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Uridine phosphorylase from Escherichia coli B.: Kinetic studies on the mechanism of catalysis
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 226, No. 2, October 15, pp. 687-692, 1933
Uridine Phosphorylase from Escherichia co/i 6.: Kinetic Studies on the Mechanism of Catalysis ALBERT0 *Dipartimento
VITA,*
CHARLES
Y. HUANG,?’
AND
GUILIO
MAGNI*
di Biologia CeUulare, Universita di Came-&o, 62032 Came&no (MC), Italy, and of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland .%%%
tL&oratory
Received
May
12, 1983, and in revised
form
July
5, 1933
Using a highly purified enzyme preparation of uridine phosphorylase from Escherichia coli B, we have performed detailed kinetic studies which include initial-velocity and product-inhibition experiments in the forward and reverse directions of the reaction. These studies indicate a rapid-equilibrium random mechanism for this enzyme with the formation of an enzyme. uracil phosphate abortive complex. Lack of formation of the enzyme. uridine . ribose-l-phosphate abortive complex suggests that the ribosyl moiety of the two ligands compete for the same binding site. The random mechanism is different from the ordered addition of substrates found for uridine phosphorylase from other sources. All the kinetic constants in the forward and reverse directions and the Keq of reaction for E. coli uridine phosphorylase are reported herein.
Uridine phosphorylase alyzes the reaction Uridine
+ phosphate
(EC 2.4.2.3) cat-
cleoside phosphorylase. Salmonella tgphimurium and Escherichia coli purine nucleoside phosphorylases have been shown to work by a sequential reaction mechanism where the nucleoside and phosphate bind to the enzyme randomly, while the purine base binds after the addition of pentose l-phosphate (7). An ordered reaction mechanism was also proposed for thymidine phosphorylase (8), a nucleoside phosphorylase showing strict specificity for the deoxyribosyl moiety. A random mechanism, however, was proposed by Krenitsky (9) for E. coli uridine phosphorylase using a partially purified enzyme preparation. In this report detailed kinetic studies, including initial-velocity and productinhibition experiments in the forward and reverse directions of catalysis, have been conducted with a homogeneous enzyme preparation. The results reveal the presence of an enzyme. uracil +phosphate abortive complex which has not been established in previous kinetic studies.
z
uracil + ribose-l-phosphate. It belongs to the class of nucleoside phosphorylases which have been shown to occur in many organisms (1,2). The enzyme, because of its role in the degradation of pyrimidine nucleosides as well as in the “salvage pathway” of nucleic acid synthesis, occupies an important position in metabolism. The uridine phosphorylase from rat liver has been characterized by initialvelocity and product-inhibition studies, showing an ordered reaction mechanism with phosphate binding first (3). A similar mechanism has been proposed for guinea pig uridine phosphorylase, rabbit thymidine phosphorylase (4), and calf spleen (5) and human erythrocytes (6) purine nu1 To whom correspondence Building 3, Room 218, NIH,
should Bethesda,
be addressed: Md. 20205. 687
0003-9861/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
688
ImM PHOSPHATET’
FIG. 1. Double-reciprocal plots with phosphate (arbitrarily designated as substrate B) as the variable substrate and uridine (substrate A) at various fixed levels: closed circles, 0.04; open circles, 0.06; closed squares, 0.0% open squares, 0.12; closed triangles, O.M), open triangles, 0.40 mM. Assay conditions were as described under Materials and Methods. Ku for phosphate is determined from the intersection point. Activity is expressed as units/ml enzyme; each ml contained 1.33 nmol uridine phosphorylase. Inset: secondary plot of ?/ intercepts vs reciprocal uridine concentrations for the determination of K, for uridine and Vf (each ml of reaction mixture contained 94 pmol of tetrameric uridine phosphorylase). MATERIALS
AND
METHODS
Hepee.’ (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), uracil, uridine, and ribose l-phosphate (dicyclohexylammonium salt) were purchased from Sigma Chemical Company; dibasic and monobasic potassium phosphate and potassium chloride were from J. T. Baker Chemical Company. Enzyne preparation Uridine phosphorylase was prepared from E. coli B through 50-&l% (NH&SO, cuts and repeated Sephadex G-150and hydroxylapatite-column steps to apparent homogeneity as judged by gel filtration, polyacrylamide gel electrophoresis, sodium dodecyl sulfate-gel electrophoresis, and isoelectro focusing experiments (10). The enzyme has a molecular weight of 125,000 and consists of four apparently identical subunits of molecular weight 31,000 (10). Enzyme assays. Phosphorolysis and synthesis of uridine were determined spectrophotometrically by the differential absorption between uridine and uracil at 280 nm (Af&~,k~ = 2.1). The reaction mixture contained 50 mM Hepes buffer, pH 7.56, 10 mM KCl, and uridine and phosphate (in the phosphorolysis direction) or uracil and ribose l-phosphate (in the synz Abbreviation used: piperazineethanesulfonic
Hepes, 4-(2-hydroxyethyl)-lacid.
