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Binding of mercury (II) to protein thiol groups: A study of proteinase K and carboxypeptidase Y
Binding of Mercury (II) to Protein Thioi Groups: A Study of Proteinase K and Carboxypeptidase Y Sven Bagger, Klaus Breddam, and Birgitte R. Byberg SB, BRB. Chemistry Department A, The Technical University of Denmark, Lyngby, Denmark.-KB. Department of Chemistry, Carlsberg Laboratory, Valby, Denmark
ABSTRACT Chemically modified enzymes have been prepared by incorporating an -Hg-L group into proteinase K and carboxypeptidase Y at the thiol groups of Cys-73 and Cys-341, respectively (L = CN- or I-). The
-S-Hg-‘kN
group has ken applied as a spectroscopic label for carbon-13 NMR spectroscopy.
INTRODUCTION The two serine proteinases carboxypeptidase Y (CPY) from baker’s yeast and proteinase K (PRK) from Tritirachium album Limber are distinguished by containing only a single cysteinyl residue with a free thiol group and by having this cysteinyl residue situated in the vicinity of the active site [l , 21. For CPY it has previously been demonstrated that useful modified enzymes can be produced by attaching various Hg(II)-complex groups to the thiol group. For example, it was found that a derivative of CPY having -S-H substituted by -S-Hg-CN was more efficient than native CPY in the enzymatic, semisynthetic conversion of porcine insulin into human insulin [3]. In the present study we have investigated the possibility of modifying PRK by the strategy previously used for CPY [4], and the -S-Hg-CN derivative of CPY has been reinvestigated using 13C NMR spectroscopy.
ABBREVIATIONS Proteinase K, PRK; PRK inactivated with Hg’+, PRK-Hg; PRK-Hg reactivated with a ligand L, PRK-Hg-L; Carboxypeptidase Y, CPY; CPY inactivated with Hg*+,
Address reprint requests and correspondence to: Sven Bagger, Chemistry Department A, Building 207, The Technical University of Denmark, DK-2800 Lyngby, Denmark. 97 Journal of Inorganic Biochemistty, 42,97- 103 (1991) 0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-01~/91/$3.~
98
S. Bagger et al.
CPY-Hg; CPY-Hg reactivated with a ligand L-phenylalanine-methyl ester, FA-Phe-OMe; MES; [Xl,, total concentration of species X. in PRK and CPY is in accordance with Refs.
L, CPY-Hg-L; N7[3-(2-furylacryloyl)2-[N-morpholino]ethanesulfonic acid. The numbering of amino acid residues 2 and 5, respectively.
MATERIALS Carboxypeptidase Y was isolated as previously described [6]. Proteinasc K tchromatographically purified, lyophilized, 27 mAnson units/mg, A ii %. I cm, 380 nm) .= 14.2) was obtained from E. Merck, FRG (Cat. No. 24568): albumin (bovine, monomer, 0.8 mol thiol ;mol protein) and K ’ “CN (99 atom ‘Z “C) from Sigma. US: FA-Phe-OMe was from Bachem, Switzerland
METHODS The enzymatic activity of PRK and its modified forms was assayed spectrophotomet rically by measuring the ester hydrolysis rate of the chromophoric substrate [7] FA-Phe-OMe. Our standard conditions were 3.3 PM enzyme. 0.27 mM FA-Phe.OMe, pH 6.4, photometric wavelength 335 nm. Enzyme concentrations were determined spectrophotometrically using A (1 7%. 1 cm, 280 nm) = 14.8 for CPY and A (l%, I cm, 280 nm) =: 14,2 for PRK; the molecular weights used were 64,000 for CPY [ 11 and 28,790 for PRK 121. Active site titrations were not performed. Kinetic parameters have been determined from initial rates. The simple Michaelis-Menten equation was fitted to the experimental data by nonlinear regression (Marquardt algorithm) using the software package Graphpad [S]. Carbon-13 NMR spectra were obtained at 125.7 MHz on a Bruker AM 500 spectrometer in 10 mm sample tubes at 300 K. Spectral conditions were normally 32 K time-domain data points, 6.5 ps pulse width (13 ps := 90” pulse), 31.250 Hz spectral width, and proton noise decoupling. Typical spectra of the enzymes consisted of 120,000-150.000 transients (1’7-22 hr). The solvent was H,O with 10% D,O added, and CH,OH (44.00 ppm) was used as internal reference. The equilibrium constant. K,, , for PRK -- Hg --. L = PRK -- Hg -t L may be derived from measurements K
_ ’
of enzyme activity [4] using
IL] . [PRK-Hg] [ PRK - Hg - I.]
