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Vol. 41, No. 6, May 1997

BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 1191-1199

D E N A T U R A T I O N O F U R I D I N E P H O S P H O R Y L A S E F R O M Escherichia coli K-12 BY GUANIDINE HYDROCHLORIDE B. I. Kurganov, 1 A. A. Burlakova, 2 V. Ya. Chernyak, 3 and V. G. Debabov 2

~A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospekt 33; Moscow 117071, Russia 2State Research Institute of Genetics and Selection of Industrial Microorganisms, Perry Dorozhny Proezd 1; Moscow 113545, Russia 3A. N. Belozersky Institute of Physico-Chemical Biology, M. V. Lomonosov Moscow State University, Moscow 119899, Russia Received October 8, 1996 Received after revision Oclober 17, 1996

S U M M A R Y : Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine

hydrochloride results in red shift of the maximum in the protein fluorescence spectrum, dissociation of the hexameric enzyme molecule into monomers, and the loss of the enzymatic activity. The initial rate of the enzyme reactivation after the dilution of the enzyme preincubated with guanidine hydrochloride has the second order with respect to protein. It is assumed that the rate of the reactivation process is limited by the reassociation of monomers possessing low enzymatic activity to dimers followed by the rapid step of hexamer formation.

Key Words: Uridine phosphorylase, denaturation by guanidine hydrochloride, renaturation.

INTRODUCTION

The molecule of uridine phosphorylase (EC 2.4.2.3) from Escherichia coli K-12 is a hexamer composed of six identical subunits each with a molecular mass of 27,5 kD (1-3). According to the X-ray data (4, 5), hexamer consists of three structurally formed dimers. Dimers are connected by the symmetry axis of the second order, whereas three dimers in hexamer are linked by the symmetry axis of the third order. When studying the behavior of uridine phosphorylase in the hydrated reversed micelles of aerosol OT in octane (6), we showed that the micelles of a definite size were able to stabilize the catalytically active monomeric, dimeric, trimeric, tetrameric, and hexameric enzyme forms.

1039-9712/97/061191-09505.00/0 1191

Copyright 9 1997 by Academic Press Australia. All rights ~f reproduction in any form reserved.

Vol. 41, No. 6, 1997

BIOCHEMISTRY and MOLECULAR BIOLOGYINTERNATIONAL

In order to obtain an additional information about the relationship between the enzymatic activity of uridine phosphorylase and its quaternary structure, in the present work we studied the enzyme inactivation and the breakdown of the oligomeric structure of the enzyme under the action of guanidine hydrochloride (GuHC1) as well as the possibility of enzyme reactivation after the treatment by GuHC1.

MATERIALS AND METHODS

Uridine and uracil were purchased from Reanal (Hungary), 2-mercaptoethanol from Merck (Germany), Sephadex G-50F from Pharmacia (Sweden), guanidine hydrochloride and 3-morpholine propane sulfonic acid (MOPS) from Fluka (Switzerland). Uridine phosphorylase was prepared from E. coli K-12 (the superproducer of the enzyme) by the method described in (7) using 1 mM 2-mercaptoethanol for protection of sulphydryl groups of the enzyme from oxidation. According to results of electrophoresis in PAAG (8), the enzyme preparation was homogeneous. Protein concentration was determined spectrophotometrically using absorbance index 0.67 cm 1 for 0. l%-solution at 280 nm (9) or by the Bradford assay (10). The enzymatic activity of uridine phosphorylase in 50 mM borate buffer, pH 7.7, was determined at 25~ by the spectrophotometric method (9); the change in absorbance at 280 nm was registered using a spectrophotometer UV-240 (Shimadzu, Japan). The reaction mixture contained 0.4 mM uridine and 100 mM inorganic phosphate. The volume of the reaction mixture was 1 ml. The enzymatic reaction was initiated by the addition of 5 ~tl of the enzyme solution to the reaction mixture. To calculate the initial rate of the enzymatic reaction, we used the difference molar absorbance index (the difference between the molar absorbance indexes of uridine and uracil at 280 nm) equal to 2106 M-l.cm l (11). Sedimentation of the solutions of uridine phosphorylase containing various concentrations of GuHC1 was carried out in a Spinco Model E analytical ultracentrifuge (Beckman, Austria) equipped with an absorption optical system at 20~ In the sedimentation experiments we used 12-mm two-sector cells and rotor An-F. Sedimentation was registered at 280 nm. Ultracentrifugation was carried on immediately after the addition of GuHC1 to the enzyme solution. The sedimentatiton coefficients were calculated with correction for viscosity and density of GuHC1 solutions (12). Protein fluorescence of the uridine phosphorylase solutions containing various concentrations of GuHC1 was measured using a fluorescence spectrophotometer MPF-4 (Hitachi, Japan) with excitation at 290 nm (20~ The enzyme concentration was 0.1 mg/ml. Computer program ORIGIN 3.5 was used for the quantitative treatment of the data obtained. RESULTS AND THEIR DISCUSSION