thesis direction) at concentrations as indicated in figure legends. The reaction was initiated by the addition of 5 ~1 protein sample containing 9.4 pm01 (tetramer) of the enzyme to a l-ml assay mixture, and the rate of disappearance or formation of uridine was followed at 230 nm using a Cary 118C spectrophotometer. One enzyme unit is defined as the amount of enzyme which catalyzes the conversion of 1 gmol of substrate/ min at 30°C. Protein concentration was determined according to Schacterle (11). RESULTS
Initial-
Velocity Studies
In the forward reaction (phosphoryolysis), when phosphate was the variable substrate and uridine the varied fixed substrate, double-reciprocal plots gave a family of straight lines intersecting at a point to the left of the ordinate above the abscissa (Fig. 1). A similar pattern was obtained when uridine was the variable substrate. In the reverse direction (nucleoside synthesis), when ribose l-phosphate was the variable substrate and uracil the varied fixed substrate, the same type of pattern
KINETIC
STUDIES
OF
URIDINE
689
PHOSPHORYLASE
2
I
44
1
2.5
-2.5
I
I
J
5.0
7.5
10
ImM RIBOSE-I-PHOSPHATE).’
FIG. 2. Initial-rate studies in the reverse direction with ribose l-phosphate (designated P) as the variable substrate and uracil (designated &) at various fixed concentrations: closed circles, 0.10; open circles, 0.15; closed squares, 0.20; open squares, 0.25; closed triangles, 0.40 mM. Kip for ribose l-phosphate, K, for uracil, and V, are determined from the primary and secondary (inset) plots. Activity is expressed as described in Fig. 1.
was also observed (Fig. 2). Double-reciprocal plots with uracil as the variable substrate yielded a similar intersecting pat-
0 0.0
tern. These intersecting plots indicate an ordered or a rapid-equilibrium random mechanism. To differentiate between these
I
I
0.2
0.4
hM
I
0.6
PHOSPHATEI“
FIG. 3. Competitive inhibition of phosphate by the product ribose l-phosphate. Linear competitive inhibition is demonstrated in the inset. Assay conditions were the same as in Fig. 1. Uridine was maintained at 0.1 mM. Ribose l-phosphate concentrations are diamonds, 0; open circles, 0.50; closed circles, 1.25, open squares, 2 mM. Activity is expressed as described in Fig. 1.
690
VITA,
HUANG,
AND
MAGNI
hibition by the product ribose l-phosphate was competitive (Fig. 3). Under the same conditions, inhibition by the other product, uracil, was noncompetitive (Fig. 4). When uridine was varied and phosphate kept at a nonsaturating level, inhibition by both uracil and ribose l-phosphate was competitive (plots not shown). Uridine syntheses. Similar product-inhibition experiments were performed in the reverse direction. All the inhibition patterns were competitive with the exception of phosphate which was noncompetitive with respect to uracil. The inhibition patterns and reactant concentrations are summarized in Table I. -0.2
0
0.2
0.4
0.6
ImM PHOSPHATEI.’
