Biochimica et Biophysica Acta, 1160 (1992) 199-205 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
199
BBAPRO 34332
Role of the reactive cysteine residue in restriction endonuclease Cfr9I Virginius Siksnys and Milda Pleckaityte Institute of Biotechnology 'Fermentas', Vilnius (Lithuania) (Received 22 April 1992) (Revised manuscript received 1 July 1992)
Key words: Restriction endonuclease Cfr9I; Thiol group; Chemical modification
Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pK a value of 8.9 _+0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pK a values of 6.3 :i: 0.15 and 8.7 + 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.
Introduction Type-II restriction endonucleases are widely used in genetic engineering experiments due to their unique capability to recognize specific nucleotide sequences in D N A and cut the phosphodiester bond at fixed locations relative to their recognition sites. Recently, we have initiated studies on the mechanism of catalysis and sequence-specific discrimination by restriction endonuclease Cfr9I, which recognizes the palindromic hexanucleotide sequence c V C C G G G , cleaving this sequence as indicated by the arrow. During the preliminary biochemical characterization of the restriction endonuclease Cfr9I we showed [1] that the enzyme completely lost the catalytic activity after incubation with N-ethylmaleimide (NEM), a selective
Correspondence to: V. Siksnys, Institute of Biotechnology, "Fermentas", Greiciuno 8, Vilnius 2028, Lithuania. Abbreviations: IA, iodoacetate; IAA, iodoacetamide; NEM, N-ethylmaleimide; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); MMTS, methyl methanethiosulfonate; NTCB, 2-nitro-5-thiocyanobenzoic acid; BSA, bovine serum albumine; DTT, dithiotreitol; EDTA, ethylenediaminetetraacetic acid, Tris-HCl, tris(hydroxymethyl)aminomethane.
sulfhydryl-modifying reagent [2]. This suggested that one or more cysteine residues might be implicated in catalysis/binding by this restriction endonuclease. On the basis of chemical modification data of restriction endonucleases by N E M it was suggested previously [3] that sulfhydryl groups were important for the activity of BamHI, PvuI, SmaI, Pst I, HindlII and AvaI, but not for that of EcoRI, Sail, BgllI, HpaI and Sst I. Recently, it was shown that restriction endonucleases Cfr9I [1], RsrI [4], EcoRV [5], Eco72I and BcnI (Siksnys, V., unpublished data) were sensitive to modification by NEM, too. We supposed that the differences in sensitivity of the restriction endonucleases to modification by N E M might reflect differences in the organisation of the active sites of various restriction endonucleases and suggested that in the restriction endonucleases sensitive to N E M cysteine residues might be located in or near the active site of the enzyme. In this paper, we report the results of chemical modification of restriction endonuclease Cfr9I by several sulfhydryl-specific reagents. On the basis of the enzyme inactivation studies by thiol-modifying reagents, substrate protection studies, pH-variation of kinetic parameters of substrate hydrolysis and substrate bind-
200 ing properties of native and modified enzyme we propose that restriction endonuclease Cfr9I contains a single reactive sulfhydryl residue which is non-essential for catalysis but might be located at or near the substrate-binding site of the enzyme. Materials and Methods
Materials. Restriction endonuclease Cfr9I, T4 polynucleotide kinase and A DNA were products of Fermentas (Vilnius). Cfr9I migrated as a single band in SDS-PAGE with a molecular mass of 35 kDa. The protein concentration was estimated according to the method of Bradford [6]. Buffer salts were obtained from Sigma, NEM, NTCB, MMTS, DTNB, IA and IAA from Fluka and agarose was supplied by Bio-Rad. [y-32p]ATP was purchased from Izotop (Leningrad). The oligodeoxynucleotide 5'-GGACCCGGGTCC-3' was synthesized using a Biosearch 8700 DNA synthesizer and purified by HPLC in our laboratory. The oligodeoxynucleotide was phosphorylated using T4 polynucleotide kinase and [y-a2p]ATP and the labeled oligodeoxynucleotide was separated on a Sephadex G-50 column. Assay of the catalytic activity of restriction endonuclease Cfr91. Aliquots (1-5/zl) of (1-5)" 10 -6 M enzyme solution were added to 40/zl of solution containing 2 /zg A DNA in a buffer of the following composition: 10 mM Tris-HC1, 5 mM MgC12, 200 mM sodium glutamate, 1 mM DTT, 100 /zg/ml BSA (pH 7.5) and incubated for a 1 h at 37°C. The reaction was quenched by addition of 20 /zl of a solution containing 0,25% Bromophenol blue, 60 mM EDTA and 50% glycerol. The cleavage products were fractionated on 0.7% agarose gels at 10 V / c m and visualised by UV-light after ethidium bromide staining. For quantitative assay of the enzyme catalytic activity in chemical modification studies, the gels were photographed under UVlight using a red filter and Micrat-N (Tasma) type film. The negatives were scanned on a Biomed laser densitometer to evaluate the relative amount of DNA in the bands.
Inactivation of restriction endonuclease Cfr9I with sulfhydryl reagents. 20 /zl aliquots of 2.3.10 - 6 M enzyme solution in 0.02 M phosphate buffer (pH 7.5) containing 0.2 M KC1 and 1 mM EDTA were incubated for 10 min at 22°C at appropriate concentration of thiol reagent (Table I), aliquots of 5 /xl were withdrawn at fixed time-intervals and residual catalytic activity of enzyme was assayed as described, except that DTT was ommitted from the reaction mixture in the case of enzyme modified with DTNB, MMTS and NTCB. In the sequential modification experiment, the enzyme was treated by MMTS or IAA, as described above and catalytic activity assayed. After 10 min expo-
sure to MMTS or IAA, N-ethylmaleinimide was added from stock solution to 10 mM, incubated for 10 min and catalytic activity assayed.
Titration of restriction endonuclease Cfr9I by DTNB. To 500/zl of 4- 10 -6 M solution of restriction endonuclease Cfr9I in 0.01 M phosphate buffer (pH 7.5) containing 0.5 mM EDTA and 0.1 M KCI and equillibrated to 25°C in a 1-ml spectrophotometer cell, 10/zl of 1.4 mM DTNB solution in the same buffer was added, thoroughly mixed and the absorbance at 412 nm recorded at fixed time-intervals on a Specord M40 spectrophotometer (Jena). The value of 13600 M -I cm-1 was used as the molar extinction coefficient for the p-nitrophenylthiolate anion [7]. To determine the extent of inactivation of restriction endonuclease Cfr9I during the titration of enzyme by DTNB, 20 #I aliquots were removed from the identical control reaction mixture and assayed for residual catalytic activity as described. Kinetics of the reaction of Cfr9I with NEM. Chemical modification of restriction endonuclease Cfr9I by NEM was carried out at 22°C in 100 /xl reaction mixture containing 10 mM Tris-HCl, 0.2 M KC1, 1 mM EDTA at appropriate pH value and 4.6.10 -6 M of enzyme. The reaction was initiated by addition of 3 Izl of 0.1 M NEM in water. Aliquots of 20 /zl were withdrawn at fixed time-intervals, 2 #1 7.2 M/3-mercaptoethanol was added to quench the reaction and assayed for the remaining catalytic activity, as presented above. The pseudo-first-order reaction-rate-constants of chemical modification of restriction endonuclease Cfr9I by NEM were calculated according to the following equation: In
At/Ao=-kt
(1)
where A t and A 0 represent the enzyme activity at times 0 and t, respectively. Second-order reactionrate-constants were determined by division of pseudofirst-order reaction-rate-constants by NEM concentration. pK a values were obtained using the non-linear regression program Enzfitter (Elsevier-Biosoft) to fit the data to the equation k = c 1 + C 2 ( 1 0 P H - p K a ) / I + 10 p H - p K a
(2)
For substrate-protection studies the enzyme was preincubated with 14.4 lzM of double-stranded oligodeoxynucleotide 5'-GGACCCGGGTCC-3' before the addition of NEM.
