Crystalline Adenylate Kinase from Carp Muscle

Eur. J. Biochem. 51,229-235 (1975)

Crystalline Adenylate Kinase from Carp Muscle Lafayette NODA, Georg E. SCHULZ, and Inge VON ZABERN Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire and Max-Planck-Institut fur Medizinische Forschung, Heidelberg

(Received August 22/0ctober 18, 1974)

1. The concentration of adenylate kinase in carp muscle is about 0.3 mg/g. An improved isolation procedure makes use of a dilute solution of the substrates, ATP and AMP, to elute the enzyme from a phosphocellulose column in overall yields of 60 % before crystallization. By the hexokinase - pH- stat assay the specific activity is 3550 units/mg. The preparation has been found to be essentially homogeneous by dodecylsulfate gel electrophoresis, isoelectrofocusing and gel filtration.

2. The molecular weight has been determined to be 22000 by several methods. The absorbance of a 1 % solution at 280 nm is 6.9 and the isoelectric point by electrofocusing is pH 5.9.

3. The crystals of carp adenylate kinase have the space group P4122 or P4,22. 4. The amino acid composition has been determined. There is no tryptophan, no cystine. There

is one amino acid residue each of cysteine and histidine which are at or close to the catalytic center. 5. Several peptides derived by tryptic hydrolysis have been isolated and identified with correspond-

ing peptides of porcine adenylate kinase. Consideration is given to histidine and cysteine being a part of the active site.

Adenylate kinase catalyzes the reaction

ATP + AMP 2ADP.

Properties and possible biological roles of this enzyme have been reviewed recently [1]. Adenylate kinase from porcine muscle yielded crystals suitable for X-ray diffraction studies [2] and its covalent [3] and three-dimensional [4] structures have been reported. In order to elucidate the phosphoryl transfer mechanism structural knowledge is indispensable. An approach to further clarification is a comparison of structural data of adenylate kinases from different species. Sequence comparisons will show where amino acid residues are exchanged and where they are conserved. In particular, variation and conservation of side chains in the active center region may give information about the mechanism. Furthermore, enzymes in a different crystal form can be expected to extend the range of possible substrate and substrate- analogue binding studies by X-ray diffraction [4]. For all these reasons we isolated, crystallized and charac- terized muscle adenylate kinase from a fish, which represents a species with considerable phylogenetic

Enzymes. Adenylate kinase (EC 2.7.4.3.) ; hexokinase (EC 2.7.1.1); trypsin (EC 3.4.21.4).

separation from mammals, the adenylate kinases of which have so far received the most attention [1, 5- 91.

MATERIALS AND METHODS

Assay for adenylate kinase was by the hexokinase - pH-stat method [lo] in a total volume of 1.5 ml. Alternatively, the activity was measured employing radioactive nucleotides and thin-la yer chromatography of the reaction products on poly(ethy1eneimine)- cellulose sheets [1 1,121. [2-3H]Adenosine 5'-monophos- phate and [2-3H]adenosine 5'-diphosphate were purchased from Amersham Buchler. Celite 503 or 535 from Johns-Manville Co. was twice washed with 2 N HCl at 80°C and thoroughly washed free of acid. Phosphocellulose was obtained from Schleicher and Schull, lot 2112 and 2136 (0.94 mequiv./g). New lots should be tested for adsorptive capacity of adenylate kinase. Sephadex G-75 was purchased from Pharmacia Fine Chemicals and ampholine from LKB Instru- ments, Inc. N-Morpholinopropane sulfonic acid, ATP, ADP, AMP, Tris and imidazole were obtained from Sigma Chemical Co. Porcine adenylate kinase was prepared as previously described [3]. Amino acid analyses, tryptic digestion in 0.5 % ammonium bi-

Eur. J. Biochem. 51 (1975)

230 Adenylate Kinase from Carp

Table 1. Isolation of udenylate kinase,from curp Starting material was 2.5 kg muscle. Protein was determined by the biuret procedure [I31 with bovine serum albumin as standard except for fractions 1V and V. These were obtained by A,,, x 1.5, to give protein concentration in mglml

Fraction Volume Protein Activity Specific Purification Activity per activity per step muscle wt

1 g MU U/mg -fold u / g I. Homogenate (centrifuged) (8.65) 16.6 2.68 35.0 (1.0) 1070 11. pH-5.0 filtrate 8.65 71.5 2.5 35.0 1 .0 1000 111. Phosphocellulose 0.581 0.112 2.12 2750 78.0 850 IV. Sephadex G-75 0.160 0.454 1.61 3550 1.3 640 a

V. Crystallization - 0.391 1.30 3320 0.94 520 ~~

a 60"/, yield based on fraction I, about 100-fold purification.

carbonate pH 8.0, carboxymethylation with iodo- [14C,]acetate as well as the separation and identifi- cation of peptides were conducted as previously described [3].

