Contribution to the study of the enzymatic activity of benzonase

Journal

of Molecular

Catalysis,

419

69 (1991) 419-427

Contribution to the study of the enzymatic activity of benzonase J. M. Moreno,

J. M. Sanchez-Montero,

tiganic and Pharmaceutical Compltiense, 28040 Madrid

Chemistry (Spain)

J. V. Sin&terra*

Department,

Fact&y

of Pharmucy,

University

and L. B. Nielsen Genetic Engineering (Received

Group, Lundtojtevqi

March 6, 1991; accepted

100, Bygning

227, 2800 Lyngby

@enmark)

May 23, 1991)

Abstract The hydrolytic activity of benzonase has been studied at different values of Mg(I1) concentration, pH, temperature and percentages of water-miscible organic solvents (DMSO, THF, ACN and DMF). The action of these parameters on the UV spectra of benzonase has been analyzed. The best experimental conditions (pH=8.0, T=37 “C, [Mg(II)] =2 mM) lead to a well-dellned conformation. This conformation is active vs. DNA and RNA. Changes in these parameters give conformational alterations which can be monitored by changes in the UV spectra. Organic solvents deactivate the enzyme by hydrophobic interaction of the lipophilic solvent molecules with the allphatic residues of the protein. DMF, the most hydrophilic solvent tested by us, gives slight deactivation of the enzyme. Benzonase hydrolyzes native DNA, heat-denatured DNA and RNA. The active site seems to be the same in all cases. Benzonase has been immobilized for the first time, retaining high enzymatic activity.

Introduction

The influence of the nature and the percentage of organic solvents on the catalytic activity of enzymes has been studied for lipases [ 1, 21, alcohol dehydrogenase [ 31 and proteases [4-61. However, little work has been done concerning nucleases, probably due to the problems associated with the presence of nucleic acids as substrates, since these macromolecules are disturbed by organic solvents and ionic strength [ 7, 8 I. During a study on the catalytic activity of nucleases, we recently reported the influence of organic water-miscible solvents on the DNase activity of native [9, lo] and insolubilized [ 11, 121 microccocal nuclease, and on the RNase activity of native spleen acid exonuclease [ 131. We report here the initial results obtained in the hydrolysis of nucleic acids in several organic-water media, using benzonase as catalyst. Benzonase is a genetically engineered endonuclease developed by Benzon Pharma A/S. The enzyme is produced in the Escherichia coli strain W3110, *Author to whom correspondence

0304-5102/91/$3.50

should be addressed.

0 1991 - Elsevier Sequoia, Lausanne

420

a mutant of the strain K12, which contains the proprietary pNUC1 production plasmid. This plasmid encodes an endonuclease normally expressed in Sewatia murcescens. Benzonase is a protein of molecular weight 30 KD with a p1 of 6.85. The enzyme is active between pH =6.0 and 10.0, and from 0 to 42 “C. A concentration of Mg(II) (l-2 mM) is required to activate the enzyme [ 141. The enzyme exhibits endonuclease activity and can hydrolyze all forms of DNA and RNA, either single- or double-stranded, linear, circular or supercoiled forms. As an endonuclease, it hydrolyzes the internal phosphodiester bond between specific nucleotides. Upon complete digestion, all the free nucleic acids present in solution are reduced to oligonucleotides of 3 to 5 bases in length [ 141. Experimental Benzonase was handed over by Dr. L. B. Nielsen (Genetic Engineering Group, Denmark). Yeast RNA type IV sodium salt and salmon testes DNA type III sodium salt were obtained from Sigma Chemical. AcetonitriIe (ACN) and dimethyl sulfoxide (DMSO) were from Scharlau (Barcelona); N,N’-dimethylformamide (DMF) and tetrahydrofuran (THF) were from Probus (Barcelona). Corn cob particles (EU-GRITS, 0.84-0.54 mm particle size, 0.083 m2 g-i) were kindly provided by Bio-Europe, Toulouse. Agarose gel beads (BioGel A-150 m, 100-200 mesh) were from Bio-Rad Laboratories (Richmond, USA). Perturbation di@zrtmce spectra The UV perturbation difference spectra of benzonase were recorded on a Shimadzu 2 100 UV-Vis spectrophotometer (1 cm pathlength cuvettes). By this method we could measure the influence in the enzyme structure of different parameters such as pH, [Mg(II)] and presence of organic solvents in the media. Enzyme concentration in the cuvette was 1 pg ml- ‘. Enzymatic activity The enzymatic activity was measured by following the increase in the absorbance at 260 run in a Shimadzu 2100 UV-Vis spectrophotometer with independently thermostatted cuvettes (1 cm pathlength) and magnetic stirring. The enzyme concentration in the cuvettes was 0.4 pg ml-‘. Nucleic acid concentrations in the cuvette varied from 5 to 90 pg ml- ‘. The reactions were carried out at diierent conditions of pH, Mg(II) concentration and presence of diverse organic solvents at 2%. The K,, and K, values were determined from kinetic data using the Lineweaver-Burk equation. Immobilization process The activation of agarose and corn cob were carried out according to the tosylation method previously described by the authors [ 15 1. The im-