pH-studies of oligodeoxynucleotide 5'-GGACCCGGGTCC-3' hydrolysis by Cfr9I. The reaction rate studies of oligodeoxynucleotide 5'-GGACCCGGGTCC-3' hydrolysis by Cfr9I were carried out at 37°C in 20/xl of reaction mixture containing 10 mM Tris-HCl, 5 mM
201 MgC12, 200 mM sodium glutamate, 1 mM DTT, 100 /zg/ml BSA at appropriate pH value and 0.02 /zM of 5'-[32p]-GGACCCGGGTCC-3 '. The reactions were initiated by addition of the enzyme to a concentration of 0.46.10 -6 M. After 5 min, 10-/zl aliquots of reaction mixture were withdrawn and spotted on DEAEcellulose thin-layer plates, which were then developed in Homomixture VI. Spots containing radioactivity were located by autoradiography. The spots corresponding to the product and substrate were scraped off the plate and their radioactivity was determined by liquid scintilation counting, pK a values were obtained using the nonlinear regression program Enzfitter (Elsevier-Biosoft) to fit the data to the following equation: v = C / 1 + 10(pK~-pH) + 10(pH pK')
(3)
where C is a pH-independent constant and pK a and pKd are pKa values of groups controlling substrate hydrolysis.
Binding studies of restriction endonuclease with DNA using gel mobility shift assay. These were performed as described in Refs. 8 and 9. The binding buffer was 10 mM Tris-HCl, 200 mM sodium glutamate, 1 mM DTT, 100 /zg/ml BSA, 0.1 mM EDTA (pH 7.5). The stock solution of restriction endonuclease Cfr9I or the solution of NEM-modified enzyme was diluted to a final concentration of 0.35 × 10 -5 M in the same binding buffer containing 0.1 nM 32p-labeled 322-bp PvuIIPvuII fragment of plasmid pUC19 containing the recognition site of restriction endonuclease Cfr9I, incubated 15 min at room temperature before adding 5 /zl of loading buffer (binding buffer with 33% glycerol and 0.1% Bromophenol blue). 5-/zl aliquots were loaded on a 6% polyacrylamide gel and subjected to electrophoresis at room temperature in TBE buffer [10]. After electrophoresis the gel was fixed in 10% acetic acid and analysed by autoradiography. Results and Discussion
Inactivation of restriction endonuclease Cfr9I by sulfhydryl-specific reagents In order to elucidate the role of cysteine residues in catalysis/binding by restriction endonuclease Cfr9I, we have studied the reaction of restriction endonuclease Cfr9I with various sulfydryl-modifyingcompounds, including alkylating reagents (iodoacetate, iodoacetamide, N-ethylmaleimide), mixed disulfide-forming sulfhydryl reagents ((5,5'-dithiobis(2-nitrobenzoic) acid, methyl methanethiosulfonate) and the cyanylating reagent 2-nitro-5-thiocyanobenzoic acid. The effect of chemical modification on the catalytic activity of restriction endonuclease Cfr9I is summarized in Table I. As can be seen from data presented in Table I, thiol blocking compounds differ regarding the effect of mod-
TABLE I Inactivation of restriction endonuclease Cfr9I by sufhydryl reagents
Reagent
Substituent
N-Ethylmaleimide
Reagent concentration (mM)
O
Catalytic activity (%)
10
0
10
0
(NEM)
Et_ i~
-
5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB)
- OOC _ ~ O2N
Iodoacetic acid
- OCCH 2 II
50
0
Methyl methane thiosulphonate (MMTS)
CH3S-
10
100
Iodoacetamide
NH2.C.CH 2-
50
100
O
0A)
O
flAA) 2-Nitro-5-thiocyanobenzoic acid (NTCB)
S-
O " CN-
0.4
40
ification on the catalytic activity of restriction endonuclease Cfr9I. Incubation of the restriction endonuclease with NEM, DTNB and IA led to a complete loss of the catalytic activity of the enzyme, suggesting that cysteine residue/residues might play a crucial role in functioning of restriction endonuclease Cfr9I. On the other hand, modification of the enzyme by MMTS and IAA had no prominent effect on the enzyme activity. Following IAA or MMTS treatment the enzyme became resistant to the inactivation by NEM (data not shown), suggesting that both IAA, MMTS and NEM react with the same thiol residue(s). Despite the differences in the chemical nature of modification agents, correlation between the structure of reaction products and the effect of modification on catalytic activity is noticeable. The first group of reagents (DTNB, NEM, IA) introduce bulky aromatic or cyclic aliphatic residues or relatively small but negatively charged residue (-CH2COO-) in the case of thiol alkylation by IA. The reagents from the second group (MMTS and IAA) upon modification of Cys-residues introduce small uncharged substituent (-CH 2 CONH 2 or CH3S-) instead of hydrogen of the thiol group, tt is reasonable to suppose that inactivation of restriction endonuclease Cfr9I by the former reagents is due to the steric or electrostatic effect created by the bulky or charged derivatives. The results of enzyme modification with NTCB are not contradicting with this conclusion, too.
202 NTCB upon reaction with sulfhydryl groups is expected [11] to introduce a small cyano group which could block thiol groups without causing any loss of enzyme activity, as it was found in the case of restriction endonuclease Cfr9I modification by MMTS. However, treatment of restriction endonuclease Cfr9I with NTCB led to a partial loss of catalytic activity. It was reported [12,13] that mixed disulfide adducts were formed during the reaction of sulfhydryl groups with NTCB, due to attack of thiol on the sulfur atom of NTCB. It is possible that the reaction of restriction endonuclease Cfr9I with NTCB procceeds by two reaction pathways leading to the formation of an active cyanylated enzyme and an inactive mixed disulfide adduct similar to the one formed during the enzyme modification by DTNB. This assumption was in agreement with experimental evidences indicating that the catalytic activity of the enzyme modified by DTNB and NTCB was co~apletely restored by treatment with flmercaptoethanol (data not shown)./3-Mercaptoethanol removes the bulky 2-nitrothiobenzoic residue in the mixed disulfide adduct of the enzyme [14], thereby restoring catalytic activity. These data indicate that restriction endonuclease Cfr9I does not contain any Cys-residue/residues essential for catalysis. However, there is a possibility that these residue/residues might be located in or near vicinity of the substrate-binding site. Chemical modification of the enzyme by bulky or charged derivatives could sterically or electrostatically hinder substrate binding thus causing the loss of catalytic activity. This assumption was further supported by the substrate protection studies and direct substrate binding experiments of the native and modified enzyme using the gel-mobility shift assay.