[I-] . ( V-u) ‘J
or
” = K,
+ [L]
u is the rate of the enzyme catalyzed reaction, [L] is the concentration of free ligand, and V is the value which u approaches asymptotically as [L] is increased. From a series of corresponding [L] and u values the maximum rate I’ and the
BINDING
0
1 mol Hg(III / 101 PRK
2
OF MERCURY(H)
TO PROTEINS
99
FIGURE 1. Inactivation of PRK with HgNO,. 3.3 PM PRK, 0.05 M MES as buffer, pH 6.4, 25”C, 0.27 mM FA-Phe-OMe as substrate. The dashed line is drawn between (0,100) and (1,O).
dissociation constant K, can be found by nonlinear regression [S]. By inspection of Eq. (1) it is seen that K, equals [L] when u = V/2; this relationship may be used to get an estimate of K,.
RESULTS PRK is inactivated by Hg2+ ions as it appears from the titration curve in Figure 1. As seen in Figures 2 and 3 addition of I- or CN- causes a reactivation of the Hg(II)-inactivated enzyme. Due to the smaller amounts of Iigand involved, the reactivation experiments with cyanide are inherently less accurate than the experiments with iodide. Note that it is a good approximation to set [II] = [I- I,, whereas [CN-] = 10e3 * [CN-1, at the pH used (pK, for HCN is 9.14). We have also tried to reactivate PRK-Hg with Br-, SCN-, and the two electroneutral ligands thiourea and imidazole, all of which are known to form strong complexes with Hg2+ (see Table 2). These attempts were unsuccessful using similar conditions as for I- and CN- and ligand/enzyme ratios up to 2000: 1. Table 1 shows the experimental kinetic parameters for native PRK and the two chemically modified forms, PRK-Hg-I and PRK-Hg-CN. The K, value for PRK-Hg-I as determined from Eq. (1) and our estimated K, value for PRK-Hg-CN are given in Table 2. To the purpose of measuring the r3C NMR spectra of CPY-Hg-i3CN and PRK-Hg-13CN, CPY-Hg and PRK-Hg were reactivated using ‘3C-enriched cyanide. Resonances assigned to enzyme-bound 13CN- were found to appear in the spectrum near the characteristic zeta-carbon peaks of Arg and Tyr. The 145-165 ppm range of the spectra is reproduced in Figures 4 and 5. Changing the pulse repetition time from 0.5 set to 2.0 set by introducing a relaxation delay of 1.5 set did not change the pattern seen in Figure 4(b), so any partial saturation of the CN- carbon is an unimportant factor in the present context. FIGURE 100
g a0 =-aJ 2: 60 .z z t:= 40 -=.+ D Y 20
I,:-
0 0
500
1000
L1rI. / urc
1500
2.
Reactivation of PRK-Hg with ICI. 3.3
PM PRK, 3.4 PM Hg’+, 0.05 M MES as buffer, pH 6.4, 25°C 0.27 mM FA-Phe-OMe as substrate. For each experimental point, PRK was incubated with HgNO, for 5 min before substrate was added. [I-], e.quals [I-] + [PRK-Hg-I]. The curve is fitted to the data points by nonlinear regression using Eq. (1) with the good approximation [I-] = [I-],. The curve fitting yields K, = 575 PM and V = 115%.