Fluorescence studies. Denaturation of uridine phosphorylase under the action of GuHC1

is accompanied by a decrease in intensity of protein fluorescence and red shift of the maximum in the emission fluorerscence spectrum (~max)" Such changes in the fluorescence spectra of uridine phosphorylase are due to the enhancement of accessibility of tryptophan residues to water surrounding during denaturation by GuHC1 (13, 14).

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Vol. 4 1 , No. 6, 1 9 9 7

The dependence of the ~vm,X value on the Guile1 concentration has a sigmoidal shape (Fig. 1) and may be described by an empiric equation which is equivalent to the Hill equation (15):

~max ----~'max,0 -1- (~'max,lim -- ~max,0) Ch / (c0h.5 q- ch), where s

and s

~'max in

are the values of

(1)

the absence and in the presence of GuHC1,

respectively; C is the concentration of GuHC1; C0.5 is the concentration of denaturant at which s

= ~m,x,0 + (~'m,x,,~m- ~'m,x,0)/2' and h is the Hill coefficient. The validity of Eq. (1) may be

interpreted as evidence that the constant of equilibrium between the native and unfolded forms of the protein is proportional to the denaturant concentration to the h power (16). The application of Eq. (1) for the analysis of the data presented in Fig. 1 results in the following values of the parameters: 7Vmax, o = 330.7 rim; ~,max,~m= 347.7 +- 0.2 nm; Co,5 = 1.18+0.01 M and h =7.8 + 0.6. Sedimentation studies. The sedimentation pattern for the native uridine phosphorylase

(Fig. 2,a) is indicative of the homogeneity of the preparation used. The sedimentation coefficient (S2o,w)was found to be equal to 8.2 + 0.1 S. In the presence of 1.18 M Guile1 the 350 .

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0 00Uv-~-

0

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.

.

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345

E e5 340

335

........ O S I

330

, 0

....................................... I 1

,

u 2

,

' 3

[GuHCI], i

Fig. 1. The dependence of the wavelength corresponding to the maximum in the fluorescence spectrum of uridine phosphorylase from Escherichia coli K-12 @re,x)on the guanidine hydrochloride concentration. Points are experimental data. Solid line is calculated from Eq. (1). The initial solution of the enzyme was prepared in 50 mM borate buffer, pH 7.7.

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a

b

=0.

Fig. 2. Influence of GuHC1 on the sedimentation of uridine phosphorylase. Concentrations of GuHC1 were: 0 M (a); 1.18 M (b); 1.3 M (c), and 2 M (d). Concentrations of the enzyme were: 1 mg/ml (a, b), 0.8 mg/ml (c), and 0.95 mg/ml (d). Rotor speeds were: 48 000 rpm (a, b, d) and 60 000 (c). Sedimentation times were: 35.3 rain (a), 69.3 min (b), 45.5 min (c), and 72.7 rain (d). Sedimentation was left to right. The initial solution of the enzyme was prepared in 50 mM borate buffer, pH 7.7.