Protective effect of substrate against inactivation of restriction endonuclease Cfr9I by NEM NEM rapidly inactivated restriction endonuclease
Cfr9I at pH 7.5 in a time-dependent manner (Fig. 1). The inactivation followed pseudo-first order kinetics. The pseudo-first-order rate-inactivation-constant at pH 7.5 was equal to 0.12 min -1 and the corresponding second-order rate-constant was 40 M-1 min-1. A plot of the logarithm of residual activity vs. time (inset in Fig. 1) was linear to where at least of 90% of the activity was lost, indicating that the inactivation appeared due to modification of a single residue (or 2 or more residues with the same reactivity towards the reagent). Preincubation of restriction endonuclease with synthetic oligodeoxynucleotide duplex 5'-GGACC C G G G T C C - 3 ' containing the recognition sequence completely protected the enzyme from inactivation by NEM (Fig. 1) indicating that in the case of restriction endonuclease Cfr9I, the cysteine residue/residues might be positioned at or near the active site of the
100
~. ~.
•
•
e
~
80 \
-~
60
~
40
CE
time, min
k
>:
•
~ o~- ,
\
~0
°
20 ,
---30
i i
20 i
0
5
i
i
10 ~5 Time, minutes
i
20
25
Fig. 1. Inactivation of restriction endonuclease Cfr91 by NEM in the absence (o) and presence (e) of 14.4 /zM oligodeoxynucleotide duplex 5'-GGACCCGGGTCC-3'. The inset shows the plot of the logarithmic dependence of enzyme inactivation in the absence of substrate. Conditions; pH 7.5, 10 mM Tris-HCl, 200 mM KCI, 1 mM EDTA. [E] = 4.6.10 -6 M, [NEM] = 3.10 -3 M, 22°C.
enzyme and may be in or in some way associated with the binding site of substrate.
Effect of modification of restriction endonuclease Cfr9I by NEM on substrate binding To examine whether the modification of restriction endonuclease Cfr9I eliminated binding of the enzyme to the DNA we tested the ability of the modified enzyme to bind a 322 bp long D N A fragment containing a single Cfr9I recognition site using a gel-mobility shift assay [8,9]. As can be seen from the data presented in Fig. 2, restriction endonuclease Cfr9I in the absence of Mg 2+ formed a complex with a 322 bp long DNA fragment containing a recognition sequence, having different electrophoretic mobility from the free DNA. However, the enzyme modified with NEM failed to form such a complex (Fig. 2, Lane 3) under the same conditions. These data demonstrate clearly that the NEM-modified enzyme loses the ability to bind DNA in the absence of Mg 2÷. However, these studies cannot eliminate the possibility that modification of restriction endonuclease Cfr9I by NEM destroys the structural integrity of the enzyme. Furthermore, it has been suggested recently that the inactivation of restriction endonucleases Rsr I [15] and EcoRV (Dr. S. Halford, personal communication) by NEM was caused by dissociation of the enzyme dimer upon modification of reactive Cys-residue located near the subunit interface. Our previous studies indicated that restriction endonuclease Cfr9I existed primarily as a dimer in solution, consisting of two identical subunits [1] and the loss of its catalytic activity on reaction with NEM could also be due to dissociation of the enzyme into subunits. To test this, a control sample of the enzyme and a sample completely inacti-
203 vated by treatment with N E M were applied to H P L C TSKgel SW3000 gel-filtration column that was previously calibrated with proteins of a known molecular weight. Two restriction endonuclease samples were found to have identical elution times, corresponding to the protein with molecular mass (70 kDa, data not shown), demonstrating that N E M inactivation is not the consequence of a subunit dissociation.
¢
2
3
Determination of the number of Cys-residues in restriction endonuclease Cfr9I by titration with 5,5'-dithiobis(2-nitrobenzoic acid) Recently, a complete primary structure of the restriction endonuclease Cfr9I has been deduced from D N A sequence data (S. Menkevicius, unpublished data), indicating the presence of four cysteine residues. We were unable to quantitate precisely the number of cysteine residues modified during reaction with N E M using a radioactive derivative of [3H]NEM, due to large experimental variations related to the incomplete and irreproducible precipitation of protein by trichloroacetic acid from diluted solution. Consequently, the modification of the enzyme with D T N B was performed to determine the n u m b e r of Cys-residues available for reaction with sulfhydryl reagents. Moreover, as indicated above, the modification of the restriction endonuclease by D T N B lead to the complete loss of the catalytic activity, as well as in the case of modification with NEM. The general features of the restriction endonuclease Cfr9I reaction with D T N B are illustrated in Fig. 3A. Concentration of T N B 2- ion which is released when D T N B reacts with sulfhydryl groups increased rapidly in the first 2 min and then remained almost constant throughout 30 min. From the concentration of T N B 2released during 30 min, the n u m b e r of sulfhydryl groups was calculated and plotted against the time of reaction. About 1 mol of D T N B reacted with 1 mol of protein of restriction endonuclease Cfr9I, indicating that among 4 sulfhydryl groups present in the protein only one sulfhydryl group reacted with DTNB. The time-course of loss of enzymatic activity corresponded to the release of T N B 2-, indicating that modification of 1 sulfhydryl group resulted in a complete loss of the catalytic activity of the enzyme, as was supposed on the basis of kinetics of enzyme inactivation by NEM. Moreover, the restriction endonuclease Cfr9I which previously reacted with N E M failed to to react with DTNB, suggesting that modification of a Cys-residue by N E M included this reaction with DTNB. On the other hand, the titration of restriction endonuclease Cfr9I in the presence of 6M of guanidinium chloride (Fig. 3B) revealed the presence of four Cys-residues, which is consistent with the number of Cys-residues deduced from the amino-acid sequence of the enzyme and indicating that the enzyme does not
L
Fig. 2. Gel-mobility shift assay on 322-bp DNA fragment. Lane 1, free DNA; lane 2, wild-type Cfr9I enzyme; lane 3, NEM-modified Cfr9I enzyme. Binding buffer; 10 mM Tris-HCl, 200 mM sodium glutamate, 1 mM DTT, 100 ~g/ml BSA, 0.1 mM EDTA (pH 7.5), final concentration of Cfr9I and the 32p-labeled 322-bp PvulI-PvulI fragment of plasmid pUC19, 0.35.10 -5 M and 0.1 nM, respectively. After incubation for 15 min, 5 /zl of loading buffer (binding buffer with 33% glycerol and 0.1% Bromophenol blue) was added and 5/zl aliquots were loaded on a 6% polyacrylamidegel and subjected to electrophoresis at room temperature in TBE buffer. After electrophoresis, the gel was fixed in 10% acetic acid and analysed by autoradiography. contain any disulfides. It seems that the three Cys-residues of restriction endonuclease Cfr9I are 'buried' in the enzyme interior and are not readily accesible to the solvent.
Determination of the pK a value of reactive Cys-residues in restriction endonuclease CfrgI In order to characterize the reactive Cys-residue located, as we suppose, at or near the substrate-binding site of restriction endonuclease Cfr9I, we have studied the p H - d e p e n d e n c e of the enzyme inactivation by NEM. We were unable to use D T N B due to its high
204 =~ l.O
~.00
S
,= ~o
A
1.0
u~
0.5
50~=.