DISCUSSION
FIG. 4. Noncompetitive inhibition of phosphate by uracil. Uridine was maintained at 0.1 mM. Kb for uracil (dissociation of uracil from the enzyme * phosphate * uracil complex, see Scheme I) is calculated from the intersection point (= -K,/KsKb). The linear nature of the noncompetitive inhibition is shown in the inset. Activity is expressed as described in Fig. 1.
two possibilities, product-inhibition Product
We have performed a detailed kinetic investigation on a highly purified preparation of uridine phosphorylase from E. coli B, including initial-velocity and product-inhibition experiments for each product-substrate pair in the forward and reverse directions of the reaction. The intersecting patterns shown in Figs. 1 and 2 exclude the classic ping-pong (12) and twosite ping-pong (13) mechanisms and favor a sequential mechanism. The product-inhibition patterns summarized in Table I are consistent with a rapid-equilibrium random mechanism with the formation of an enzyme. uracil * phosphate abortive
we carried out a series of experiments.
Inhibition
Studies
Phosphorolysis of uridine. With variable concentrations of phosphate and a constant, nonsaturating level of uridine, inTABLE SUMMARY
Reaction
Pi, Pi, Ud, Ud,
Nucleoside synthesis
u, 0.5 u, 0.5 R-l-P, 1 R-l-P, 1
n Experimental U = uracil, R-l-P
OF PRODUCT-INHIBITION
Constant substrate Cm@
Phosphorolysis
conditions = ribose
I
10 10 0.1 0.1
were as described l-phosphate.
Variable substrate (mM) Ud Ud Pi Pi
(0.025-0.30) (0.025-0.30) (2-20) (2-20)
R-l-P (0.5-2) R-l-P (0.5-2) U (0.06-0.40) u (0.06-0.60) under
Materials
STUDIES’
Product inhibitor bM)
5Pe of inhibition
u (O-0.40) R-l-P (O-3) R-l-P (O-2) u (O-0.60)
Competitive Competitive Competitive Non-competitive
Ud Pi Ud Pi
Competitive Competitive Competitive Non-competitive
(o-0.30) (O-40) (o-0.20) (O-40)
and Methods;
Pi = phosphate,
Ud = uridine,
KINETIC
complex. The proposed kinetic lustrated in Scheme I:
STUDIES
OF
URIDINE
mary and secondary plots and are compiled in Table II. These constants permit the Keq for the uridine phosphorylase reaction (at pH 7.56, 30°C) to be estimated according to the Haldane relationship:
model is il-
The Keg so obtained falls in the range 0.54-0.61. Our findings are essentially in agreement with those of Krenitsky (9), obtained with a partially purified enzyme preparation, except that we have established the presence of an enzyme. uracil . phosphate abortive complex as evidenced by the noncompetive inhibition of uracil vs phosphate in both forward and reverse directions. The absence of the enzyme. uridine . ribose lphosphate abortive complex is not unexpected because the ribosyl moiety of these two compounds presumably would compete for the same binding site. It appears that the uracil and phosphate sites, being separated by the ribose site, do not exert effects on each other’s binding. The agree-
where A = uridine, B = phosphate, P = ribose l-phosphate, and Q = uracil. All the kinetic constants are defined in Scheme I. The kinetic equation describing Scheme I is V
iig=
kf(AB/&&) - kdPQ/KipKq) 1 + A/Kti + B/K6 + &?/K,K, + P/K, + Q/Kti + PQ/K;&*
All the kinetic been determined
[l]
+ BQ/Ksb
constants in Eq. [l] have from our data using priTABLE
II
KINETIC CONSTANTS FOR E. coli URIDINE Reaction Uridine
synthesis
Kinetic
(A)
Phosphate
Nucleoside
PHOSPHORYLASE
Reactant
Phosphorolysis
691
PHOSPHORYLASE
constant’ 0.094 0.054
Kit% Kl (B)
Ribose
l-phosphate
Uracil
(Q)
Kib Kb Kb
mM mM
11.1 6.25 9.9
mM
12.0b
s-i
1.00 0.29
mM rnM
0.57
mM
(P)
mrvi mM
0.15 mM 0.51 mM 5.5b
kb “Experimental Scheme I. b Calculated
conditions per subunit,
are described assuming
four
under identical
Materials monomers.
and Methods.