sedimentation pattern (Fig. 2,b), apart from the fast moving boundary which corresponds to the hexameric form of the enzyme (s2o,w= 8.2 +_0.1 S), includes a slowly moving boundary with the sedimentation coefficient equal to 2.6 _+0.1 S. In accordance with an empiric relationship (17), the sedimentation coefficient for globular proteins is proportional to the molecular mass to the 2/3 power. From this relationship it follows that the sedimentation coefficient for the monomeric form of uridine phosphorylase with a molecular mass of 27.5 kD should be 2.5 S. Thus, the slowly moving boundary in Fig. 2,b (S2o,w= 2.6 _+0.1 S) corresponds to the monomeric form of the enzyme. In the presence of 1.3 M GuHC1 the sedimentation pattern consists of two boundaries (Fig. 2,c) one of which corresponds to the hexameric enzyme form (s~o., = 8.2 + 0.1 S) and other moves as a monomer (S2o,w= 2.8 _+ 0.1 S). When the solution contains 2 M GuHC1, the enzyme sediments as a monomer (s2o.w= 2.8 _+0.1 S; Fig. 2,d). 1194

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Influence of guanidine hydrochloride on the enzymatic activity of uridine phosphorylase. Figure 3 shows the dependence of the residual activity of uridine phosphorylase (A/Ao) preincubated with GuHC1 for 3.5 h on the concentration of denaturant (curve 1). At high concentrations of GuHC1 (above 2 M) the enzymatic activity falls to the limiting value other than zero: (A/Ao)~m = 0.06. This level of the residual activity remains constant up to 6 M GuHC1. When concentration of GuHC1 is above 2 M, uridine phosphorylase exists as a monomer. Thus, one can assume that the (A/Ao)~im value corresponds to the enzymatic activity of the monomeric form of the enzyme in the solutions with high concentrations of GuHC1. The fact that the monomeric form of uridine phosphorylase reveals the catalytic activity was demonstrated by us earlier when we studied the behavior of uridine phosphorylase in the hydrated reversed micelles of aerosol OT in octane (6).

I

I

I

1.o

Q

~0.5

0.0

,

0

1

i

2

'-'

i

i

3

[GuHCI], i Fig. 3. Dependence of the relative enzymatic activity (A/Ao) of uridine phosphorylase preincubated with GuHC1 for 3.5 h at 20~ on the denaturant concentration in the.absence of the specific ligands (1) and in the presence of 5 mM uridine (2), 5 mM uracil (3), and 100 mM inorganic phosphate (4). The initial solution of the enzyme was prepared in 50 mM borate buffer, pH 8.1. The final concentration of the enzyme in the mixture with GuHC1 was 0.23 mg/ml. When testing the enzymatic activity, 5 ~tl of the enzyme solution was added to the reaction mixture (the total volume was 1 ml).

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The data presented in Fig. 3 indicate that uridine and uracil (the substrate and product of the uridine phosphorylase reaction) stabilize the protein molecule. The protective effect of the ligands may be characterized by the denaturant concentration (C0.5) at which A/A o = (A/Ao)~r, + [1 - (A/Ao)~JI2:1.7 and 1.5 M respectively for uridine and uracil used in concentrations equal to 5 mM (curves 2.and 3). At the same time, inorganic phosphate (the second substrate of the forward reaction) reveals destabilizing effect: C0.5 = 0.6 M at 100 mM inorganic phosphate (curve 4).