'~
2
;2
-~
- ,---t
i--
o
1
o.o[ 0
10
........ 20 30
Q3
5 10 Time, minutes
Time. minutes
15
reactivity. NEM inactivated the enzyme more slowly than DTNB, permitting determination of the reactionrate-constants. The inactivation of the restriction endonuclease by NEM was dependent on the pH value of the reaction mixture and accelerated with increasing pH (Fig. 4A). This would be consistent with a reaction mechanism involving an unprotonated thiolate residue reacting by
800
>
60
>. 600 ~J {D co ~o
E
7 4O
400
2O
20C
~o co oD
0 0
0.6
0.4 c'r"
0.2
i
I
I
I
6
7
8
9
pH Fig. 5. Effect of pH on the reaction rate of oligodeoxynucleotide duplex 5 ' - G G A C C C G G G T C C - 3 ' hydrolysis by restriction endonuclease Cfr9I. Conditions: 10 m M Tris-HCl, 5 m M MgCI2, 200 m M sodium glutamate, 1 m M DTT, 1 0 0 / ~ g / m l BSA, [E] = 0.46.10 -6 M, 5'-[32 P ] - G G A C C C G G G T C C - 3 ' ] = 2 . 1 0 - 8 M.
nucleophilic addition to NEM. The data on pH-dependence of the inactivation rate constant (Fig. 4B) allowed us to determine the pK a value of the group involved in the modification reaction and controlling the catalytic activity of the enzyme. The determined value of 8.9 was in a good agreement with the known values of pKa of sulfhydryl groups in proteins [16].
pH-dependence of kinetic parameters of substrate hydrolysis by restriction endonuclease Cfr9I
100 80
> >
Fig. 3. The reaction of restriction endonuclease Cfr9I with DTNB. The extent of reaction was monitored at 412 n m by release of 5-thio-2-nitrobenzoic acid anion, the value of 13600 M -1 cm - I [7] was used as the molar extinction coefficient for the p-nitrophenylthiolate anion. The a m o u n t of T N B 2- released is calculated per mol of the protein subunit. (A): 1, Time-course of the restriction endonuclease Cfr9I reaction with D T N B under non-denaturing conditions; 2, time-course of enzyme inactivation. Conditions: [ E ] = 4.10 - 6 M, [ D T N B ] = 8 . 1 0 - 6 M, 0.01 M phosphate buffer (pH 7.5), 0.5 m M E D T A , 0.1 M KCI, 25°C. (B): 1, Time-course of the restriction endonuclease Cfr9I reaction with D T N B under non-denaturing conditions: [ E ] = 4 . 1 0 - 6 M, D T N B = 2 . 6 - 1 0 -5 M, 0.01 M phosphate buffer (pH 7.5), 0.5 m M E D T A , 0.1 M KCI, 25°C; 2, time-course of the restriction endonuclease Cfr9I reaction with D T N B in the presence of 6 M of guanidinium chloride. Conditions are the same as in 1, except for the presence of 6 M guanidinium chloride.
°
0.8
c3
5 Time,
~0 15 minutes
20
7.5
8.0
8.5
g.O
9.5
pH
Fig. 4. Effect of pH on the inactivation rate of restriction endonuclease Cfr9I by NEM. (A): Time-course of enzyme inactivation at different pH values. Conditions: 10 m M Tris-HCl, 200 m M KCI, 1 m M E D T A , [ E ] = 4.6.10 -6 M, [ N E M ] = 3.10 -3 M, T = 22°C. (B): Effect of pH on the second-order rate-constant of inactivation of restriction endonuclease Cfr9I by NEM. Conditions as in Fig.2. The second-order rate-constants were calculated by dividing the pseudofirst-order inactivation-rate-constant by the concentration of N E M used. The solid curve is the best fit to the expermental points according to Eqn. 2.
pH-dependence studies of kinetic parameters of the enzymatic reaction could give additional information regarding the groups on the enzyme or substrate involved in either binding or catalysis. We have studied the pH-dependence of the reaction rate of oligodeoxynucleotide duplex 5'-GGACCCGGGTCC-3' hydrolysis by restriction endonuclease Cfr9I (Fig. 5). The hydrolysis rate of the synthetic oligodeoxynucleotide duplex by Cfr9I showed its maximum value at pH 7.5 which decreased on either side of this maximum. The reaction rate dependence of the substrate hydrolysis by Cfr9I vs. pH was bell-shape-dependent and revealed two residues with pK a values of 6.3 + 0.15 and 8.7 + 0.15, controlling the catalytic activity of the enzyme. The pK a value of 6.3 + 0.15 might be assigned to Hisor Glu-residues. The pK a value of the group controlling the alkaline limb of the substrate hydrolysis is similar to the pK a value of the cysteine residue modified by NEM.
205
Conclusions Our results demonstrate that restriction endonuclease Cfr9I contains a reactive Cys-residue which is not essential for the catalytic activity of the enzyme. However, these data provide a strong evidence that this Cys-residue is located near or within the active site of the enzyme. Additional studies will be required to localize the reactive Cys-residue in the sequence and establish the particular role played by this cysteine residue in the functioning of restriction endonuclease
Cfr9I. Acknowledgements The authors are indebted to Ms. R. Lukauskaite for purification of the enzyme, Mrs. Z. Maneliene for the oligodeoxynucleotide synthesis, Mrs. L. Petrauskiene for technical assistance, Mr. T. Tarvainis for computational work and Ms. Z. Lenkaite for the help with the manuscript. The authors are appreciate to Dr. Halford for sharing unpublished data and critical comments, Prof. F. Eckstein for valuable discussion on the subject and Prof. A. Janulaitis, Dr. Butkus, Dr. R. Roberts and Dr. S. Klimasauskas for critical reading of the manuscript.
References 1 Siksnys, V., Pleckaityte, M., Lukauskaite, R., Butkus, A. and Janulaitis, A. (1990) in Abstracts of the 20th FEBS Meeting, Budapest, p. 207.
2 Lunblad, R.L. and Noyes, C.M. (1984) Chemical reagents for protein modification, Vol. 1, pp. 55-93, CRC Press, Boca Raton, FL. 3 Wells, R.D., Klein, R.D. and Singleton, C.K. (1981) in The Enzymes (Boyer, P.D., ed.), Vol. 14, pp. 166-167, Academic Press, New York. 4 Aiken, C. and Gumport, R.I. (1988) Nucleic Acids Res. 16, 7901-7916. 5 Luke, P.A., McCallum, S.A. and Halford, S.E. (1987) In Gene amplification and analysis (Chirikjian, J.G., ed.), Vol. 5, pp. 187-207, Elsevier, New York. 6 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 7 Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 72-77. 8 Fried, M.G. and Crothers, D.M. (1981) Nucleic Acids Res. 9, 6505-6525. 9 Taylor, J.D., Badcoe, 1.G., Clarke, A.R. and Halford, S.E. (1991) Biochemistry 30, 8743-8753. 10 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, a Laboratory Manual, 2nd Edn., CSHL Press, Cold Spring Harbor, NY. 11 Degani, Y., Neumann, H. and Patchornik, A. (1970) J. Am. Chem. Soc. 92, 6969-6971. 12 Degani, Y. and Patchornik, A. (1974) Biochemistry 13, 1-11. 13 Kindmann, L.A. and Jencks, W.P. (1981) Biochemistry 20, 51835187. 14 Vanaman, T.C. and Stark, G.R. (1970) J. Biol. Chem. 245, 35653573. 15 Aiken, C.R., Fisher, E.W and Gumport, R.I. (1991) J. Biol. Chem. 266, 19063-19069. 16 Torchinsky, Y.M. (1981) Sulfur in proteins (Metzler, D., ed.), Pergamon, New York.
