biol. Chem., 21o (1954) 203. 11 j . F. THOMPSON, D. H. TURNER AND R. K. GERING, Federation Proc., 21 (1962) 5. 12 H. WAELSCH, in S. P. COLOWICK AND ~T. O. KAPLAN, Methods in Enzymology, Vol. 2, Academic Press, New York, 1955, p. 267. 18 A. JEANES, C. S. \VISE AND R. J. DIMLER, Anal. Chem., 23 (1951) 415 . 14 M. GOLDENBERG, M. FABER, E. J. ALSTON AND E. C. CHARGAFF, Science, lO9 (1949) 534. 1~ ~V. H. ELLIOTT, Biochem. J., 49 (1951) lO6. 18 H. WAELSCH, in W. D. MCELROY AND B. GLASS, Phosphorus Metabolism, Vol. 2, J o h n s H o p k i n s Press, Baltimore, 1952, p. lO9. 1T j . F. TI~OMPSON, C. J. MORRIS, W, N. ARNOLD AND D. H. TURNER, in J. T. HOLDEN, Amino Acid Pools, Distribution, Formation and Function of Free Amino Acids, Elsevier, A m s t e r d a m , 1962, P. 54.
Received December I6th, 1963 P r e s e n t address: Scripps Clinic and Research F o u n d a t i o n , La Jolla, Calif. (U.S.A.) "* Research Career D e v e l o p m e n t Awardee (GM-3I I5-K-3), U.S. Public H e a l t h Service.
Biochim. Biophys. Acts, 81 (1964) 618-62o
PN 10O93
The phosphorylation of Tris by alkaline phosphatase Solutions of Tris and its conjugate acid (pKa = 8.o) are frequently used as buffers in the study of enzyme-catalyzed reactions. In studies with alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) (Escherichia coli), it has been found that the enzyme activity increases with the concentration of Tris buffer much Biochim. Biophys. Acts, 81 (1964) 620-623
PRELIMINARY NOTES
62I
faster than it increases with the ionic strength of other salts such as NaC1 and Mg2SO4 (refs. I, 2). This unexpected behavior of Tris suggested to us that a transphosphorylation reaction may occur, especially since the substrate used in these experiments was p-nitrophenyl phosphate. Transphosphorylation has been shown with other acceptors using other phosphatases 3-6, but has not yet been demonstrated for Tris or for the enzyme from E. coli, nor has an increase in the rate of utilization of substrate been reported in these cases. If transphosphorylation should occur, we would be able in this case to reach theoretical conclusions concerning the mechanism and kinetics of the reaction, as well as to explain the enhanced activity in the presence of Tris. We show here that a lively transphosphorylation does occur. The amount of Tris phosphate that is formed is on the order of 25 times more than the amount of phosphate ester that would be in equilibrium with Tris and phosphate. According to the transfer theory of hydrolytic enzymes, we should expect a phosphoryl enzyme as an intermediate in the hydrolysis of phosphate esters, and in fact there is some evidence for such an intermediate ~ n. We will discuss the hydrolysis of p-nitrophenyl phosphate by phosphatase in the presence of Tris in terms of the transfer theory, although it will be clear that our demonstration that transphosphorylation occurs is independent of any theory. A reasonable scheme for the hydrolytic process, including transphosphorylation is: kl
ka
E + S ~ E S - - + E " + P~ k, \ Y/~5 Pa
I,,4X P4