421

mobilization of benzonase was carried out at 4 “C for 3 h with slow stirring in the optimum buffer for the enzyme (pH =8.0). The percentage of immobilized enzyme was determined by the difference between the initial activity of the native enzyme and the activity of the filtrate of the immobilization process.

Results

and discussion

Influence of pH, iVg(ll) and organic solvents on the enzyme structure In Fig. 1 we show the influence of pH on the enzyme structure. Strong hypsochromic and hypochromic effects are observed in the Uv spectra of benzonase when the pH decreases. This finding can be related to strong conformational changes which would bury the Tyr unit in zones inaccessible to the media. The marked change in the UV spectra at pH G pl (6.85) [ 14) can be explained by the fact that the enzyme is not active at these pHs. In Fig. 2 we show that the progressive increase in Mg(I1) concentration decreases the absorptivity of UV spectra of the enzyme. This is in contrast to observations by other authors for endonuclease S. aur-e-usin the presence of Ca(I1) [ 10, 161. This effect is related to a change in the benzonase structure that affects the active site of the enzyme, such that the enzyme displays an optimum value of Mg(II) concentration of 2 mM. Therefore, we can conclude that Mg(I1) is a cofactor for the enzyme.

270

Fig. 1. UV spectra pH=5.15; [enz]=l

260

290

of benzonsse at (-) pH =8.0, (-o-) r+g ml-‘; T=37 “C; [Mg(I1)]=2 mM.

300

pH = 7.0, (-o-)

pH=6.0,

(-o-)

*g 260

270

Fig.

290

280

0

,

(nm) 2.

UV

[Mg(II)]=9

TABLE

spectra of benzonase at (-) mM; [enz]=l pg ml-‘; T=37

[Mg(II)] = 1 mM, (+) “C; pH=8.0.

Wg(WI=2

mW C-o-1

1

Influence of organic solvents in the UV spectra of benzonase” Solvent

%82

(2943)

(ml

ACN DMF DMSO THF “pH=8.0;

283.0 284.5 284.0 284.5 284.0 [enz] = 1 &ml;

%QO

mg-’

cm-‘)

(ml mg-’

18.5 15.0 14.5 10.0 14.0 T=37

“C; [Mg(II)]=2

7.5 6.5 7.0 4.5 6.0

%!82kQO

cm-‘) 2.47 2.31 2.07 2.22 2.33

mM.

Table 1 indicates that the organic solvents produced a small bathochromic and hypochromic effects on the UV spectra of benzonase. These processes have been related in other enzymes [ 17, 18 ] to conformational changes due to the decrease in polarity of the media by the addition of solvents with a dielectric constant lower than that of water. This effect induces an alteration in the ionization degree of the Tyr of the protein that is analyzed by UV spectroscopy. This change is attributed to an alteration in the hydrogen bonds and hydrophobic bonds between the enzyme and the lipophilic solvents. Therefore, we suggest that benzonase activity is strictly dependent on pH, [Mg(II)] and the dielectric constant of the medium. The action of these effecters on Tyr is marked; thus, these residues must be accessible to the medium in active form at the enzyme.