Renaturation of uridine phosphorylase denaturated by guanidine hydrochloride. Figure 4,a shows that dilution of the enzyme preincubated with 2 M GuHC1 for 10 rain by the buffer containing 100 mM inorganic phosphate results in the partial recovery of the enzymatic activity. It should be noted that we did not observe the recovery of the enzymatic activity in the absence of inorganic phosphate. The analysis of the kinetic curves showed that the time of half-conversion for the reactivation process decreases with increasing protein concentration. Therefore one can assume that the enzyme reactivation includes the kinetically significant steps of the association of certain oligomeric forms of the enzyme. If the reactivation process is an association of two inactive enzyme forms into the active oligomer, the initial rate of the reactivation process should be proportional to the enzyme concentration [E] 0 squared. The calculations of the initial rate of the reactivation process (w0) at various concentrations of uridine phosphorylase indicate that the reactivation rate has the second order with respect to the enzyme. The linear character of the dependence of wo on [E]Zo (Fig. 4,b) confirms this conclusion. Since in 2 M GuHC1 the initial enzyme form is monomer, we assume that the rate-limiting step of the reactivation process is the association of monomers possessing low enzymatic activity to dimers followed by fast association of dimers to hexamers: k 2M

> D slowly

~- hexamer

(2)

quickly

(k is the rate constant of the second order). If the formation of dimer from monomers proceeds as an irreversible reaction and at the initial instance all the enzyme exists in the monomeric form, the increase in the portion of the dimeric form (F) with time follows the equation (18):

F=2k[elotl(l+ 2 k [ e l 0 t) where [e] o is the initial molar concentration of the enzyme calculated on monomer.

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Vol. 41, No. 6, 1997

I

I

I

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

L) W

6

3

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1000

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2000

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200

w

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400

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600

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Fig. 4. Reactivation of uridine phosphorylase preincubated with 2 M GuHCI for 10 min at 20~ The dependence of the enzymatic rate catalyzed by uridine phosphorylase v (in ~tmoles of the substrate/1 per 1 sec) on the time after the dilution of the enzyme solution containing the denaturant at 20~ (a) and the dependence of the initial rate of the reactivation of ufidine phosphorylase wo (in nmoles of the enzyme/t per 1 sec) on the enzyme concentration squared ([El o is the molar enzyme concentration calculated on hexamer) (b). The initial solution of the enzyme was prepared in 50 mM MOPS, pH 7.5. The enzyme solution (0.25 mg/ml) in 2 M GuHC1 was diluted by MOPS containing t00 mM inorganic phosphate down to the final concentration: 1.25 ~tg/ml (1); 2.5 ~tg/ml (2), and 5 gg/ml (3). Dotted lines are the levels of the enzymatic activity corresponding to the native enzyme.

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'1

I

I

1.0

O ................... + +~,"~ []

o

u +

~o.s

0

~r-

o

A

~ ~ ' ~ - ' " - " ~ - ' ~

~

[

]

+ - 1

o 1:3

o-2 A-3 ~-4 0.0

i

0

I

100

~

|

t

I

200 [e]0.t, gM.sec

300

Fig. 5. Checking of the applicability of Eq. (4) for description of the kinetics of reactivation of uridine phosphorylase preincubated with 2 M GuHC1 (curves I-3) and 3 M GuHC1 (curve 4) for 10 min. The final concentrations of the enzyme were: 1.25 gg/ml (1); 2.5 gg/ml (2); 5 ~tg/ml (3), and 2.6 gg/ml (4). The enzymatic activity of uridine phosphorylase related to the activity of the native enzyme (AIAo) is shown as a function of the product [e]ot where [el o is the initial molar concentration of the enzyme calculated on monomer and t is time.

Taking into account this equation, we can obtain the following expression for the time-dependent change in the relative enzymatic activity (A/A o is the activity divided on the enzymatic activity of the native enzyme):

2[(A/Ao) -(A/Ao)o]k (A/Ao) [el ot A/Ao = (A/Ao)o + 1 + 2 k (A/Ao) [el o t

'

(4)

where (A/Ao)o and (A/Ao)~ are the values of A/A o at t = 0 and t --> oo (when deriving this equation, we assumed that the final oligomeric form of the reactivation process is active hexamer). If the values of (A/Ao)| for all the kinetic curves are identical, the experimental points should fall on a common curve in the coordinates {A/Ao; [e]ot }. Figure 5 demonstrates the kinetic data presented in these coordinates. Apart from the data taken from Fig. 4, we included the kinetic curve obtained for reactivation of uridine phosphorylase preincubated in 3 M GuHCI for 10 min. Fitting the kinetic data to Eq. (4) has given the following values of the generalized