199
BBAPRO 34332
Role of the reactive cysteine residue in restriction endonuclease Cfr9I Virginius Siksnys and Milda Pleckaityte Institute of Biotechnology 'Fermentas', Vilnius (Lithuania) (Received 22 April 1992) (Revised manuscript received 1 July 1992)
Key words: Restriction endonuclease Cfr9I; Thiol group; Chemical modification
Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pK a value of 8.9 _+0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pK a values of 6.3 :i: 0.15 and 8.7 + 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.
Introduction Type-II restriction endonucleases are widely used in genetic engineering experiments due to their unique capability to recognize specific nucleotide sequences in D N A and cut the phosphodiester bond at fixed locations relative to their recognition sites. Recently, we have initiated studies on the mechanism of catalysis and sequence-specific discrimination by restriction endonuclease Cfr9I, which recognizes the palindromic hexanucleotide sequence c V C C G G G , cleaving this sequence as indicated by the arrow. During the preliminary biochemical characterization of the restriction endonuclease Cfr9I we showed [1] that the enzyme completely lost the catalytic activity after incubation with N-ethylmaleimide (NEM), a selective
Correspondence to: V. Siksnys, Institute of Biotechnology, "Fermentas", Greiciuno 8, Vilnius 2028, Lithuania. Abbreviations: IA, iodoacetate; IAA, iodoacetamide; NEM, N-ethylmaleimide; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); MMTS, methyl methanethiosulfonate; NTCB, 2-nitro-5-thiocyanobenzoic acid; BSA, bovine serum albumine; DTT, dithiotreitol; EDTA, ethylenediaminetetraacetic acid, Tris-HCl, tris(hydroxymethyl)aminomethane.
sulfhydryl-modifying reagent [2]. This suggested that one or more cysteine residues might be implicated in catalysis/binding by this restriction endonuclease. On the basis of chemical modification data of restriction endonucleases by N E M it was suggested previously [3] that sulfhydryl groups were important for the activity of BamHI, PvuI, SmaI, Pst I, HindlII and AvaI, but not for that of EcoRI, Sail, BgllI, HpaI and Sst I. Recently, it was shown that restriction endonucleases Cfr9I [1], RsrI [4], EcoRV [5], Eco72I and BcnI (Siksnys, V., unpublished data) were sensitive to modification by NEM, too. We supposed that the differences in sensitivity of the restriction endonucleases to modification by N E M might reflect differences in the organisation of the active sites of various restriction endonucleases and suggested that in the restriction endonucleases sensitive to N E M cysteine residues might be located in or near the active site of the enzyme. In this paper, we report the results of chemical modification of restriction endonuclease Cfr9I by several sulfhydryl-specific reagents. On the basis of the enzyme inactivation studies by thiol-modifying reagents, substrate protection studies, pH-variation of kinetic parameters of substrate hydrolysis and substrate bind-
200 ing properties of native and modified enzyme we propose that restriction endonuclease Cfr9I contains a single reactive sulfhydryl residue which is non-essential for catalysis but might be located at or near the substrate-binding site of the enzyme. Materials and Methods
Materials. Restriction endonuclease Cfr9I, T4 polynucleotide kinase and A DNA were products of Fermentas (Vilnius). Cfr9I migrated as a single band in SDS-PAGE with a molecular mass of 35 kDa. The protein concentration was estimated according to the method of Bradford [6]. Buffer salts were obtained from Sigma, NEM, NTCB, MMTS, DTNB, IA and IAA from Fluka and agarose was supplied by Bio-Rad. [y-32p]ATP was purchased from Izotop (Leningrad). The oligodeoxynucleotide 5'-GGACCCGGGTCC-3' was synthesized using a Biosearch 8700 DNA synthesizer and purified by HPLC in our laboratory. The oligodeoxynucleotide was phosphorylated using T4 polynucleotide kinase and [y-a2p]ATP and the labeled oligodeoxynucleotide was separated on a Sephadex G-50 column. Assay of the catalytic activity of restriction endonuclease Cfr91. Aliquots (1-5/zl) of (1-5)" 10 -6 M enzyme solution were added to 40/zl of solution containing 2 /zg A DNA in a buffer of the following composition: 10 mM Tris-HC1, 5 mM MgC12, 200 mM sodium glutamate, 1 mM DTT, 100 /zg/ml BSA (pH 7.5) and incubated for a 1 h at 37°C. The reaction was quenched by addition of 20 /zl of a solution containing 0,25% Bromophenol blue, 60 mM EDTA and 50% glycerol. The cleavage products were fractionated on 0.7% agarose gels at 10 V / c m and visualised by UV-light after ethidium bromide staining. For quantitative assay of the enzyme catalytic activity in chemical modification studies, the gels were photographed under UVlight using a red filter and Micrat-N (Tasma) type film. The negatives were scanned on a Biomed laser densitometer to evaluate the relative amount of DNA in the bands.
Inactivation of restriction endonuclease Cfr9I with sulfhydryl reagents. 20 /zl aliquots of 2.3.10 - 6 M enzyme solution in 0.02 M phosphate buffer (pH 7.5) containing 0.2 M KC1 and 1 mM EDTA were incubated for 10 min at 22°C at appropriate concentration of thiol reagent (Table I), aliquots of 5 /xl were withdrawn at fixed time-intervals and residual catalytic activity of enzyme was assayed as described, except that DTT was ommitted from the reaction mixture in the case of enzyme modified with DTNB, MMTS and NTCB. In the sequential modification experiment, the enzyme was treated by MMTS or IAA, as described above and catalytic activity assayed. After 10 min expo-
sure to MMTS or IAA, N-ethylmaleinimide was added from stock solution to 10 mM, incubated for 10 min and catalytic activity assayed.
Titration of restriction endonuclease Cfr9I by DTNB. To 500/zl of 4- 10 -6 M solution of restriction endonuclease Cfr9I in 0.01 M phosphate buffer (pH 7.5) containing 0.5 mM EDTA and 0.1 M KCI and equillibrated to 25°C in a 1-ml spectrophotometer cell, 10/zl of 1.4 mM DTNB solution in the same buffer was added, thoroughly mixed and the absorbance at 412 nm recorded at fixed time-intervals on a Specord M40 spectrophotometer (Jena). The value of 13600 M -I cm-1 was used as the molar extinction coefficient for the p-nitrophenylthiolate anion [7]. To determine the extent of inactivation of restriction endonuclease Cfr9I during the titration of enzyme by DTNB, 20 #I aliquots were removed from the identical control reaction mixture and assayed for residual catalytic activity as described. Kinetics of the reaction of Cfr9I with NEM. Chemical modification of restriction endonuclease Cfr9I by NEM was carried out at 22°C in 100 /xl reaction mixture containing 10 mM Tris-HCl, 0.2 M KC1, 1 mM EDTA at appropriate pH value and 4.6.10 -6 M of enzyme. The reaction was initiated by addition of 3 Izl of 0.1 M NEM in water. Aliquots of 20 /zl were withdrawn at fixed time-intervals, 2 #1 7.2 M/3-mercaptoethanol was added to quench the reaction and assayed for the remaining catalytic activity, as presented above. The pseudo-first-order reaction-rate-constants of chemical modification of restriction endonuclease Cfr9I by NEM were calculated according to the following equation: In
At/Ao=-kt
(1)
where A t and A 0 represent the enzyme activity at times 0 and t, respectively. Second-order reactionrate-constants were determined by division of pseudofirst-order reaction-rate-constants by NEM concentration. pK a values were obtained using the non-linear regression program Enzfitter (Elsevier-Biosoft) to fit the data to the equation k = c 1 + C 2 ( 1 0 P H - p K a ) / I + 10 p H - p K a
(2)
For substrate-protection studies the enzyme was preincubated with 14.4 lzM of double-stranded oligodeoxynucleotide 5'-GGACCCGGGTCC-3' before the addition of NEM.