Kinetic
constants
5-l
are defined
in
692
VITA,
HUANG,
ment between Kib (11.1 mM) and Kb (9.9 mM) and between KQ (0.57 mM) and Kb (0.51 m&f) supports this notion. REFERENCES 1. FRIEDKIN, M., AND KALCKAR, H. (1961) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), 2nd ed., Vol. 5, pp. 237-255, Academic Press, New York. 2. PARKS, R. E., JR., AND AGARWAL, R. P. (1972) iu The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 7, pp. 483-514, Academic Press, New York. 3. KRAUT, A., AND YAMADA, E. W. (1971) J. Biol Chem 246,2021-2030. 4. KRENITSKY, T. A. (1968) J. Biol Chem 243,28712875.
AND
MAGNI
5. KRENITSKY, T. A. (1967) Mol Phavmmm! 3, 526536. 6. KIM, B. Y., CHA, S., AND PARKS, R. E., JR., (1968) J. B&x! Chem 243,1763-1770. 7. JENSEN, K. F. (1976) Eur. J. Bioehem 61,377-386. 8. SCHWARTZ, M. (1971) Eur. J. Biochem 21, 191198. 9. KRENITSKY, T. A. (1976) Biochen~ Biophys. Actu 429, 352-358. 10. VITA, A., PULCINI, A., AND MAGNI, G. (1981) ItaL J. B&hem 30,498~502. 11. SCHA~TERLE, G. R., AND POLLACK, R. L. (1973) And Biochem 51, 654-655. 12. CLELAND, W. W. (1963) Biochem Biophvs. Acta 67, 104-137. 13. NORTHROP, D. B. (1969) J. Bid Chem 244,58085819.
Uridine Phosphorylase from Escherichia co/i 6.: Kinetic Studies on the Mechanism of Catalysis ALBERT0 *Dipartimento
VITA,*
CHARLES
Y. HUANG,?’
AND
GUILIO
MAGNI*
di Biologia CeUulare, Universita di Came-&o, 62032 Came&no (MC), Italy, and of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland .%%%
tL&oratory
Received
May
12, 1983, and in revised
form
July
5, 1933
Using a highly purified enzyme preparation of uridine phosphorylase from Escherichia coli B, we have performed detailed kinetic studies which include initial-velocity and product-inhibition experiments in the forward and reverse directions of the reaction. These studies indicate a rapid-equilibrium random mechanism for this enzyme with the formation of an enzyme. uracil phosphate abortive complex. Lack of formation of the enzyme. uridine . ribose-l-phosphate abortive complex suggests that the ribosyl moiety of the two ligands compete for the same binding site. The random mechanism is different from the ordered addition of substrates found for uridine phosphorylase from other sources. All the kinetic constants in the forward and reverse directions and the Keq of reaction for E. coli uridine phosphorylase are reported herein.
Uridine phosphorylase alyzes the reaction Uridine
+ phosphate
(EC 2.4.2.3) cat-
cleoside phosphorylase. Salmonella tgphimurium and Escherichia coli purine nucleoside phosphorylases have been shown to work by a sequential reaction mechanism where the nucleoside and phosphate bind to the enzyme randomly, while the purine base binds after the addition of pentose l-phosphate (7). An ordered reaction mechanism was also proposed for thymidine phosphorylase (8), a nucleoside phosphorylase showing strict specificity for the deoxyribosyl moiety. A random mechanism, however, was proposed by Krenitsky (9) for E. coli uridine phosphorylase using a partially purified enzyme preparation. In this report detailed kinetic studies, including initial-velocity and productinhibition experiments in the forward and reverse directions of catalysis, have been conducted with a homogeneous enzyme preparation. The results reveal the presence of an enzyme. uracil +phosphate abortive complex which has not been established in previous kinetic studies.