RESULTS Isolution of Adenylute Kinase from Carp Muscle

The procedure in the main simply involves phosphocellulose chromatography with elution by the use of substrates ATP and AMP, but in the following considerable details are given with the aim of repro- ducibility. All steps were carried out in a cold room (2-4 C) or in an ice bath. For the experiments reported here two lots of carps obtained from the fresh water streams of the Midwest and the East Coast of North America have been used. No differences between the preparations were seen. After killing carp, the muscle was immediately removed to polyethylene bags and cooled in ice. Frozen muscle has been stored in a deep freeze for several weeks with slight loss of activity. For the extraction of frozen muscle (2.5 kg) broken pieces were warmed only until pieces could be cut with a knife (about 15-20 min in the draft of a hood). More thorough thawing resulted in decreased enzymatic activity. The frozen pieces were ground in a motor-driven grinder (about 3-mm holes) and quickly stirred with water previously adjusted to 13 "C and equal to 3 times the weight of ground muscle. The final temperature of the mixture was 1 - 3 "C. The homogenate was stirred for 30 min and a small sample was centrifuged for assay (fraction 1, Table 1). The pH of the homogenate was adjusted to 5.0 with 5 N acetic acid and the mixture was stirred for 10 min. A clear solution was obtained by mixing the homogenate with acid-washed Celite equal to 1/10 the weight of muscle and filtering through a 50-cm Buchner funnel prepared with a thin layer of acid-washed Celite. (Clarification may also be attained by centrifugation.) The reddish colored clear filtrate is fraction 11.

Phosphocellulose was cycled and equilibrated with 0.1 M sodium acetate pH 5.0 and evenly packed 5 cm high in a 25-cm internal diameter (490 cm2) Buchner funnel or preferably a tabletop filter serving as a column. A floating, porous polyethylene disc was found helpful to break up the stream of solution being added and thus prevent the cellulose from being disturbed. A polyethylene bottle used as a float and fitted with a sealed glass tube tapered to seat against a constriction in a larger glass tube to serve as a valve to control the liquid level, together with a siphon from a reservoir were helpful to obviate need for constant manual additions of liquid to the column. Gravity flow of250-350ml/min (0 .5 -0 .7 rn l~c rn -~ xmin-') was maintained by a liquid head about 5 cm above the phosphocellulose and an effective liquid head up to about 60 cm contributed by tubing (3 - 5-mm internal diameter) attached to the effluent end of the Buchner funnel serving as a column.

The schedule of development of the column after adsorption of the enzymic activity was (a) 1 column volume (2.5 I) 0.10 M sodium acetate, pH 5.0, (b) 2 column volumes (5 1) 0.10 M sodium acetate pH 5.7 and (c) 4 column volumes (10 1) 0.050 M N-morpholinopropane sulfonic acid adjusted to pH 7.0 with NaOH. During the washing several peaks of proteins are eluted from the column and at the end the effluent should be light yellow or nearly colorless. The phosphocellulose in the funnel was sucked moderately dry and turned out on a sheet of polyethylene. The lower colorless layer generally compris- ing about one-third of the total phosphocellulose was removed. Layers of the increasingly colored phosphocellulose were suspended in 0.050 M sodium N-morpholinopropanesulfonate, pH 7.0 and packed into an 8.7-cm diameter (60 cm2) column generally to a height of about 24 cm. This column was connected to a peristaltic pump (about 30 ml/min, 0.5 ml x cm-' x min- ') and 20-ml aliquots were collected. Washing of the phosphocellulose was continued with 0.020 M sodium N-morpholinopropanesulfonate, pH 7.0