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values of the parameters: (A/Ao)o = 0.05 _+0.01; (A/Ao)o~= 0.56 _+0.03 and k -- (3.0 + 0.3)'104 -1

-1

M .s . Thus, under the conditions studied the recovery of the enzymatic activity proceeds to a level corresponding to 56% from the activity of the native enzyme. The results obtained testifies that incubation of uridine phosphorylase from E. coli K-12 with GuHC1 produces monomers which am able to reassociation after the dilution of the enzyme solution. This study was funded by Grant 96-04-10016C from the Russian Foundation for Fundamental Research.

REFERENCES 1. Cook, W.L, Kosalka, G.W., Hall, W.W., Narayana, S.V.L., and Ealick, S.E. (1987) J. Biol. Chem., 262, 2852-2853. 2. Tsuprun, V.L., Tagunova, I.L., Lin'kova, E.V., and Mironov, A.S. (1991) Biokhimiya, 56, 930-934 (In Russian). 3. Walton, L., Richards, C.A., and Elwell, L.P. (1989) Nucleic Acids Res., 17, 6741. 4. Morgunova, E.Yu., Mikhailov, A.M., Komissarov, A.A., Mao, Ch., Linkova, E.V., Mironov, A.S., Popov, A.N., Armstrong, Sh.R., Burlakova, A.A., Romanova, D.V., Blagova, E.V., Srnirnova, E.A., Dehabov, V.G., and Ealick, S.E. (1995) Krystallografiya, 40, 672-680 (In Russian). 5. Morgunova, E.Yu., Mikhailov, A.M., Popov, A.N., Blagova, E.V., Smirnova, E.A., Vainshtein, B.K., Mao, Ch., Armstrong, Sh.R., Ealick, S.E., Komissarov, A.A., Linkova, E.V., Burlakova, A.A., Mironov, A.S., and Debabov, V.G. (1995) FEBS Lett., 367, 183187. 6. Burlakova, A.A., Kurganov, B.I., Chebotareva, N.A., Debabov, B.G. (1996) Biol. Membrany, 13, 504-511 (In Russian). 7. Mikhailov, A.M., Smirnova, E.A., Tsuprun, V.L., Tagunova, I.V., Vainshtein, B.K., Linkova, E.V., Komissarov, A.A., Siprashvili, Z.Z., and Mironov, A.S. (1992) Biochem. Int., 26, 607-615. 8. Laemmli, U.K. (1970) Nature, 277, 680-685. 9. Leer, J.C., Hammer-Jespersen, K., and Schwartz, M. (1977) Eur. J. Biochem. 75, 217-224. 10. Bradford, M.M. (1976) Anal. Biochem., 2, 248-254. 11. Dawson, R.M.C., Elliott, D.C., Elliott, W.H., and Jones, K.M. (1986) Data for Biochemical Research, pp. 88-98, Oxford. 12. Kawahara K. and Tanford Ch. (1966) J. Biol. Chem., 241, 3228-3232. 13. Burstein, ~.,A. (1977) Advances in Science and Technology. Biophysics, Vol. 7, p. 190, VINITI, Moscow (In Russian). 14. Demchenko, A.P. (1988) Luminescence and Dynamics of Protein Structure, pp. 62-64, Naukova Dumka, Kiev. 15. Sugrobova, N.P., Gurevich, V.M., Chebotareva, N.A., and Kurganov, B.I. (1979) Biokhimiya, 44, 424-431 (In Russian). 16. Tanford, Ch. (1970) Advances in Protein Chemistry, 24, 60. 17. Bowen, T.J. (1971) An Introduction to Ultracentrifugation, Chapter 7, Wiley-Interscience, London. 18. Kurganov, B.I. (1982) Allosteric Enzymes. Kinetic Behaviour, p. 209, Chichester, John Wiley and Sons. 1199