z
uracil + ribose-l-phosphate. It belongs to the class of nucleoside phosphorylases which have been shown to occur in many organisms (1,2). The enzyme, because of its role in the degradation of pyrimidine nucleosides as well as in the “salvage pathway” of nucleic acid synthesis, occupies an important position in metabolism. The uridine phosphorylase from rat liver has been characterized by initialvelocity and product-inhibition studies, showing an ordered reaction mechanism with phosphate binding first (3). A similar mechanism has been proposed for guinea pig uridine phosphorylase, rabbit thymidine phosphorylase (4), and calf spleen (5) and human erythrocytes (6) purine nu1 To whom correspondence Building 3, Room 218, NIH,
should Bethesda,
be addressed: Md. 20205. 687
0003-9861/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
688
ImM PHOSPHATET’
FIG. 1. Double-reciprocal plots with phosphate (arbitrarily designated as substrate B) as the variable substrate and uridine (substrate A) at various fixed levels: closed circles, 0.04; open circles, 0.06; closed squares, 0.0% open squares, 0.12; closed triangles, O.M), open triangles, 0.40 mM. Assay conditions were as described under Materials and Methods. Ku for phosphate is determined from the intersection point. Activity is expressed as units/ml enzyme; each ml contained 1.33 nmol uridine phosphorylase. Inset: secondary plot of ?/ intercepts vs reciprocal uridine concentrations for the determination of K, for uridine and Vf (each ml of reaction mixture contained 94 pmol of tetrameric uridine phosphorylase). MATERIALS
AND
METHODS
Hepee.’ (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), uracil, uridine, and ribose l-phosphate (dicyclohexylammonium salt) were purchased from Sigma Chemical Company; dibasic and monobasic potassium phosphate and potassium chloride were from J. T. Baker Chemical Company. Enzyne preparation Uridine phosphorylase was prepared from E. coli B through 50-&l% (NH&SO, cuts and repeated Sephadex G-150and hydroxylapatite-column steps to apparent homogeneity as judged by gel filtration, polyacrylamide gel electrophoresis, sodium dodecyl sulfate-gel electrophoresis, and isoelectro focusing experiments (10). The enzyme has a molecular weight of 125,000 and consists of four apparently identical subunits of molecular weight 31,000 (10). Enzyme assays. Phosphorolysis and synthesis of uridine were determined spectrophotometrically by the differential absorption between uridine and uracil at 280 nm (Af&~,k~ = 2.1). The reaction mixture contained 50 mM Hepes buffer, pH 7.56, 10 mM KCl, and uridine and phosphate (in the phosphorolysis direction) or uracil and ribose l-phosphate (in the synz Abbreviation used: piperazineethanesulfonic
Hepes, 4-(2-hydroxyethyl)-lacid.
thesis direction) at concentrations as indicated in figure legends. The reaction was initiated by the addition of 5 ~1 protein sample containing 9.4 pm01 (tetramer) of the enzyme to a l-ml assay mixture, and the rate of disappearance or formation of uridine was followed at 230 nm using a Cary 118C spectrophotometer. One enzyme unit is defined as the amount of enzyme which catalyzes the conversion of 1 gmol of substrate/ min at 30°C. Protein concentration was determined according to Schacterle (11). RESULTS
Initial-
Velocity Studies
In the forward reaction (phosphoryolysis), when phosphate was the variable substrate and uridine the varied fixed substrate, double-reciprocal plots gave a family of straight lines intersecting at a point to the left of the ordinate above the abscissa (Fig. 1). A similar pattern was obtained when uridine was the variable substrate. In the reverse direction (nucleoside synthesis), when ribose l-phosphate was the variable substrate and uracil the varied fixed substrate, the same type of pattern
KINETIC
STUDIES
OF
URIDINE
689
PHOSPHORYLASE
2
I
44
1
2.5
-2.5
I
I
J
5.0
7.5
10
ImM RIBOSE-I-PHOSPHATE).’