pH-studies of oligodeoxynucleotide 5'-GGACCCGGGTCC-3' hydrolysis by Cfr9I. The reaction rate studies of oligodeoxynucleotide 5'-GGACCCGGGTCC-3' hydrolysis by Cfr9I were carried out at 37°C in 20/xl of reaction mixture containing 10 mM Tris-HCl, 5 mM
201 MgC12, 200 mM sodium glutamate, 1 mM DTT, 100 /zg/ml BSA at appropriate pH value and 0.02 /zM of 5'-[32p]-GGACCCGGGTCC-3 '. The reactions were initiated by addition of the enzyme to a concentration of 0.46.10 -6 M. After 5 min, 10-/zl aliquots of reaction mixture were withdrawn and spotted on DEAEcellulose thin-layer plates, which were then developed in Homomixture VI. Spots containing radioactivity were located by autoradiography. The spots corresponding to the product and substrate were scraped off the plate and their radioactivity was determined by liquid scintilation counting, pK a values were obtained using the nonlinear regression program Enzfitter (Elsevier-Biosoft) to fit the data to the following equation: v = C / 1 + 10(pK~-pH) + 10(pH pK')
(3)
where C is a pH-independent constant and pK a and pKd are pKa values of groups controlling substrate hydrolysis.
Binding studies of restriction endonuclease with DNA using gel mobility shift assay. These were performed as described in Refs. 8 and 9. The binding buffer was 10 mM Tris-HCl, 200 mM sodium glutamate, 1 mM DTT, 100 /zg/ml BSA, 0.1 mM EDTA (pH 7.5). The stock solution of restriction endonuclease Cfr9I or the solution of NEM-modified enzyme was diluted to a final concentration of 0.35 × 10 -5 M in the same binding buffer containing 0.1 nM 32p-labeled 322-bp PvuIIPvuII fragment of plasmid pUC19 containing the recognition site of restriction endonuclease Cfr9I, incubated 15 min at room temperature before adding 5 /zl of loading buffer (binding buffer with 33% glycerol and 0.1% Bromophenol blue). 5-/zl aliquots were loaded on a 6% polyacrylamide gel and subjected to electrophoresis at room temperature in TBE buffer [10]. After electrophoresis the gel was fixed in 10% acetic acid and analysed by autoradiography. Results and Discussion
Inactivation of restriction endonuclease Cfr9I by sulfhydryl-specific reagents In order to elucidate the role of cysteine residues in catalysis/binding by restriction endonuclease Cfr9I, we have studied the reaction of restriction endonuclease Cfr9I with various sulfydryl-modifyingcompounds, including alkylating reagents (iodoacetate, iodoacetamide, N-ethylmaleimide), mixed disulfide-forming sulfhydryl reagents ((5,5'-dithiobis(2-nitrobenzoic) acid, methyl methanethiosulfonate) and the cyanylating reagent 2-nitro-5-thiocyanobenzoic acid. The effect of chemical modification on the catalytic activity of restriction endonuclease Cfr9I is summarized in Table I. As can be seen from data presented in Table I, thiol blocking compounds differ regarding the effect of mod-
TABLE I Inactivation of restriction endonuclease Cfr9I by sufhydryl reagents
Reagent
Substituent
N-Ethylmaleimide
Reagent concentration (mM)
O
Catalytic activity (%)
10
0
10
0
(NEM)
Et_ i~
-
5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB)
- OOC _ ~ O2N
Iodoacetic acid
- OCCH 2 II
50
0
Methyl methane thiosulphonate (MMTS)
CH3S-
10
100
Iodoacetamide
NH2.C.CH 2-
50
100
O
0A)
O
flAA) 2-Nitro-5-thiocyanobenzoic acid (NTCB)
S-
O " CN-
0.4
40
ification on the catalytic activity of restriction endonuclease Cfr9I. Incubation of the restriction endonuclease with NEM, DTNB and IA led to a complete loss of the catalytic activity of the enzyme, suggesting that cysteine residue/residues might play a crucial role in functioning of restriction endonuclease Cfr9I. On the other hand, modification of the enzyme by MMTS and IAA had no prominent effect on the enzyme activity. Following IAA or MMTS treatment the enzyme became resistant to the inactivation by NEM (data not shown), suggesting that both IAA, MMTS and NEM react with the same thiol residue(s). Despite the differences in the chemical nature of modification agents, correlation between the structure of reaction products and the effect of modification on catalytic activity is noticeable. The first group of reagents (DTNB, NEM, IA) introduce bulky aromatic or cyclic aliphatic residues or relatively small but negatively charged residue (-CH2COO-) in the case of thiol alkylation by IA. The reagents from the second group (MMTS and IAA) upon modification of Cys-residues introduce small uncharged substituent (-CH 2 CONH 2 or CH3S-) instead of hydrogen of the thiol group, tt is reasonable to suppose that inactivation of restriction endonuclease Cfr9I by the former reagents is due to the steric or electrostatic effect created by the bulky or charged derivatives. The results of enzyme modification with NTCB are not contradicting with this conclusion, too.
202 NTCB upon reaction with sulfhydryl groups is expected [11] to introduce a small cyano group which could block thiol groups without causing any loss of enzyme activity, as it was found in the case of restriction endonuclease Cfr9I modification by MMTS. However, treatment of restriction endonuclease Cfr9I with NTCB led to a partial loss of catalytic activity. It was reported [12,13] that mixed disulfide adducts were formed during the reaction of sulfhydryl groups with NTCB, due to attack of thiol on the sulfur atom of NTCB. It is possible that the reaction of restriction endonuclease Cfr9I with NTCB procceeds by two reaction pathways leading to the formation of an active cyanylated enzyme and an inactive mixed disulfide adduct similar to the one formed during the enzyme modification by DTNB. This assumption was in agreement with experimental evidences indicating that the catalytic activity of the enzyme modified by DTNB and NTCB was co~apletely restored by treatment with flmercaptoethanol (data not shown)./3-Mercaptoethanol removes the bulky 2-nitrothiobenzoic residue in the mixed disulfide adduct of the enzyme [14], thereby restoring catalytic activity. These data indicate that restriction endonuclease Cfr9I does not contain any Cys-residue/residues essential for catalysis. However, there is a possibility that these residue/residues might be located in or near vicinity of the substrate-binding site. Chemical modification of the enzyme by bulky or charged derivatives could sterically or electrostatically hinder substrate binding thus causing the loss of catalytic activity. This assumption was further supported by the substrate protection studies and direct substrate binding experiments of the native and modified enzyme using the gel-mobility shift assay.