BINDING
OF MERCURY(B)
TO PROTEINS
101
FIGURE 5. 13C NMR spectra of PRK-Hg-13CN. (a): 1.00 mM (2.96 pmol) PRK, 39 mM MES, pH 6.1, 2.96 pmol HgNO, , 2.92 pm01 K13CN; the spectrum was
measured immediately after preparation of the sample. (b): 1
I60
I
150
ppm
The same as (a) after 47 d at 7°C. (c): The same as (a) after 82 d at 7°C; the enzymatic activity was found to be zero.
Addition of excess i3CN- to the sample solution used for spectrum (b) in Figure 4 did not change the appearance or position of the peak at 150.6 ppm but a new peak due to H13CN was seen at 112 ppm. In a blind experiment it was assured that the characteristic broad resonance in the 145- 165 ppm region was absent in a solution without enzyme but in other respects with the same composition as the sample solution for spectrum (b) in Figure 4. When cysteine was added to the NMR sample solution of CPY-Hg-13CN, the -Hg-13CN peak disappeared and a peak at 112 ppm due to H13CN appeared instead. This indicates that Hg(II) is removed from the enzyme and cyanide is liberated under formation of a Hg(II)/cysteine complex. Likewise it was found that addition of another thiol ligand, 2-mercapto-ethanol, to a solution of the inactive PRK-Hg, caused a full recovery (96%) of the activity of native PRK. To obtain satisfactory NMR spectra a minimum enzyme concentration of about 1 mM was required. However, saturated solutions of the commercial PRK preparation in pure water and in 0.05 M MES (pH 6.4) proved to be only 0.60 mM and 0.53 mM, respectively. It was found that the solid preparation contained 51% (w/w) calcium acetate, added for stabilization. To remove the excess Ca’+, a suspension in water was dialyzed against an aqueous 5 mM CaCl, solution, and a subsequent ultrafiltration allowed preparation of a 1.49 mM PRK solution to be used for the preparation of the NMR sam le. R NMR spectra of PRK-HgCN are shown in Figure 5. As discussed later, the two peaks at 151.7 and 148.6 ppm in spectrum 5(a) are assigned to -S-Hg-13CN. From spectra 5(b) and 5(c) it appears that the latter peak decreases in time and finally disappears. It was found that bovine serum albumin monomer, a protein which contains a single free cysteinyl thiol group like PRK and CPY, also exhibited the characteristic additional peak (at 152 ppm) when treated with Hg*+ and 13CN-. This corroborates the assignment of the -S-Hg-13CN resonance.
102 S. Bagger et al.
DISCUSSION Addition of one equivalent of Hg*+ to PRK in solution reduces the enzymatic activity to zero. In the inactive enzyme, PRK-Hg, Hg2+ is assumed to bind to the thiol group of Cys-73. This is in accordance with an x-ray crystallographic study of PRK crystals soaked in HgCl, [2]. Considering the position of Cys-,73 and active-site His-69 in the three-dimensional structure of the enzyme [2. 121 it is likely that Hg(I1) forms an -S-Hg-imidazole bridge between Cys-73 and His-69. Our results indicate that the reactivation of PRK-Hg with I or CN is an effect of the formation of an -S-Hg-1 or -S-Hg-CN group at Cys-73. The regaining of enzymatic activity is explained by the release of the catalytically active His-69 from Hg(II) in the above-mentioned bridge al; a result of substitution by the added reactivating ligand, As seen in Table 2, the applied ligands are generally found to bind more strongly to CPY-Hg than to PRK-Hg. The presence of the broad peak from the -S-Hg-“CN label in the spectra of CPY-Hg-‘3CN and PRK-Hg-‘3CN proves that the reactivation hl cyanide is not simply due to a removal of Hg(I1) from the enzymes. Consequently. it can be concluded that I does not remove Hg(I1) either, rince it has iess affinity to Hg(I1) than CN (see Table 2). In the case of CPY the latter i,nnclusion has already been drawn previously [4] from different evidence. PRK is known to undergo autolysis in solution 1131. Our tentative interpretation of the changes in the NMR spectra shown in Figure 5 is that the decreasing resonance at 148.6 ppm originates from unimpaired PRK-Hg-CN while the resonance at 151.7 ppm is due to degradation products containing the .-S-Hg-CN group
CONCLUSION Proteinase K which has been inhibited by treatment with Hg ” can be reactivated by I and CN- under formation of active, modified enzymes containing the groups -S-Hg-I or -S-Hg-CN in the substrate recognition region. The -S-Hg-13CN group was found to be a convenient NMR spectroscopic label. It has the “C resonance located in a region of the spectrum where no protein resonances occur and may be useful in studies of other enzymes and proteins having cysteinyl residues accessible for modification. The Danish Natural Science Research Council provided the 500 MHz Braker spectrotneter. We thank Klaus Bock and Christian Pedersen ~for v&able heip with rhe N.MR meusurements.