423

InJh.ence of MgQI) and pH on the enzyme activity The influence of the concentration of Mg(I1) on the nuclease activity of the enzyme was measured under optimal condition (pH = 8.0) according to the literature [ 141. The data in Table 2 suggest that the active conformation of benzonase which degrades native DNA, RNA and heat-denatured DNA is the same for each substrate and requires [Mg(II)] = 2 mM as cofactor (Table 2). Since the nucleic acid structure does not seem to be altered by an increase in [Mg(II)] (7, 13, 19, 201, the observed enhancement of enzymatic activity must be related to the enzyme requirement for Mg(I1) cofactor. This might be related to conformational changes of the enzyme triggered by Mg(I1) as described above (Fig. 2) - which could stabilize the active conformation of benzonase by electrostatic bonds with the charged residues of the protein. Amounts of Mg(II) greater than 2 mM would produce a strong electrostatic interaction with the protein chain, inducing a rigidity of the protein structure that would decrease the nuclease activity and explain (from the structural point view) the experimental data described in the literature for the optimum conditions for benzonase hydrolysis [ 14). Table 2 reveals that the enzyme exhibits greater activity for native DNA compared to other substrates. This indicates a greater specificity of benzonase to double-stranded nucleic acids compared to single-stranded. This specificity is in contrast to that observed for endonuclease S. aureus [ 201 which primarily hydrolyzes heat-denatured DNA [ 16, 211. The greater values of KC, vs. denatured DNA than vs. RNA would be related to a chemospecificity for deoxyribonucleotides, due to the low degree of renaturation of heat-denatured DNA occurring at room temperature [2 11. On the other hand, our data indicate that the enzymatic kinetic values are not consistent with a Michaelis-Menten model, because K, and KC, both TABLE

2

Influence of [Mg(II)] on the nuclease activity of benzonase* Substrate

[Mg(W 1 bw

Km (mg N.A. ml-‘)

K eat (mg N.A. ml-’ min-’ mg enz-‘)

RNA RNA RNA RNA

0 1 2 9

0.082 0.153 0.517 0.155

22.9 30.9 211.6 57.6

DNA DNA DNA DNA

0 1 2 9

0.284 0.350 1.442 0.228

102.0 266.3 584.1 96.2

DNA desn. DNA desn.

1 2

0.202 0.763

137.5 315.7

‘N’NA.=nucleic pg ml-‘.

acid, enz=enzyme;

[enz] = 0.4 pg ml-‘;

pH=8.0;

T=37

“C; [N.A.]=5

to 90

424

increase and decrease in the same directions (Table 2). This finding might be attributed to the fact that the measured processes are: (i) the interaction nucleic acid-Mg(l1); (ii) the interaction nucleic acid-Mg(II)-enzyme; (iii) hydrolysis. The first step is not significant [ 201 but the second one must be important. This fact has been reported for other enzymes, e.g. ribonuclease A or endonuclease S. aureu.s[21, 221. The influence of pH on the nuclease activity of benzonase is shown in Fig. 3. It is observed that the optimum value is pH = 8.0, in both cases. Therefore, the same enzyme conformation is active vs. DNA and RNA, and the same active site might be involved for both substrates. On the other hand, the optimum temperature is 37 “C in all cases (Fig. 4). Influence of organic media on the nuclease activity (DMSO), N,N’Several organic solvents, viz. dimethyl sulfoxide dimethylformamide (DMF’), tetrahydrofuran (THF) and acetonitrile (ACN), were chosen according to their different physicochemical properties [23]. The influence of the nature of these organic solvents at 2% in the presence of Mg(I1) (2 mM) is shown in Table 3. The enzyme activity in the presence of different solvents is similar with the exception of DMF. These results can be explained assuming that the

6

0

P

Fig. 3. Influence of pH in the nuclease activity of benzonase; [enz] = 0.4 pg ml-‘; [Mg(II)] = 2 mM.

T=37

“C;

425

%Actv

t’

20

35

Fig. 4. Influence of temperature pH-8.0; [Mg(II)]=2 mM.