L. Noda, G. E. Schulz, and I. von Zabern 231

(about 1 column volume) until the absorbance at 280 nm was less than 0.050. To elute the adenylate kinase, 0.2 mM ATPl0.2 mM AMP in the 0.020 M buffer adjusted to pH 7.0 was used followed by about 1 column volume of the same buffer. The enzyme appears as a sharp peak (280 nm) on the leading edge of a broad plateau representing the absorbance of the adenine nucleotides. Aliquots of the effluent containing more than about 500 enzyme units/ml were collected (fraction 111) and precipitated by addition of solid ammonium sulfate to 90% saturation and adjustment of pH to 6.1. The precipitated protein was allowed to flocculate overnight and collected as a paste (centrifuged at 10000 rev./min for 30 min).

The paste from two 2.5-kg preparations was dis- solved in a minimal volume of 0.10 M imidazole- H,SO,, pH 7.0, cleared by centrifugation and a total volume not to exceed 20 ml was applied to a Sephadex G-75 column (5.2 x 160 cm) equilibrated with the same buffer. A flow rate of about 1.5 ml/min (0.071 ml xcm-2xmin- ' ) was used. A small yellow inert protein peak preceded the activity peak which was collected in 15-ml aliquots. Fractions having 1000 U/ml or greater were collected and precipitated at 90 % saturation of ammonium sulfate (enzyme grade). After the addition of salt the suspension was brought to pH 6.1 with 2 N sulfuric acid. The protein was collected as a paste by centrifugation.

In our experience crystallization did not lead to increased purity when the specific activity after the Sephadex step was already very high. Crystallization was carried out near 0°C at an initial protein concentration of 30 to 35 mg/ml, ammonium sulfate about 0.62 saturation and in the presence of buffer (imidazole) with final adjustment of pH to 6.0. After crystallization has proceeded ammonium sulfate concentration is increased in several steps of 1 % saturation.

The isolation procedure reported here (Table 1) using a combination of the substrates, ATP and AMP, instead of AMP alone as reported for porcine adenylate kinase [3] has made possible far more effective and efficient isolation of the enzyme in phosphocellulose chromatography. Further improvement of method has been the use of a short large-diameter column for the adsorption and washing steps followed by re- packing into a column of the more usual dimensions.

Homogeneity of Enzyme Preparation

From a Sephadex G-75 column the single sym- metrical activity peak showed uniform specific activity. In isoelectrofocusing using pH 5 - 8 ampholyte, no evidence was found for contaminating protein. Dodecylsulfate gel electrophoresis [14] were run and

Table 2. Physical and kineticproperties ofcarp adenylate kinuse

Property Value Basis

Molecular weight

Isoelectric point

K m (ATPI

22 000 Sephadex G-75 [15], identity with porcine enzyme

electrophoresis, identity with porcine enzyme

analysis and also - SH titrations

pH 5.9 electrofocusing, ampholine pH 5 to 8

2.03 x M assay by direct determination

1 .I4 x M of products by thin- layer chromatography

22 000 dodecytsulfate gel

6.9 protein by amino acid

tracings were made on a Gilford scanner. Tracings as well as visual inspection showed only a single component.

It may be concluded that the preparation of adenylate kinase from carp is pure.

Physical Proper ties

Behavior of the carp enzyme as observed during isolation procedures indicates the similarity of adenylate kinases isolated from the muscle of carp and those from rabbit, pig and man with respect to acid stability and solubility in ammonium sulfate solutions. On Sephadex G-75 column chromatography the elution volumes of the carp and porcine enzymes were the same. In dodecylsulfate gel electrophoresis of porcine and carp enzymes individually and in equal mixture only single identical bands were found. These results lead to the conclusion of the equality within the limitations of experiment of the molecular weights of the carp and the porcine enzymes (21700). Some physical properties of carp adenylate kinase are summarized in Table 2.

Properties of the Crystals

For X-ray studies carp adenylate kinase crystals have been grown in 0.1 M Tris - maleate pH 6.1,2.4 M ammonium sulfate and at about 20 mg protein/ml to a size of about 500pm3. Crystals have been stored prior to use by transfer to a solution composed of 0.1 M Tris-maleate 3.0 M ammonium sulfate pH 6.4. The shape of the crystals is an octahedron that is slightly shortened along one of the diagonals (Fig. 1 A) suggesting 422 symmetry with the four-fold axis along the short diagonal.