REFERENCES 1. 2. 3. 4.
K. C. K. K.
Breddam, Carlsberg Res. Comm. 51, 83 (1986). Betzel, G. P. Pal, and W. Saenger, Eur. J. Biochem. 178. 155 (1988) Breddam and J. T. Johansen, Carlsberg Res. Comm. 49. 463 (1983). Breddam, Carlsberg Rex Comm. 48, 9 i1983'~.
BINDING OF MERCURY(I1)
TO PROTEINS
103
5. K. Breddam and I. Svendsen, Carlsberg Res. Comm. 49, 639 (1984). 6. J. T. Johansen, K. Breddam, and M. Ottesen, Carlsberg Res. Comm. 41, 1 (1976). 7. J. Feder and J. M. Schuck, Biochemistry 9, 2784 (1970). 8. H. J. Motulsky, Graphpad, IS1 Software, Philadelphia, 1987. 9. Stability Constants, Special Publication No. 17, The Chemical Society, London, 1964. 10. Stability Constants, Special Publication No. 25, The Chemical Society, London, 1971. 11. Stability Constants of Metal-Zen Complexes, IUPAC Chemical Data Series No. 21, Pergamon, Oxford, 1982. 12. C. Betzel, G. P. Pal, M. Struck, K.-D. Jany,and W. Saenger, FEBS Let?. 197, 105 (1986). 13. J. Bajorath, W. Saenger, and G. P. Pal, Biochim. Biophys. Acta 954, 176 (1988).
Received July 28, 1990; accepted October IO, .I990
ABSTRACT Chemically modified enzymes have been prepared by incorporating an -Hg-L group into proteinase K and carboxypeptidase Y at the thiol groups of Cys-73 and Cys-341, respectively (L = CN- or I-). The
-S-Hg-‘kN
group has ken applied as a spectroscopic label for carbon-13 NMR spectroscopy.
INTRODUCTION The two serine proteinases carboxypeptidase Y (CPY) from baker’s yeast and proteinase K (PRK) from Tritirachium album Limber are distinguished by containing only a single cysteinyl residue with a free thiol group and by having this cysteinyl residue situated in the vicinity of the active site [l , 21. For CPY it has previously been demonstrated that useful modified enzymes can be produced by attaching various Hg(II)-complex groups to the thiol group. For example, it was found that a derivative of CPY having -S-H substituted by -S-Hg-CN was more efficient than native CPY in the enzymatic, semisynthetic conversion of porcine insulin into human insulin [3]. In the present study we have investigated the possibility of modifying PRK by the strategy previously used for CPY [4], and the -S-Hg-CN derivative of CPY has been reinvestigated using 13C NMR spectroscopy.
ABBREVIATIONS Proteinase K, PRK; PRK inactivated with Hg’+, PRK-Hg; PRK-Hg reactivated with a ligand L, PRK-Hg-L; Carboxypeptidase Y, CPY; CPY inactivated with Hg*+,
Address reprint requests and correspondence to: Sven Bagger, Chemistry Department A, Building 207, The Technical University of Denmark, DK-2800 Lyngby, Denmark. 97 Journal of Inorganic Biochemistty, 42,97- 103 (1991) 0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-01~/91/$3.~
98
S. Bagger et al.
CPY-Hg; CPY-Hg reactivated with a ligand L-phenylalanine-methyl ester, FA-Phe-OMe; MES; [Xl,, total concentration of species X. in PRK and CPY is in accordance with Refs.
