An essential tryptophan residue for rabbit muscle creatine kinase

Biochimica et Biophysica Acta 830 (1985) 59-63 Elsevier

59

BBA 32249

An essential tryptophan residue for rabbit muscle creatine kinase Hai-Meng Zhou and Chen-Lu Tsou * Institute of Biophysics, Academia Sinica, Beijing (China) (Received February 4th, 1985)

Key words: Creatine kinase: Chemical modification: Tryptophan residue: (Rabbit muscle)

The tryptophan residues in rabbit muscle creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) have been modified by dimethyl(2-hydroxy-5-nitrobenzyi) sulfonium bromide after reversible protection of the reactive SH groups. The modification of two tryptophan ?esidues as measured by spectrophotometric titration leads to complete loss of enzymatic activity. Control experiments show that reversible protection of the reactive SH groups as S-sulfonates followed by reduction results in nearly quantitative recovery of enzyme activity. The presence of a 410 nm absorption maximum and the decrease in fluorescence of the modified enzyme indicate the modification of tryptophan residues. At the same time, SH determinations after reduction of the modified enzyme show that the reagent has not affected the protected SH groups. Quantitative treatment of the data (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of substrates partially protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.

Introduction

Creatine kinase (ATP : creatine N-phosphotransferase, EC 2.7.3.2.) is an important enzyme in energy metabolism and hence has been extensively studied. Attempts at mapping the side-chain groups essential for its activity have been largely centered on its reactive SH groups [1-5]. Modification of two reactive SH groups of this dimeric enzyme by some reagents leads to complete inactivation, whereas modification by some other reagents results only in partial inactivation. Moreover, quantitative assessment [6] of the data reported by previous workers [3,5] indicates that among the two reactive SH groups, the modification of only one per activity unit of the molecule is responsible for the decrease in * To whom correspondence should be addressed. Abbreviation: DTNB, 5,5'-dithiobis(2-nitrobenzoicacid).

activity observed. On the other hand, chemical modification of its tyrosine [7], histidine [8], lysine [9] and arginine [10] residues have also been reported. Attempting to modify the tryptophan residues of this enzyme, Rooustan et al. [5] reported that the tryptophan-indole group was not affected by treatment with 2-hydroxy-5-nitrobenzyl bromide. We have now shown that the tryptophan residues are modified by the water-soluble reagent dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide [11] and that one of them is essential for the catalytic activity of this enzyme. Materials and Methods Purification of rabbit muscle creatine kinase, protein concentration determinations and the assay of creatine kinase activity were as described before [12]. DTNB and dimethyl-(2-hydroxy-5nitrobenzyl)sulfonium bromide were Sigma prod-

0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

60 ucts, dithiothreitol was from Serva, and urea was a local analytical reagent recrystallized according to Marangos and Constantinides [13] before use. All other reagents were local products of analytical grade used without purification. Protection of the reactive SH groups of the enzyme was carried out as described by F a t t o u m et al. [7], except that the sodium salt of tetrathionate was used instead of the potassium salt. Modification of the tryptophan residues of the protected enzyme was carried out in 0.05 M Tris acetate buffer (pH 8.0)/1 mM E D T A with an excess of dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide, After 30 min reaction at 4°C, the reaction mixture was passed through a Sephadex G-25 column pre-equilibrated with the same buffer to remove the sulfonium bromide in excess. The modified enzyme was then reduced by dithiothreitol and then again passed through the Sephadex G-25 column to remove dithiothreitol in excess. Control experiments in which the enzyme was oxidized by tetrathionate followed by reduction with dithiothreitol resulted in quantitative activity recovery. The reactive SH groups of the enzyme was determined according to Ellman [14] in 0.05 M Tris-acetate b u f f e r / 1 m M EDTA. The total number of SH groups of the enzyme was determined by the same method in the presence of 8 M urea. The extent of tryptophan modification by the sulfonium bromide was determined by the method of Barman and Koshland [15] as the same group was introduced as when 2-hydroxy-5-nitrobenzyl bromide was used. Absorbance at 410 nm was measured and the a m o u n t of 2-hydroxy-5-nitrobenzyl groups introduced calculated with the absorbance coefficient 18000 cm -~ . M ~. A Cary 219 spectrophotometer and a Hitachi M P F - 4 spectrofluorometer were used for absorbance and fluoresence measurements.

fected. The completely thiothreitol reactive SH

original activity of the enzyme can be recovered after reduction with ditogether with the regeneration of the groups (Table I).