Protective effect of substrate against inactivation of restriction endonuclease Cfr9I by NEM NEM rapidly inactivated restriction endonuclease
Cfr9I at pH 7.5 in a time-dependent manner (Fig. 1). The inactivation followed pseudo-first order kinetics. The pseudo-first-order rate-inactivation-constant at pH 7.5 was equal to 0.12 min -1 and the corresponding second-order rate-constant was 40 M-1 min-1. A plot of the logarithm of residual activity vs. time (inset in Fig. 1) was linear to where at least of 90% of the activity was lost, indicating that the inactivation appeared due to modification of a single residue (or 2 or more residues with the same reactivity towards the reagent). Preincubation of restriction endonuclease with synthetic oligodeoxynucleotide duplex 5'-GGACC C G G G T C C - 3 ' containing the recognition sequence completely protected the enzyme from inactivation by NEM (Fig. 1) indicating that in the case of restriction endonuclease Cfr9I, the cysteine residue/residues might be positioned at or near the active site of the
100
~. ~.
•
•
e
~
80 \
-~
60
~
40
CE
time, min
k
>:
•
~ o~- ,
\
~0
°
20 ,
---30
i i
20 i
0
5
i
i
10 ~5 Time, minutes
i
20
25
Fig. 1. Inactivation of restriction endonuclease Cfr91 by NEM in the absence (o) and presence (e) of 14.4 /zM oligodeoxynucleotide duplex 5'-GGACCCGGGTCC-3'. The inset shows the plot of the logarithmic dependence of enzyme inactivation in the absence of substrate. Conditions; pH 7.5, 10 mM Tris-HCl, 200 mM KCI, 1 mM EDTA. [E] = 4.6.10 -6 M, [NEM] = 3.10 -3 M, 22°C.
enzyme and may be in or in some way associated with the binding site of substrate.
Effect of modification of restriction endonuclease Cfr9I by NEM on substrate binding To examine whether the modification of restriction endonuclease Cfr9I eliminated binding of the enzyme to the DNA we tested the ability of the modified enzyme to bind a 322 bp long D N A fragment containing a single Cfr9I recognition site using a gel-mobility shift assay [8,9]. As can be seen from the data presented in Fig. 2, restriction endonuclease Cfr9I in the absence of Mg 2+ formed a complex with a 322 bp long DNA fragment containing a recognition sequence, having different electrophoretic mobility from the free DNA. However, the enzyme modified with NEM failed to form such a complex (Fig. 2, Lane 3) under the same conditions. These data demonstrate clearly that the NEM-modified enzyme loses the ability to bind DNA in the absence of Mg 2÷. However, these studies cannot eliminate the possibility that modification of restriction endonuclease Cfr9I by NEM destroys the structural integrity of the enzyme. Furthermore, it has been suggested recently that the inactivation of restriction endonucleases Rsr I [15] and EcoRV (Dr. S. Halford, personal communication) by NEM was caused by dissociation of the enzyme dimer upon modification of reactive Cys-residue located near the subunit interface. Our previous studies indicated that restriction endonuclease Cfr9I existed primarily as a dimer in solution, consisting of two identical subunits [1] and the loss of its catalytic activity on reaction with NEM could also be due to dissociation of the enzyme into subunits. To test this, a control sample of the enzyme and a sample completely inacti-
203 vated by treatment with N E M were applied to H P L C TSKgel SW3000 gel-filtration column that was previously calibrated with proteins of a known molecular weight. Two restriction endonuclease samples were found to have identical elution times, corresponding to the protein with molecular mass (70 kDa, data not shown), demonstrating that N E M inactivation is not the consequence of a subunit dissociation.
¢
2
3
Determination of the number of Cys-residues in restriction endonuclease Cfr9I by titration with 5,5'-dithiobis(2-nitrobenzoic acid) Recently, a complete primary structure of the restriction endonuclease Cfr9I has been deduced from D N A sequence data (S. Menkevicius, unpublished data), indicating the presence of four cysteine residues. We were unable to quantitate precisely the number of cysteine residues modified during reaction with N E M using a radioactive derivative of [3H]NEM, due to large experimental variations related to the incomplete and irreproducible precipitation of protein by trichloroacetic acid from diluted solution. Consequently, the modification of the enzyme with D T N B was performed to determine the n u m b e r of Cys-residues available for reaction with sulfhydryl reagents. Moreover, as indicated above, the modification of the restriction endonuclease by D T N B lead to the complete loss of the catalytic activity, as well as in the case of modification with NEM. The general features of the restriction endonuclease Cfr9I reaction with D T N B are illustrated in Fig. 3A. Concentration of T N B 2- ion which is released when D T N B reacts with sulfhydryl groups increased rapidly in the first 2 min and then remained almost constant throughout 30 min. From the concentration of T N B 2released during 30 min, the n u m b e r of sulfhydryl groups was calculated and plotted against the time of reaction. About 1 mol of D T N B reacted with 1 mol of protein of restriction endonuclease Cfr9I, indicating that among 4 sulfhydryl groups present in the protein only one sulfhydryl group reacted with DTNB. The time-course of loss of enzymatic activity corresponded to the release of T N B 2-, indicating that modification of 1 sulfhydryl group resulted in a complete loss of the catalytic activity of the enzyme, as was supposed on the basis of kinetics of enzyme inactivation by NEM. Moreover, the restriction endonuclease Cfr9I which previously reacted with N E M failed to to react with DTNB, suggesting that modification of a Cys-residue by N E M included this reaction with DTNB. On the other hand, the titration of restriction endonuclease Cfr9I in the presence of 6M of guanidinium chloride (Fig. 3B) revealed the presence of four Cys-residues, which is consistent with the number of Cys-residues deduced from the amino-acid sequence of the enzyme and indicating that the enzyme does not
L
Fig. 2. Gel-mobility shift assay on 322-bp DNA fragment. Lane 1, free DNA; lane 2, wild-type Cfr9I enzyme; lane 3, NEM-modified Cfr9I enzyme. Binding buffer; 10 mM Tris-HCl, 200 mM sodium glutamate, 1 mM DTT, 100 ~g/ml BSA, 0.1 mM EDTA (pH 7.5), final concentration of Cfr9I and the 32p-labeled 322-bp PvulI-PvulI fragment of plasmid pUC19, 0.35.10 -5 M and 0.1 nM, respectively. After incubation for 15 min, 5 /zl of loading buffer (binding buffer with 33% glycerol and 0.1% Bromophenol blue) was added and 5/zl aliquots were loaded on a 6% polyacrylamidegel and subjected to electrophoresis at room temperature in TBE buffer. After electrophoresis, the gel was fixed in 10% acetic acid and analysed by autoradiography. contain any disulfides. It seems that the three Cys-residues of restriction endonuclease Cfr9I are 'buried' in the enzyme interior and are not readily accesible to the solvent.
Determination of the pK a value of reactive Cys-residues in restriction endonuclease CfrgI In order to characterize the reactive Cys-residue located, as we suppose, at or near the substrate-binding site of restriction endonuclease Cfr9I, we have studied the p H - d e p e n d e n c e of the enzyme inactivation by NEM. We were unable to use D T N B due to its high
204 =~ l.O
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S
,= ~o
A
1.0
u~
0.5
50~=.