L, CPY-Hg-L; N7[3-(2-furylacryloyl)2-[N-morpholino]ethanesulfonic acid. The numbering of amino acid residues 2 and 5, respectively.
MATERIALS Carboxypeptidase Y was isolated as previously described [6]. Proteinasc K tchromatographically purified, lyophilized, 27 mAnson units/mg, A ii %. I cm, 380 nm) .= 14.2) was obtained from E. Merck, FRG (Cat. No. 24568): albumin (bovine, monomer, 0.8 mol thiol ;mol protein) and K ’ “CN (99 atom ‘Z “C) from Sigma. US: FA-Phe-OMe was from Bachem, Switzerland
METHODS The enzymatic activity of PRK and its modified forms was assayed spectrophotomet rically by measuring the ester hydrolysis rate of the chromophoric substrate [7] FA-Phe-OMe. Our standard conditions were 3.3 PM enzyme. 0.27 mM FA-Phe.OMe, pH 6.4, photometric wavelength 335 nm. Enzyme concentrations were determined spectrophotometrically using A (1 7%. 1 cm, 280 nm) = 14.8 for CPY and A (l%, I cm, 280 nm) =: 14,2 for PRK; the molecular weights used were 64,000 for CPY [ 11 and 28,790 for PRK 121. Active site titrations were not performed. Kinetic parameters have been determined from initial rates. The simple Michaelis-Menten equation was fitted to the experimental data by nonlinear regression (Marquardt algorithm) using the software package Graphpad [S]. Carbon-13 NMR spectra were obtained at 125.7 MHz on a Bruker AM 500 spectrometer in 10 mm sample tubes at 300 K. Spectral conditions were normally 32 K time-domain data points, 6.5 ps pulse width (13 ps := 90” pulse), 31.250 Hz spectral width, and proton noise decoupling. Typical spectra of the enzymes consisted of 120,000-150.000 transients (1’7-22 hr). The solvent was H,O with 10% D,O added, and CH,OH (44.00 ppm) was used as internal reference. The equilibrium constant. K,, , for PRK -- Hg --. L = PRK -- Hg -t L may be derived from measurements K
_ ’
of enzyme activity [4] using
IL] . [PRK-Hg] [ PRK - Hg - I.]
[I-] . ( V-u) ‘J
or
” = K,
+ [L]
u is the rate of the enzyme catalyzed reaction, [L] is the concentration of free ligand, and V is the value which u approaches asymptotically as [L] is increased. From a series of corresponding [L] and u values the maximum rate I’ and the
BINDING
0
1 mol Hg(III / 101 PRK
2
OF MERCURY(H)
TO PROTEINS
99
FIGURE 1. Inactivation of PRK with HgNO,. 3.3 PM PRK, 0.05 M MES as buffer, pH 6.4, 25”C, 0.27 mM FA-Phe-OMe as substrate. The dashed line is drawn between (0,100) and (1,O).
dissociation constant K, can be found by nonlinear regression [S]. By inspection of Eq. (1) it is seen that K, equals [L] when u = V/2; this relationship may be used to get an estimate of K,.