232

4 +

Adenylate Kinase from Carp

t 500 urn I

A 6 Fig. 1. Crystals of carp adenylate kinase. The space group is P4, 0,22. (A) The shape of the crystals is an octahedron slightly shortened along the four-fold axis. The directions of

the four-fold and two-fold axes are indicated. (B) X-ray precession photograph of the Okl-plane, that is the plane which contains the four-fold axis and a two-fold axis

Single crystals were examined by X-ray diffraction on a precession camera. The diffraction patterns show that the molecules have crystallized in one of the two enantiomorphic space groups P4,22 or P4,22 with the four-fold axis along the short diagonal. This corre-

cupied single-site mercury derivative [4]. This should be an easy handle to the solution of the heavy atom localization problem.

Amino Acid Composition sponds well with the observed crystal shape (Fig. 1 A). The lengths of the crystal axes are 8.6 nm x 8.6 nm x 12.8 nm, giving a calculated unit cell volume of 946 nm3.

The number of molecules in an asymmetric unit of the unit cell was calculated [16] from the molecular weight, 22000, the protein density (taken as 1.35g/cm3), the storage solution density (1.20 g/cm3) and crystal density (1.27 0.01 g/cm3). The crystal density was determined by sedimentation of crystals in a toluene - carbon tetrachloride density gradient calibrated with droplets of cesium chloride solutions of known density. The formula yields 2.16 as the number of molecules per asymmetric unit, i.e. 2 to the nearest whole number. Using 2.0, the crystal volume per molecular mass is V, = 0.0026 nm3/dalton, which corresponds well to values found with other protein crystals [17].

Obviously the structural analysis of this enzyme would be facilitated if a crystal form with one molecule per asymmetric unit could be found. The present crystals, however, do show advantages for X-ray analysis. (a) They are large; (b) the diffraction pattern extends to high resolution, i.e. the molecules are well ordered in the crystal; (c) the molecule contains only one -SH group corresponding to Cys-25 of the porcine enzyme and thus ought to give a highly oc-

In Table 3 are given the amino acid composition of the enzyme from carp and for comparison that of pig [3]. The amino acid composition of the enzyme isolated from carp and from the muscle of mammals is notable with respect to the absence of tryptophan. By the spectroscopic method of Edelhoch [19] 0.3 residue tryptophan was calculated. After tryptic hydrolysis and two-dimensional separation of peptides on paper, the Ehrlich stain for tryptophan was negative. Similarly, no tryptophan was detected in amino acid analysis after hydrolysis with p-toluene sulfonic acid [18].

Most notably there is only one residue each of histidine and cysteine. By comparison the porcine enzyme has two residues each of cysteine and histidine. Individual amino acids of considerably greater number of residues in carp than in the porcine enzyme include the hydrophobic residues, alanine and isoleucine and the hydrophilic aspartate residue. On the other hand in the carp enzyme the hydrophilic threonine residue is much less than in the porcine enzyme.

Active-Site Peptides

Three tryptic peptides from carp adenylate kinase have been isolated and identified on the basis of


L. Noda, G. E. Schulz, and I. von Zabern 233

Table 3. Amino acid composition of carp adenylate kinase The molecular weight was taken as 22000. Data on porcine adenylate kinase [3] are included for comparison

Amino acid Residues per molecule in

carp Pig -~

measured integer

Aspartate/asparagine Threonine Serine Glutamate/glutamine Proline Glycine Alanine Valine Methionine Isoleucine Leu c i n e Tyrosine Phen ylalanine Lysine Histidine Arginine C ysteine Tryptophan

Total

16.6 7.7 8.6

25.8 6.3

18.0 16.6 17.4 4.9

15.4 16.8 9.0 2.8

22.9 0.9 8.9 1.0" 0.0b

17 8 9

26 6

18 11 17 5

15 17 9 3

23 1 9 1 0

13 14 11 25 6

19 8

17 6 9

18 7 5

21 2

11 2 0

20 1 194

a Relative to 1.0 residue of histidine. Determined as cysteic acid after performic acid oxidation,

By p-toluenesulfonic acid hydrolysis [IS].