FIG. 2. Initial-rate studies in the reverse direction with ribose l-phosphate (designated P) as the variable substrate and uracil (designated &) at various fixed concentrations: closed circles, 0.10; open circles, 0.15; closed squares, 0.20; open squares, 0.25; closed triangles, 0.40 mM. Kip for ribose l-phosphate, K, for uracil, and V, are determined from the primary and secondary (inset) plots. Activity is expressed as described in Fig. 1.
was also observed (Fig. 2). Double-reciprocal plots with uracil as the variable substrate yielded a similar intersecting pat-
0 0.0
tern. These intersecting plots indicate an ordered or a rapid-equilibrium random mechanism. To differentiate between these
I
I
0.2
0.4
hM
I
0.6
PHOSPHATEI“
FIG. 3. Competitive inhibition of phosphate by the product ribose l-phosphate. Linear competitive inhibition is demonstrated in the inset. Assay conditions were the same as in Fig. 1. Uridine was maintained at 0.1 mM. Ribose l-phosphate concentrations are diamonds, 0; open circles, 0.50; closed circles, 1.25, open squares, 2 mM. Activity is expressed as described in Fig. 1.
690
VITA,
HUANG,
AND
MAGNI
hibition by the product ribose l-phosphate was competitive (Fig. 3). Under the same conditions, inhibition by the other product, uracil, was noncompetitive (Fig. 4). When uridine was varied and phosphate kept at a nonsaturating level, inhibition by both uracil and ribose l-phosphate was competitive (plots not shown). Uridine syntheses. Similar product-inhibition experiments were performed in the reverse direction. All the inhibition patterns were competitive with the exception of phosphate which was noncompetitive with respect to uracil. The inhibition patterns and reactant concentrations are summarized in Table I. -0.2
0
0.2
0.4
0.6
ImM PHOSPHATEI.’
DISCUSSION
FIG. 4. Noncompetitive inhibition of phosphate by uracil. Uridine was maintained at 0.1 mM. Kb for uracil (dissociation of uracil from the enzyme * phosphate * uracil complex, see Scheme I) is calculated from the intersection point (= -K,/KsKb). The linear nature of the noncompetitive inhibition is shown in the inset. Activity is expressed as described in Fig. 1.
two possibilities, product-inhibition Product
We have performed a detailed kinetic investigation on a highly purified preparation of uridine phosphorylase from E. coli B, including initial-velocity and product-inhibition experiments for each product-substrate pair in the forward and reverse directions of the reaction. The intersecting patterns shown in Figs. 1 and 2 exclude the classic ping-pong (12) and twosite ping-pong (13) mechanisms and favor a sequential mechanism. The product-inhibition patterns summarized in Table I are consistent with a rapid-equilibrium random mechanism with the formation of an enzyme. uracil * phosphate abortive
we carried out a series of experiments.
Inhibition
Studies
Phosphorolysis of uridine. With variable concentrations of phosphate and a constant, nonsaturating level of uridine, inTABLE SUMMARY
Reaction
Pi, Pi, Ud, Ud,
Nucleoside synthesis
u, 0.5 u, 0.5 R-l-P, 1 R-l-P, 1
n Experimental U = uracil, R-l-P
OF PRODUCT-INHIBITION
Constant substrate Cm@
Phosphorolysis
conditions = ribose
I
10 10 0.1 0.1
were as described l-phosphate.