RESULTS PRK is inactivated by Hg2+ ions as it appears from the titration curve in Figure 1. As seen in Figures 2 and 3 addition of I- or CN- causes a reactivation of the Hg(II)-inactivated enzyme. Due to the smaller amounts of Iigand involved, the reactivation experiments with cyanide are inherently less accurate than the experiments with iodide. Note that it is a good approximation to set [II] = [I- I,, whereas [CN-] = 10e3 * [CN-1, at the pH used (pK, for HCN is 9.14). We have also tried to reactivate PRK-Hg with Br-, SCN-, and the two electroneutral ligands thiourea and imidazole, all of which are known to form strong complexes with Hg2+ (see Table 2). These attempts were unsuccessful using similar conditions as for I- and CN- and ligand/enzyme ratios up to 2000: 1. Table 1 shows the experimental kinetic parameters for native PRK and the two chemically modified forms, PRK-Hg-I and PRK-Hg-CN. The K, value for PRK-Hg-I as determined from Eq. (1) and our estimated K, value for PRK-Hg-CN are given in Table 2. To the purpose of measuring the r3C NMR spectra of CPY-Hg-i3CN and PRK-Hg-13CN, CPY-Hg and PRK-Hg were reactivated using ‘3C-enriched cyanide. Resonances assigned to enzyme-bound 13CN- were found to appear in the spectrum near the characteristic zeta-carbon peaks of Arg and Tyr. The 145-165 ppm range of the spectra is reproduced in Figures 4 and 5. Changing the pulse repetition time from 0.5 set to 2.0 set by introducing a relaxation delay of 1.5 set did not change the pattern seen in Figure 4(b), so any partial saturation of the CN- carbon is an unimportant factor in the present context. FIGURE 100
g a0 =-aJ 2: 60 .z z t:= 40 -=.+ D Y 20
I,:-
0 0
500
1000
L1rI. / urc
1500
2.
Reactivation of PRK-Hg with ICI. 3.3
PM PRK, 3.4 PM Hg’+, 0.05 M MES as buffer, pH 6.4, 25°C 0.27 mM FA-Phe-OMe as substrate. For each experimental point, PRK was incubated with HgNO, for 5 min before substrate was added. [I-], e.quals [I-] + [PRK-Hg-I]. The curve is fitted to the data points by nonlinear regression using Eq. (1) with the good approximation [I-] = [I-],. The curve fitting yields K, = 575 PM and V = 115%.
BINDING
OF MERCURY(B)
TO PROTEINS
101
FIGURE 5. 13C NMR spectra of PRK-Hg-13CN. (a): 1.00 mM (2.96 pmol) PRK, 39 mM MES, pH 6.1, 2.96 pmol HgNO, , 2.92 pm01 K13CN; the spectrum was
measured immediately after preparation of the sample. (b): 1
I60
I
150
ppm
The same as (a) after 47 d at 7°C. (c): The same as (a) after 82 d at 7°C; the enzymatic activity was found to be zero.
Addition of excess i3CN- to the sample solution used for spectrum (b) in Figure 4 did not change the appearance or position of the peak at 150.6 ppm but a new peak due to H13CN was seen at 112 ppm. In a blind experiment it was assured that the characteristic broad resonance in the 145- 165 ppm region was absent in a solution without enzyme but in other respects with the same composition as the sample solution for spectrum (b) in Figure 4. When cysteine was added to the NMR sample solution of CPY-Hg-13CN, the -Hg-13CN peak disappeared and a peak at 112 ppm due to H13CN appeared instead. This indicates that Hg(II) is removed from the enzyme and cyanide is liberated under formation of a Hg(II)/cysteine complex. Likewise it was found that addition of another thiol ligand, 2-mercapto-ethanol, to a solution of the inactive PRK-Hg, caused a full recovery (96%) of the activity of native PRK. To obtain satisfactory NMR spectra a minimum enzyme concentration of about 1 mM was required. However, saturated solutions of the commercial PRK preparation in pure water and in 0.05 M MES (pH 6.4) proved to be only 0.60 mM and 0.53 mM, respectively. It was found that the solid preparation contained 51% (w/w) calcium acetate, added for stabilization. To remove the excess Ca’+, a suspension in water was dialyzed against an aqueous 5 mM CaCl, solution, and a subsequent ultrafiltration allowed preparation of a 1.49 mM PRK solution to be used for the preparation of the NMR sam le. R NMR spectra of PRK-HgCN are shown in Figure 5. As discussed later, the two peaks at 151.7 and 148.6 ppm in spectrum 5(a) are assigned to -S-Hg-13CN. From spectra 5(b) and 5(c) it appears that the latter peak decreases in time and finally disappears. It was found that bovine serum albumin monomer, a protein which contains a single free cysteinyl thiol group like PRK and CPY, also exhibited the characteristic additional peak (at 152 ppm) when treated with Hg*+ and 13CN-. This corroborates the assignment of the -S-Hg-13CN resonance.