Modification of the tryptophan residues The enzyme with its reactive SH groups protected by tetrathionate oxidation was then treated with different amounts of the sulfonium bromide and the modified enzyme preparations with different amounts of 2-hydroxy-5-nitrobenzyl groups introduced were then reduced with an excess of dithiothreitol to regenerate the reactive SH groups. The protection of the reactive SH groups from attack by the sulfonium bromide has been effective and these SH groups can be fully regenerated upon reduction again by dithiothreitol (Table I). However, the enzyme with its reactive SH groups regenerated showed decreased activity depending on the a m o u n t of tryptophan residues modified. The relation of the 2-hydroxy-5-nitrobenzyl groups introduced and the activity remaining is shown in Fig. 1 as a Tsou plot [6]. It can be seen from Fig. 1 that the modification of two of the eight tryptophan residues [16] of creatine kinase led to the complete inactivation of the enzyme. Moreover, the linearity of the plot shows that a m o n g the two tryptophan

TABLE I EFFECTIVE PROTECTION OF ACTIVE SITE SH BY TETRATHIONATE DURING DIMETHYL(2-HYDROXY-5NITROBENZYL)SULFONIUM BROMIDE TREATMENT Enzyme

Relative activity

Activesite Total SH a SH

Reversible protection of the reactive SH groups

100 Native enzyme Enzyme oxidized by 0.8-+0.4 tetrathionate Oxidized enzyme reduced 99 -+5 h by dithothreitol 2-hydroxy-5-nitro0.4_+0.1 benzyl enzyme 2-hydroxy-5-nitrobenzyl enzyme reduced by 0.4+0.1 dithiothreitol

Treatment of native creatine kinase with N a tetrathionate at a 400-fold molar excess led to the oxidation of the two reactive SH groups and complete inactivation of the enzyme, whereas the buried SH groups of this enzyme were not af-

a Total SH was determined by DTNB in the presence of 8 M urea. b Sometimes activity recovery over 100% was obtained probably due to partial oxidation of active site SH of the native enzyme.

Results

1.73_+0.046.20_+0.15 0.08-+0.05 4.80-+0.12 1.72_+0.05 6.35_+0.20 0.08_+0.05 4.71_+0.30 1.74+0.05 6.32_+0.25

61 tion peak at 403 nm. It is also to be noted that modification of the t r y p t o p h a n residues results in a decrease in a b s o r p t i o n at the protein peak, 280

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Fig. I. Effect of the introduction of 2-hydroxy-5-nitrobenzyl groups to creatine kinase on its activity. Fraction of creatine kinase activity remaining (a) plotted against the number of 2-hydroxy-5-nitrobenzylgroups introduced per enzyme molecule (m). For details see text.

residues modified only one is essential for the activity of the enzyme. The significance of this will be discussed later.

The absorbance and fluorescence spectra of the 2-hydroxy-5-nitrobenzyl enzyme It has been reported by K o s h l a n d [17] that c h y m o t r y p s i n with its t r y p t o p h a n residues modified by 2-hydroxy-5-nitrobenzyl b r o m i d e has an a b s o r p t i o n m a x i m u m at 410 nm. Fig. 2 shows that the 2 - h y d r o x y - 5 - n i t r o b e n z y l - m o d i f i e d c r e t i n e kinase also has a n a b s o r p t i o n m a x i m u m at the same wavelength. The reagent itself has an absorp-

The intrinsic fluorescence emission spectra of the native and the modified enzyme are shown in Fig. 3. As an excitation wavelength of 290 n m was used, the decrease in fluorescence shows essentially the modification of the t r y p t o p h a n residues. At a large excess of the reagent more than two t r y p t o p h a n residues were modified and the modified enzyme has its emission m a x i m u m slightly blue-shifted toward 330 nm, suggesting that the t r y p t o p h a n residues relatively close to the surface of the molecule have been modified. N o gross c o n f o r m a t i o n a l change has occurred a n d the unmodified t r y p t o p h a n residues remain buried.

Reaction of dimethyl-( 2-hydroxv-5-nitrobenzvl)sulfonium bromide with ribonuclease A If the s u l f o n i u m b r o m i d e is indeed specific for t r y p t o p h a n and SH groups [11], treatment of b o v i n e pancreatic ribonuclease A with an excess of this reagent should result in no i n t r o d u c t i o n of 2-hydroxy-5-nitrobenzyl group into this protein

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Fig. 2. Absorption spectra of dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide, creatine kinase and the modified enzyme. Spectra were measured at a constant temperature of 25°C in 0.05 M Tris acetate buffer (pH 8.0). (l) native enzyme, 7.0 p.M; (2) modified enzyme, 6.3 #M; (3) dimethyl(2-hydroxy5-nitrobenzyl)sulfoniumbromide, 13.5 ~M.

310

330

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Fig. 3, Fluorescence emission spectra of native and the modified enzyme. Experimental conditions as for Fig. 2 except that the enzyme concentration was 0.03 ~M. The excitation wavelength was 290 nm. (a) native enzyme; (b) modified enzyme, 2.10 mol of 2-hydroxy-5-nitrobenzyl/mol of enzyme; (c) modified enzyme, 3.25 mol of 2-hydroxy-5-nitrobenzyl/mol of enzyme.