'~
2
;2
-~
- ,---t
i--
o
1
o.o[ 0
10
........ 20 30
Q3
5 10 Time, minutes
Time. minutes
15
reactivity. NEM inactivated the enzyme more slowly than DTNB, permitting determination of the reactionrate-constants. The inactivation of the restriction endonuclease by NEM was dependent on the pH value of the reaction mixture and accelerated with increasing pH (Fig. 4A). This would be consistent with a reaction mechanism involving an unprotonated thiolate residue reacting by
800
>
60
>. 600 ~J {D co ~o
E
7 4O
400
2O
20C
~o co oD
0 0
0.6
0.4 c'r"
0.2
i
I
I
I
6
7
8
9
pH Fig. 5. Effect of pH on the reaction rate of oligodeoxynucleotide duplex 5 ' - G G A C C C G G G T C C - 3 ' hydrolysis by restriction endonuclease Cfr9I. Conditions: 10 m M Tris-HCl, 5 m M MgCI2, 200 m M sodium glutamate, 1 m M DTT, 1 0 0 / ~ g / m l BSA, [E] = 0.46.10 -6 M, 5'-[32 P ] - G G A C C C G G G T C C - 3 ' ] = 2 . 1 0 - 8 M.
nucleophilic addition to NEM. The data on pH-dependence of the inactivation rate constant (Fig. 4B) allowed us to determine the pK a value of the group involved in the modification reaction and controlling the catalytic activity of the enzyme. The determined value of 8.9 was in a good agreement with the known values of pKa of sulfhydryl groups in proteins [16].
pH-dependence of kinetic parameters of substrate hydrolysis by restriction endonuclease Cfr9I
100 80
> >
Fig. 3. The reaction of restriction endonuclease Cfr9I with DTNB. The extent of reaction was monitored at 412 n m by release of 5-thio-2-nitrobenzoic acid anion, the value of 13600 M -1 cm - I [7] was used as the molar extinction coefficient for the p-nitrophenylthiolate anion. The a m o u n t of T N B 2- released is calculated per mol of the protein subunit. (A): 1, Time-course of the restriction endonuclease Cfr9I reaction with D T N B under non-denaturing conditions; 2, time-course of enzyme inactivation. Conditions: [ E ] = 4.10 - 6 M, [ D T N B ] = 8 . 1 0 - 6 M, 0.01 M phosphate buffer (pH 7.5), 0.5 m M E D T A , 0.1 M KCI, 25°C. (B): 1, Time-course of the restriction endonuclease Cfr9I reaction with D T N B under non-denaturing conditions: [ E ] = 4 . 1 0 - 6 M, D T N B = 2 . 6 - 1 0 -5 M, 0.01 M phosphate buffer (pH 7.5), 0.5 m M E D T A , 0.1 M KCI, 25°C; 2, time-course of the restriction endonuclease Cfr9I reaction with D T N B in the presence of 6 M of guanidinium chloride. Conditions are the same as in 1, except for the presence of 6 M guanidinium chloride.
°
0.8
c3
5 Time,
~0 15 minutes
20
7.5
8.0
8.5
g.O
9.5
pH
Fig. 4. Effect of pH on the inactivation rate of restriction endonuclease Cfr9I by NEM. (A): Time-course of enzyme inactivation at different pH values. Conditions: 10 m M Tris-HCl, 200 m M KCI, 1 m M E D T A , [ E ] = 4.6.10 -6 M, [ N E M ] = 3.10 -3 M, T = 22°C. (B): Effect of pH on the second-order rate-constant of inactivation of restriction endonuclease Cfr9I by NEM. Conditions as in Fig.2. The second-order rate-constants were calculated by dividing the pseudofirst-order inactivation-rate-constant by the concentration of N E M used. The solid curve is the best fit to the expermental points according to Eqn. 2.
pH-dependence studies of kinetic parameters of the enzymatic reaction could give additional information regarding the groups on the enzyme or substrate involved in either binding or catalysis. We have studied the pH-dependence of the reaction rate of oligodeoxynucleotide duplex 5'-GGACCCGGGTCC-3' hydrolysis by restriction endonuclease Cfr9I (Fig. 5). The hydrolysis rate of the synthetic oligodeoxynucleotide duplex by Cfr9I showed its maximum value at pH 7.5 which decreased on either side of this maximum. The reaction rate dependence of the substrate hydrolysis by Cfr9I vs. pH was bell-shape-dependent and revealed two residues with pK a values of 6.3 + 0.15 and 8.7 + 0.15, controlling the catalytic activity of the enzyme. The pK a value of 6.3 + 0.15 might be assigned to Hisor Glu-residues. The pK a value of the group controlling the alkaline limb of the substrate hydrolysis is similar to the pK a value of the cysteine residue modified by NEM.
205
Conclusions Our results demonstrate that restriction endonuclease Cfr9I contains a reactive Cys-residue which is not essential for the catalytic activity of the enzyme. However, these data provide a strong evidence that this Cys-residue is located near or within the active site of the enzyme. Additional studies will be required to localize the reactive Cys-residue in the sequence and establish the particular role played by this cysteine residue in the functioning of restriction endonuclease
Cfr9I. Acknowledgements The authors are indebted to Ms. R. Lukauskaite for purification of the enzyme, Mrs. Z. Maneliene for the oligodeoxynucleotide synthesis, Mrs. L. Petrauskiene for technical assistance, Mr. T. Tarvainis for computational work and Ms. Z. Lenkaite for the help with the manuscript. The authors are appreciate to Dr. Halford for sharing unpublished data and critical comments, Prof. F. Eckstein for valuable discussion on the subject and Prof. A. Janulaitis, Dr. Butkus, Dr. R. Roberts and Dr. S. Klimasauskas for critical reading of the manuscript.
References 1 Siksnys, V., Pleckaityte, M., Lukauskaite, R., Butkus, A. and Janulaitis, A. (1990) in Abstracts of the 20th FEBS Meeting, Budapest, p. 207.
2 Lunblad, R.L. and Noyes, C.M. (1984) Chemical reagents for protein modification, Vol. 1, pp. 55-93, CRC Press, Boca Raton, FL. 3 Wells, R.D., Klein, R.D. and Singleton, C.K. (1981) in The Enzymes (Boyer, P.D., ed.), Vol. 14, pp. 166-167, Academic Press, New York. 4 Aiken, C. and Gumport, R.I. (1988) Nucleic Acids Res. 16, 7901-7916. 5 Luke, P.A., McCallum, S.A. and Halford, S.E. (1987) In Gene amplification and analysis (Chirikjian, J.G., ed.), Vol. 5, pp. 187-207, Elsevier, New York. 6 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 7 Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 72-77. 8 Fried, M.G. and Crothers, D.M. (1981) Nucleic Acids Res. 9, 6505-6525. 9 Taylor, J.D., Badcoe, 1.G., Clarke, A.R. and Halford, S.E. (1991) Biochemistry 30, 8743-8753. 10 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, a Laboratory Manual, 2nd Edn., CSHL Press, Cold Spring Harbor, NY. 11 Degani, Y., Neumann, H. and Patchornik, A. (1970) J. Am. Chem. Soc. 92, 6969-6971. 12 Degani, Y. and Patchornik, A. (1974) Biochemistry 13, 1-11. 13 Kindmann, L.A. and Jencks, W.P. (1981) Biochemistry 20, 51835187. 14 Vanaman, T.C. and Stark, G.R. (1970) J. Biol. Chem. 245, 35653573. 15 Aiken, C.R., Fisher, E.W and Gumport, R.I. (1991) J. Biol. Chem. 266, 19063-19069. 16 Torchinsky, Y.M. (1981) Sulfur in proteins (Metzler, D., ed.), Pergamon, New York.