102 S. Bagger et al.
DISCUSSION Addition of one equivalent of Hg*+ to PRK in solution reduces the enzymatic activity to zero. In the inactive enzyme, PRK-Hg, Hg2+ is assumed to bind to the thiol group of Cys-73. This is in accordance with an x-ray crystallographic study of PRK crystals soaked in HgCl, [2]. Considering the position of Cys-,73 and active-site His-69 in the three-dimensional structure of the enzyme [2. 121 it is likely that Hg(I1) forms an -S-Hg-imidazole bridge between Cys-73 and His-69. Our results indicate that the reactivation of PRK-Hg with I or CN is an effect of the formation of an -S-Hg-1 or -S-Hg-CN group at Cys-73. The regaining of enzymatic activity is explained by the release of the catalytically active His-69 from Hg(II) in the above-mentioned bridge al; a result of substitution by the added reactivating ligand, As seen in Table 2, the applied ligands are generally found to bind more strongly to CPY-Hg than to PRK-Hg. The presence of the broad peak from the -S-Hg-“CN label in the spectra of CPY-Hg-‘3CN and PRK-Hg-‘3CN proves that the reactivation hl cyanide is not simply due to a removal of Hg(I1) from the enzymes. Consequently. it can be concluded that I does not remove Hg(I1) either, rince it has iess affinity to Hg(I1) than CN (see Table 2). In the case of CPY the latter i,nnclusion has already been drawn previously [4] from different evidence. PRK is known to undergo autolysis in solution 1131. Our tentative interpretation of the changes in the NMR spectra shown in Figure 5 is that the decreasing resonance at 148.6 ppm originates from unimpaired PRK-Hg-CN while the resonance at 151.7 ppm is due to degradation products containing the .-S-Hg-CN group
CONCLUSION Proteinase K which has been inhibited by treatment with Hg ” can be reactivated by I and CN- under formation of active, modified enzymes containing the groups -S-Hg-I or -S-Hg-CN in the substrate recognition region. The -S-Hg-13CN group was found to be a convenient NMR spectroscopic label. It has the “C resonance located in a region of the spectrum where no protein resonances occur and may be useful in studies of other enzymes and proteins having cysteinyl residues accessible for modification. The Danish Natural Science Research Council provided the 500 MHz Braker spectrotneter. We thank Klaus Bock and Christian Pedersen ~for v&able heip with rhe N.MR meusurements.
REFERENCES 1. 2. 3. 4.
K. C. K. K.
Breddam, Carlsberg Res. Comm. 51, 83 (1986). Betzel, G. P. Pal, and W. Saenger, Eur. J. Biochem. 178. 155 (1988) Breddam and J. T. Johansen, Carlsberg Res. Comm. 49. 463 (1983). Breddam, Carlsberg Rex Comm. 48, 9 i1983'~.
BINDING OF MERCURY(I1)
TO PROTEINS
103
5. K. Breddam and I. Svendsen, Carlsberg Res. Comm. 49, 639 (1984). 6. J. T. Johansen, K. Breddam, and M. Ottesen, Carlsberg Res. Comm. 41, 1 (1976). 7. J. Feder and J. M. Schuck, Biochemistry 9, 2784 (1970). 8. H. J. Motulsky, Graphpad, IS1 Software, Philadelphia, 1987. 9. Stability Constants, Special Publication No. 17, The Chemical Society, London, 1964. 10. Stability Constants, Special Publication No. 25, The Chemical Society, London, 1971. 11. Stability Constants of Metal-Zen Complexes, IUPAC Chemical Data Series No. 21, Pergamon, Oxford, 1982. 12. C. Betzel, G. P. Pal, M. Struck, K.-D. Jany,and W. Saenger, FEBS Let?. 197, 105 (1986). 13. J. Bajorath, W. Saenger, and G. P. Pal, Biochim. Biophys. Acta 954, 176 (1988).
Received July 28, 1990; accepted October IO, .I990