where E is the enzyme, S is the substrate p-nitrophenyl phosphate, E' is the phosphoryl enzyme intermediate, P1 is p-nitrophenol, P2 is phosphoric acid, Pa is Tris phosphate, and ROH is Tris. This simple scheme is quantitatively appropriate within the theory if [Pll, [P2~ and [Pal are low, and if ROH (which is in high concentration) does not have an appreciable binding constant with either the enzyme or the phosphoryl enzyme. The relative amounts of p-nitrophenol and phosphate are determined by ksIROH 1 and h 4 (the concentration of water is included in k4). If transphosphoryla~ion occurs (ks[ROH 1 # 0), the amount of p-nitrophenol produced at any time will exceed the amount of phosphate. Our results indicate that k5 is slightly lower than k 4. Whether there will be an increase in the rate of utilization of substrate (increase in the rate of formation of p-nitrophenol) depends upon the relative values of k3 and k 4. If k.~ << ka, there will be no change in the rate of utilization of substrate in the presence of Tris, but since the substrate which reacts will be partitioned into two products, the rate of appearance of phosphate will be correspondingly decreased. I f on the other hand, ka is comparable to or greater than k4, i.e. if the dephosphorylation of the enzyme is a rate-controlling step, the rate of utilization of substrate will be increased in the presence of Tris. The relative values of k3 and k4 determine whether there will be a decrease in the rate of formation of phosphate with increasing concentration of Tris; if k3 >> k 4 there will be no decrease, but if k3 and k 4 are approximately equal there vdll be a decrease. Biochim. Biophys. Acta, 81 (1964) 620 623
622
PRELIMINARY
NOTES
W e have m e a s u r e d the r a t e of a p p e a r a n c e of p - n i t r o p h e n o l a n d p h o s p h a t e as a function of Tris concentration between o.I a n d 1.6 M at 25 °, p H 8.0 a n d I M ionic strength, held c o n s t a n t b y suitable a d d i t i o n s of NaC1. A t high ionic strength, the enzyme a c t i v i t y is insensitive to changes in ionic s t r e n g t h 1. I n a t y p i c a l experiment, the reaction was i n i t i a t e d b y the a d d i t i o n of o.I ml aqueous solution of alkaline p h o s p h a t a s e ( W o r t h i n g t o n Biochemical Co.) to I. I ml of a r e a c t i o n m i x t u r e containing Tris (Sigma) (pH 8.o), a n d p - n i t r o p h e n y l p h o s p h a t e (Sigma) at 25 °. F i n a l concent r a t i o n s in 1.2 ml of reaction m i x t u r e : I I M Tris, 7.86 mM p - n i t r o p h e n y l phosphate, I O . 4 # g enzyme per ml. Controls of (I) p - n i t r o p h e n y l p h o s p h a t e a n d Tris a n d (2) enzyme a n d Tris were run concurrently. The reaction was s t o p p e d after 3 min b y the a d d i t i o n of I ml of 5 N H2SO 4. I - m l aliquots of the reaction m i x t u r e were p i p e t t e d (I) into a 5-ml v o l u m e t r i c flask for the d e t e r m i n a t i o n of p h o s p h a t e b y the m e t h o d of DRYER et al. 1~, in which the final concentration of H2SO 4 was I N, a n d (2) into a I o - m l v o l u m e t r i c flask for the d e t e r m i n a t i o n of p-nitrophenol. P h o s p h a t e was 0.8C
0.7( o.6c
,/o/
p-Nitrophenof
-/
:x 0.5C
0.40
"°--°~°-~ 0.2
0.4
°~o~.Phosp o 0.6
0.8
1.0
h et e ~ 1.2 1.4 1.6 Tris (M)
Fig. i. Rate of liberation of p-nitrophenol and phosphate as a function of Tris concentration. The ionic strength was maintained at 1 M by suitable additions of NaC]. The basic assay mixture contained 7.9 mM p-nitrophenyl phosphate, lO.4,ug of enzyme per ml, and Tris buffer, pH = 8.0, 25 °. d e t e r m i n e d from the a b s o r b a n c y at 77 ° m/~, a n d p - n i t r o p h e n o l (diluted to volume w i t h I M Tris) from the a b s o r b a n c y at 41o m # (A = 1.62. lO 4 (ref. 8), p H 8.o, in I M Tris) w i t h a Zeiss s p e c t r o p h o t o m e t e r . Our results are shown in Figs. i a n d 2. F i r s t , we note t h a t there is a m a r k e d t r a n s p h o s p h o r y l a t i o n i n d i c a t e d b y the fact t h a t more n i t r o p h e n o l is formed t h a n p h o s p h a t e , a n d