TABLE

T

!50

in the nuclease activity of benzonase;

[enz] = 0.4 pg ml-‘;

3

Influence of organic solvents in the nuclease activity of benzonase” Substrate

Solvent

Properties

(296)

-

RNA RNA RNA RNA RNA

DMSO THF ACN DMF

DNA DNA DNA

DMSO THF

-

Kll

K EM

(mg N.A. ml-‘)

(mg NA. ml-’ min-’ mg enz-‘)

E

log P

78.5 46.6 7.9 37.6 36.7

- 0.970 0.744 - 0.340 - 1.803

0.517 0.350 0.307 0.130 0.433

211.6 45.5 70.5 45.6 144.7

78.5 46.6 7.9

- 0.970 - 0.744

1.442 0.110 0.116

584.1 16.9 35.7

‘N.A.=nucleic acid, enz=enzyme; T=37 “C; [Mg(II)]=2 mM.

[enz]=0.4

pg ml-‘;

[N.A.]=5

to 90 Kg ml-‘;

pH=8.0;

enzyme is deactivated by the presence of organic solvents, producing conformational changes that do not allow the positive effect of Mg(I1). Therefore, the behaviour of benzonase vs. organic water-miscible solvents is similar to that observed with other nucleases [ 13, 211 except in the case of DMF. The explanation for this finding could be that the active conformation is markedly

426

disturbed by hydrophobic bonds between the organic solvent molecule and. the amino acid residue. These interactions might take place with aliphatic amino acids, because small changes are observed in the UV spectra (Table 1) where only the conformational changes related to Phe and ‘&I- can be recorded. DMF is the most hydrophilic solvent, and thus the hydrophobic interactions would be minor. Therefore, this organic solvent displays the least denaturing effect and exhibits the highest value of Kc, (Table 3). Unfortunately a more detailed structural discussion cannot be carried out because the active site of benzonase is not yet well known. Finally, if we examine the results of the influence of pH and temperature (Figs. 3 and 4) on the RNase and DNase activities of the enzyme, we can see small differences in the alteration that both nuclease activities undergo. This fact can be related to the presence of an active site with slight conformational differences in the enzyme-substrate binding zone. A similar fact have been postulated to endonuclease S. VZLUWUS [ 211, which hydrolyzes DNA and RNA at the same pH, but whose behaviour us. temperature and [ Ca(I1) ] is slightly different. Immobilization of enzyme The immobilization of benzonase was carried out for the llrst time using the tosylation method [ 151. The results obtained are shown in Table 4. The percentage of enzyme immobilized is small, perhaps due to the small number of e-NH2 groups from lysine residues, because the isoelectric point ot the enzyme (PI= 6.85) suggests that the NH2 groups are free. Nielsen and Hansen [24] tried to immobilize the enzyme on Sephacryl S-1000R by reductive amination, and did not observe any residual activity in the enzymatic derivative. This value, in our case, is very good (75 and 100% remaining activity) (Table 4). The smaller value of residual activity in the case of agarose in comparison to corn cob can be correlated with diffusional and steric problems of the agarose matrix that obstruct diffusion of the nucleic acids at the active site of the immobilized enzyme into the agarose matrix. These enzyme molecules would be inactive. Only the enzyme immobilized on the surface would be active, as in the case of the enzyme immobilized on corn cob, whose internal structure is inaccessible to the substrate and to the enzyme.

TABLE

4

Summary support

corn cob agarose

of insolubilized

process

of benzonase

support area

46 Supp. actv. (pmol Tos

Insol. enzyme

Residual activity

(m’ g-9

g SUPP-9

(%I

(%I

0.85 17.0

3.6 6.6

13.5 15.0

100 75

427

Finally, benzonase immobilized on both kind of supports exhibits optimum value of pH, Mg(I1) concentration and temperature identical to the native enzyme; furthermore, it undergoes deactivation in the presence of organic solvents, as does the native enzyme (ZO]. Therefore it can be concluded that our immobilization method does not alter the active site of benzonase.

Acknowledgements The present work was supported by the Spanish C.I.C.Y.T. (Grant BIO 88-0246).

References 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

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