Variable substrate (mM) Ud Ud Pi Pi
(0.025-0.30) (0.025-0.30) (2-20) (2-20)
R-l-P (0.5-2) R-l-P (0.5-2) U (0.06-0.40) u (0.06-0.60) under
Materials
STUDIES’
Product inhibitor bM)
5Pe of inhibition
u (O-0.40) R-l-P (O-3) R-l-P (O-2) u (O-0.60)
Competitive Competitive Competitive Non-competitive
Ud Pi Ud Pi
Competitive Competitive Competitive Non-competitive
(o-0.30) (O-40) (o-0.20) (O-40)
and Methods;
Pi = phosphate,
Ud = uridine,
KINETIC
complex. The proposed kinetic lustrated in Scheme I:
STUDIES
OF
URIDINE
mary and secondary plots and are compiled in Table II. These constants permit the Keq for the uridine phosphorylase reaction (at pH 7.56, 30°C) to be estimated according to the Haldane relationship:
model is il-
The Keg so obtained falls in the range 0.54-0.61. Our findings are essentially in agreement with those of Krenitsky (9), obtained with a partially purified enzyme preparation, except that we have established the presence of an enzyme. uracil . phosphate abortive complex as evidenced by the noncompetive inhibition of uracil vs phosphate in both forward and reverse directions. The absence of the enzyme. uridine . ribose lphosphate abortive complex is not unexpected because the ribosyl moiety of these two compounds presumably would compete for the same binding site. It appears that the uracil and phosphate sites, being separated by the ribose site, do not exert effects on each other’s binding. The agree-
where A = uridine, B = phosphate, P = ribose l-phosphate, and Q = uracil. All the kinetic constants are defined in Scheme I. The kinetic equation describing Scheme I is V
iig=
kf(AB/&&) - kdPQ/KipKq) 1 + A/Kti + B/K6 + &?/K,K, + P/K, + Q/Kti + PQ/K;&*
All the kinetic been determined
[l]
+ BQ/Ksb
constants in Eq. [l] have from our data using priTABLE
II
KINETIC CONSTANTS FOR E. coli URIDINE Reaction Uridine
synthesis
Kinetic
(A)
Phosphate
Nucleoside
PHOSPHORYLASE
Reactant
Phosphorolysis
691
PHOSPHORYLASE
constant’ 0.094 0.054
Kit% Kl (B)
Ribose
l-phosphate
Uracil
(Q)
Kib Kb Kb
mM mM
11.1 6.25 9.9
mM
12.0b
s-i
1.00 0.29
mM rnM
0.57
mM
(P)
mrvi mM
0.15 mM 0.51 mM 5.5b
kb “Experimental Scheme I. b Calculated
conditions per subunit,
are described assuming
four
under identical
Materials monomers.
and Methods.
Kinetic
constants
5-l
are defined
in
692
VITA,
HUANG,
ment between Kib (11.1 mM) and Kb (9.9 mM) and between KQ (0.57 mM) and Kb (0.51 m&f) supports this notion. REFERENCES 1. FRIEDKIN, M., AND KALCKAR, H. (1961) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), 2nd ed., Vol. 5, pp. 237-255, Academic Press, New York. 2. PARKS, R. E., JR., AND AGARWAL, R. P. (1972) iu The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 7, pp. 483-514, Academic Press, New York. 3. KRAUT, A., AND YAMADA, E. W. (1971) J. Biol Chem 246,2021-2030. 4. KRENITSKY, T. A. (1968) J. Biol Chem 243,28712875.
AND
MAGNI
5. KRENITSKY, T. A. (1967) Mol Phavmmm! 3, 526536. 6. KIM, B. Y., CHA, S., AND PARKS, R. E., JR., (1968) J. B&x! Chem 243,1763-1770. 7. JENSEN, K. F. (1976) Eur. J. Bioehem 61,377-386. 8. SCHWARTZ, M. (1971) Eur. J. Biochem 21, 191198. 9. KRENITSKY, T. A. (1976) Biochen~ Biophys. Actu 429, 352-358. 10. VITA, A., PULCINI, A., AND MAGNI, G. (1981) ItaL J. B&hem 30,498~502. 11. SCHA~TERLE, G. R., AND POLLACK, R. L. (1973) And Biochem 51, 654-655. 12. CLELAND, W. W. (1963) Biochem Biophvs. Acta 67, 104-137. 13. NORTHROP, D. B. (1969) J. Bid Chem 244,58085819.