second, t h a t the r a t e of f o r m a t i o n of p h o s p h a t e decreases, b u t n o t v e r y r a p i d l y . F i n a l l y , we n o t e t h a t the r a t e of u t i l i z a t i o n of s u b s t r a t e increases in t h e presence of Tris; a result a l r e a d y found b y others 1. The r a t i o of n i t r o p h e n o l to phosp h a t e increases l i n e a r l y w i t h the c o n c e n t r a t i o n of Tris up to a b o u t I M. I n t e r m s of the scheme, these results i n d i c a t e t h a t ks, k4, a n d k 3 have c o m p a r a b l e values; ks/k4is o.93 a n d k3/k 4 is a b o u t 3, e v a l u a t e d from equations derived from the scheme. The first r a t i o can b y given with considerable confidence because a n y r e a c t i o n scheme is certain to contain t e r m s which are essentially the same as our k 5 a n d k 4. The second r a t i o depends more upon the scheme a n d even t h o u g h we t h i n k the scheme is essentiall y correct, we are not certain t h a t the r e l a t i v e l y small decrease in p h o s p h a t e f o r m a t i o n with high Tris c o n c e n t r a t i o n is not a consequence of t h e p a r t i c i p a t i o n of Tris in the r e a c t i o n in some m a n n e r n o t c o n t e m p l a t e d in t h e scheme. Tris in high c o n c e n t r a t o n m a y exert a non-specific i n h i b i t o r y action, or it m a y inhibit b y zinc chelation. F o r
Biochim. Biophvs. Acla, 8I (1964) 62o-623
PRELIMINARY NOTES
623
these reasons it is better to consider the value of 3 for ratio k3/k 4 as a minimum one
i.e. k3/k4 >- about 3.
/
2.5
2.0
o
./'/
1.5
/
/ 1.£ ./o
I 0.2
I 0.4
I 0.6
I 0.8
I 1.0
[ I I 1.2 1.4 1.6 Tris (hi)
Fig. 2. R a t i o of n i t r o p h e n o l to p h o s p h a t e as a f u n c t i o n of Tris c o n c e n t r a t i o n , a t ionic s t r e n g t h of i M. See l e g e n d to Fig. I.
The fact that Tris is not an inert component of the medium with respect to alkaline phosphatase reactions, will in some cases require a reevaluation of experimental results. The turnover number 13, 3200 molecules of p-nitrophenyl phosphate cleaved per molecule of enzyme at 25 °, i M Tris, p H 8.0, includes both hydrolysis and transphosphorylation, and should be reduced to about 19oo to correspond to hydrolysis in the absence of Tris. This work was supported by the Division of Research Grants and Fellowships of the National Institutes of Health, Grant B-573 (C I5), b y United States Public Health Service and Research Career Award GM-K3-I5, o12, and by National Science Foundation Grant 18926.
Departments of Biochemistry and Neurology, Columbia University College of Physicians and Surgeons, New York, N.Y. (U.S.A.)
JEAN DAYAN IRWIN B. WILSON
1 D. J. PLOCKE AND B. L. VALLEE, Biochemistry, i (1962) lO39. 2 A. GAREN AND C. LEVINTHAL, Biochim. Biophys. Acta, 38 (196o) 47 o. 3 j . APPLEYARD, Biochem. J., 42 (I948) 569. * B. AXELROD, J. Biol. Chem., 172 (1948) I. 5 0 . MEYERHOF AND H. GREEN, J. Biol. Chem., 183 (195 o) 377. 6 R. K. MORTON, Biochem. J., 7 ° (1958) 139. 7 S. S. STEIN AND D. E. KOSHLAND, Arch. Biochem., 39 (1952) 229-23 o. 8 L. ENGSTEOM AND G. ANGREN, Acta Chem. Scan&, 12 (1958) 357. 9 L. ENOSTEOM, Bioehim. Biophys. Acta, 52 (1961) 49. 10 H. GREENBERG AND D. NACHMANSOHN, Biochem. Biophys. Res. Commun., 7 (1962) 186. 11 J. H. SCHWARTZ AND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1996. 12 R. L. DRYER, A. R. TAMMES AND G. J. ROUTH, J. Biol. Chem., 225 (1957) 177. 13 D. J. PLOCKE. C. LEVINTHAL AND B. L. VALLEE, Biochemistry, i (1962) 373.
Received December ISth, 1963 Biochim. Biophys. Acta, 81 (i964) 62o-623