62

which contains no SH group and no tryptophan residue, as has been shown for the hydrophobic reagent 2-hydroxy-5-nitrobenzyl bromide [18]. Ribonuclease A was reduced and converted to the S-sulfonates [19] first at all its cysteine residues followed by treatment with an excess of sulfonium bromide. S p e c t r o p h o t o m e t r i c determinations showed that the modified protein contained 0.3 2-hydroxy-5-nitrobenzyl group per protein molecule. After reduction with an excess of dithiothreitol, the reduced protein contained only 0.09 2-hydroxy-5-nitrobenzyl group per molecule. The above results clearly show that the protection with S-sulfonate is effective to prevent the reaction of this reagent with the sulfur atoms of the cysteine residues. Moreover, as ribonuclease A contains all the amino acids except tryptophan, the fact that only a small amount of 2-hydroxy-5-nitrobenzyl group was introduced, the greater part of which can be removed by reduction with an excess of dithiothreitol, illustrates the specificity of dimethyl(2-hydroxy-5-nitrobenzyl)sul fonium bromide with tryptophan under the conditions employed in the present study. 1.0

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Fig. 4. Substrate protection of the modification of creatine kinase by d i m e t h y l ( 2 - h y d r o x y - 5 - n i t r o b e n z y l ) s u l f o n i u m bromide. Conditions as described in the materials and methods section except that during modification a mixture of the substrates containing creatine, 20 mM; Mg 2+, 0.65 mM; ADP, 0.65 mM; and nitrate 20 mM were also present. Curves 1 and 2, left-hand ordinate, activity remaining in the presence and absence of the substrates respectively. Curves 3 and 4, righthand ordinate, 2-hydroxy-5-nitrobenzyl (HNB) groups introduced per enzyme molecule in the presence and absence of the substrates respectively; abscissa, molar ratio of the sulfonium bromide added to the enzyme during modification.

The specificity of this reagent has also been shown by the finding that the native and the modified creatine kinase had essentially the same amino acid composition except for tryptophan.

Protection by substrates It is known that nitrate is an effective transition state analog inhibitor of creatine kinase. A mixture of creatine, ADP, Mg 2+, and nitrate show marked protection Of this enzyme against both the amount of tryptophan residues modified and the inactivation by dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (Fig. 4). The above results would seem to suggest that the essential tryptophan residue is situated at the active site of this enzyme. The substrates when employed separately gave very little protection. Discussion The results presented in the above sections show clearly that the modification of one essential tryptophan residue at the active site of creatine kinase by the water-soluble reagent dimethyl 2-hydroxy5-nitrobenzyl sulfonium bromide leads to the complete inactivation of this enzyme. The failure of Roustan et al. [5] to modify creatine kinase with either 2-hydroxy-5-nitrobenzyl bromide or 2methoxy-5-nitrobenzyl bromide suggests that the essential tryptophan residue is situated at a hydrophilic site of the enzyme molecule which is inaccessible to the poorly soluble reagents but is open to attack by the easily soluble sulfonium bromide. However, even with a large excess of the sulfonium bromide less than half of the tryptophan residues of the enzyme were modified suggesting that the unreactive residues are probably deeply buried in the interior of the enzyme. The fact that the modified enzyme has decreased fluorescence but with an emission peak slightly blue-shifted toward 330 nm indicates the modification of tryptophan residues near the surface of the enzyme molecule. All the above results are in accord with the suggestion [12,20,21] based on comparisons of denaturation and inactivation rates in guanidine and urea solutions that the active site of this enzyme is situated at an exposed and easily accessible region of the molecule. Horton and Koshland [18] have shown that in

63 neutral and acidic solutions, 2-hydroxy-5-nitrobenzyl b r o m i d e reacts only with the indole group of t r y p t o p h a n a n d SH group of cysteine for a n u m b e r of proteins. This has now been shown to be also the case for the water-soluble dimethyl 2-hydroxy-5-nitrobenzyl s u l f o n i u m b r o m i d e as ill u s t r a t e d by the e x p e r i m e n t s with b o v i n e ribonuclease A. O u r results also show the protection of the SH group with S-sulfonate is effective. As creatine kinase is a dimeric enzyme, the fact that a m o n g the t r y p t o p h a n residues modified, only one is essential for its activity would seem to require some explanation. Each s u b u n i t of creatine kinase carries a reactive SH group a n d chemical modification of these groups has long been shown to affect the activity of this enzyme markedly [1-4]. Q u a n t i t a t i v e analysis [6] of data given in the literature [3,5] also shows that only one of the SH groups is essential per activity unit of this dimeric enzyme. These findings can be simply explained by assuming that the m i n i m a l activity unit of the enzyme is the m o n o m e r . However, Degani a n d Degani [4] have recently presented some most interesting results to suggest that the two s u b u n i t s of this enzyme are arranged asymmetrically a n d that they are not functionally identical, implying that the enzyme has to be in the dimeric state to be active. If this is indeed the case, it is possible that the two s u b u n i t s are arranged in such a way that they c o n t r i b u t e asymmetrically to form a single active site for the dimeric enzyme. T h a t a t r y p t o p h a n residue is situated at or near the active site of creatine kinase has earlier been suggested by Vasak et al. [22] based o n the effect of A D P o n the fluorescence a n d N M R properties of the enzyme. Their results would seem to show the presence of t r y p t o p h a n residue(s) at or near the A D P - b i n d i n g site, b u t this residue is not necessarily essential for the activity of the enzyme. This has now been definitely shown to be the case.

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