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 VITA,* CHARLES Y. HUANG,?’ AND GUILIO MAGNI*

*Dipartimento di Biologia CeUulare, Universita di Came-&o, 62032 Came&no (MC), Italy, and tL&oratory of Biochemistry, National Heart, Lung, and Blood Institute,

National Institutes of Health, Bethesda, Maryland .%%%

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 (EC 2.4.2.3) cat- alyzes the reaction

Uridine + phosphate z

uracil + ribose-l-phosphate.

It belongs to the class of nucleoside phos- phorylases which have been shown to occur in many organisms (1,2). The enzyme, be- cause of its role in the degradation of py- rimidine nucleosides as well as in the “sal- vage pathway” of nucleic acid synthesis, occupies an important position in metab- olism. The uridine phosphorylase from rat liver has been characterized by initial- velocity 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 thymi- dine phosphorylase (4), and calf spleen (5) and human erythrocytes (6) purine nu-

1 To whom correspondence should be addressed:

Building 3, Room 218, NIH, Bethesda, Md. 20205.

cleoside phosphorylase. Salmonella tgphi- murium and Escherichia coli purine nu- cleoside phosphorylases have been shown to work by a sequential reaction mecha- nism 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 re- action mechanism was also proposed for thymidine phosphorylase (8), a nucleoside phosphorylase showing strict specificity for the deoxyribosyl moiety. A random mech- anism, however, was proposed by Krenit- sky (9) for E. coli uridine phosphorylase using a partially purified enzyme prepa- ration.

In this report detailed kinetic studies, including initial-velocity and product- inhibition experiments in the forward and reverse directions of catalysis, have been conducted with a homogeneous en- zyme preparation. The results reveal the presence of an enzyme. uracil + phosphate abortive complex which has not been es- tablished in previous kinetic studies.

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-piperazineethane- sulfonic acid), uracil, uridine, and ribose l-phosphate

(dicyclohexylammonium salt) were purchased from Sigma Chemical Company; dibasic and monobasic po-

tassium 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-150- and hydroxyl-

apatite-column steps to apparent homogeneity as judged by gel filtration, polyacrylamide gel electro- phoresis, 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 di- rection) or uracil and ribose l-phosphate (in the syn-

z Abbreviation used: Hepes, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid.

thesis direction) at concentrations as indicated in fig-

ure 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 (phosphory- olysis), when phosphate was the variable substrate and uridine the varied fixed sub- strate, double-reciprocal plots gave a fam- ily of straight lines intersecting at a point to the left of the ordinate above the ab- scissa (Fig. 1). A similar pattern was ob- tained when uridine was the variable sub- strate. 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 PHOSPHORYLASE 689

2

44 I -2.5

1 I I J

2.5 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-recip- tern. These intersecting plots indicate an rocal plots with uracil as the variable sub- ordered or a rapid-equilibrium random strate yielded a similar intersecting pat- mechanism. To differentiate between these

0 I I I

0.0 0.2 0.4 0.6

hM 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 com- petitive (plots not shown).

Uridine syntheses. Similar product-in- hibition experiments were performed in the reverse direction. All the inhibition patterns were competitive with the excep- tion of phosphate which was noncompet- itive 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.’

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, we carried out a series of product-inhibition experiments.

Product Inhibition Studies

Phosphorolysis of uridine. With variable concentrations of phosphate and a con- stant, nonsaturating level of uridine, in-

We have performed a detailed kinetic investigation on a highly purified prepa- ration of uridine phosphorylase from E. coli B, including initial-velocity and prod- uct-inhibition experiments for each prod- uct-substrate pair in the forward and re- verse directions of the reaction. The in- tersecting patterns shown in Figs. 1 and 2 exclude the classic ping-pong (12) and two- site ping-pong (13) mechanisms and favor a sequential mechanism. The product-in- hibition patterns summarized in Table I are consistent with a rapid-equilibrium random mechanism with the formation of an enzyme. uracil * phosphate abortive

DISCUSSION

TABLE I

SUMMARY OF PRODUCT-INHIBITION STUDIES’

Constant Product

substrate Variable inhibitor 5Pe of Reaction Cm@ substrate (mM) bM) inhibition

Phosphorolysis Pi, 10 Ud (0.025-0.30) u (O-0.40) Competitive

Pi, 10 Ud (0.025-0.30) R-l-P (O-3) Competitive

Ud, 0.1 Pi (2-20) R-l-P (O-2) Competitive

Ud, 0.1 Pi (2-20) u (O-0.60) Non-competitive

Nucleoside u, 0.5 R-l-P (0.5-2) Ud (o-0.30) Competitive

synthesis u, 0.5 R-l-P (0.5-2) Pi (O-40) Competitive

R-l-P, 1 U (0.06-0.40) Ud (o-0.20) Competitive

R-l-P, 1 u (0.06-0.60) Pi (O-40) Non-competitive

n Experimental conditions were as described under Materials and Methods; Pi = phosphate, Ud = uridine,

U = uracil, R-l-P = ribose l-phosphate.

KINETIC STUDIES OF URIDINE PHOSPHORYLASE 691

complex. The proposed kinetic model is il- lustrated in Scheme I:

where A = uridine, B = phosphate, P = ri- bose 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;&* + BQ/Ksb [l]

All the kinetic constants in Eq. [l] have been determined from our data using pri-

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:

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 prepara- tion, except that we have established the presence of an enzyme. uracil . phosphate abortive complex as evidenced by the non- competive inhibition of uracil vs phosphate in both forward and reverse directions. The absence of the enzyme. uridine . ribose l- phosphate abortive complex is not unex- pected 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 sep- arated by the ribose site, do not exert ef- fects on each other’s binding. The agree-

TABLE II

KINETIC CONSTANTS FOR E. coli URIDINE PHOSPHORYLASE

Reaction Reactant Kinetic constant’

Nucleoside synthesis

Phosphorolysis Uridine (A)

Phosphate (B)

Kit%

Kl

Kib

Kb Kb

Ribose l-phosphate (P)

Uracil (Q)

kb

0.094 mM

0.054 mM

11.1 mM

6.25 mrvi

9.9 mM

12.0b s-i

1.00 mM 0.29 rnM

0.57 mM 0.15 mM

0.51 mM

5.5b 5-l

“Experimental conditions are described under Materials and Methods. Kinetic constants are defined in Scheme I.

b Calculated per subunit, assuming four identical monomers.

692 VITA, HUANG, AND MAGNI

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.

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