identity of fingerprint spots and chemical information provided by the sequence determination of the porcine enzyme [3]. The cysteine peptide (peptide B) was identified by its radioactivity from labelling with iodo- [l4C2]acetic acid, the histidine peptide (peptide A) by the Pauly stain, and peptide C, by its distinguishing yellow color with cadmium- ninhydrin stain and location of fingerprint spot. The amino acid composition and other properties of the carp peptides A, B, and C (Table 4) show great similarity with corresponding data on the tryptic fragments of porcine adenylate kinase which include His-36, Cys-25 and Asp-93. On the basis of the amino acid analysis peptide A has serine substituted for threonine of the porcine His-36 peptide and in peptide C tyrosine is substituted for phenylalanine of the porcine Asp-93 peptide. It is found that the isolated peptide B from the carp enzyme has residues corresponding to the porcine Cys-25 peptide. Thus, with two conservative amino acid substitutions each of which could be accounted for by one base exchange in the DNA, the three peptides of carp adenylate kinase correspond in amino acid composition to the peptides which have been sequenced for the porcine enzyme.

Table 4. Amino acids of trypticpeptides of carp adenylate kinase

Amino acid Peptide A Peptide B Peptide C

Carboxymethylcysteine Aspartic acid

Threonine Serine Glutamic acid

Proline Glycine Isoleucine Leucine Tyrosine Lysine Histidine Arginine

+ asparagine

+ glutamine

1.1 0.9 1.7

1.9

3.3 2.2

0.9 1.0

+" 1 .0

0.9

2.3 1.1

1 .0 2.1 1 .0 1.0 1.9

0.8

1 .0

Total 13 6 9

Properties

Electrophoretic 0 to mobility - 0.04 + 0.33 0

Charge at pH 6.5 < + I - 1 0 Color with cadmium-

ninhydrin reagent orange- yellow yellow red

purification BAWP BAWP BAWP Methods of 6.5 6.5 6.5

1.9 1.9 1.9 1.9

Mobility in descending chromatography 0.72 0.16 0.73

Assumed corresponding segment in porcine adenylate kinase (residue numbers[3]) 32-44 22-27 89-97

a The peptide was radioactive. This accounts for one residue of ['4C]carboxymethylcy~teine since carp muscle adenylate kinase contains only one single cysteine.

The methods of purification are abbreviated thus: 1.9 and 6.5, high-voltage paper electrophoresis at pH 1.9 and 6.5, respectively; BAWP, descending chromatography in solvent butan-1-01 - acetic acid - water - pyridine. The electrophoretic mobilities refer to pH 6.5 and are relative to aspartic acid. The charge at pH 6.5 was determined according to Offord [20]. The mobility in descending chromatography was measured relative to the marker dye rosaniline hydrochloride.

Kinetic Constants

Michaelis constants for the substrates have been measured by direct determination of the reaction products by thin-layer chromatography. Values found for the substrates are given in Table 2.


234 Adenylate Kinase from Carp

DISCUSSION

A comparison of fingerprints and amino acid compositions showed that the carp enzyme is different from the porcine enzyme, the three-dimensional structure of which has been elucidated [4]. Never- theless, one has to expect that the chain fold [21] and that the residues in the catalytic center are conserved 1221. Similarity of kinetic constants and identical effective inhibition by diadenosine pentaphosphate (H. Schirmer, private communication, 1974) corro- borate the latter assumption. Therefore, we shall discuss the carp enzyme in connection with the porcine enzyme. As will be shown in the following section, the data on carp adenylate kinase confirm the location which has been proposed for the active center in the porcine enzyme [4].

It was shown by nuclear magnetic resonance [23] that one of the two histidines in the porcine enzyme is at the catalytic center. In addition, chemical modifi- cation studies [8] indicate that one of the three histidines in the rabbit enzyme might be essential for enzymatic activity. Since the two histidines in the porcine enzyme are far apart from each other [4], there remained an ambiguity in the location of the catalytic center. This can now be resolved with the histidine-containing peptide A of the carp enzyme (Table 4). Except for a serine-threonine exchange the peptide A composition fits the amino acid sequence around His-36 of the porcine enzyme and is at variance with the sequence around the other histidine [3]. Since residues at the catalytic center are expected to be conserved during evolution, the only histidine of the carp enzyme should correspond to the histidine in the catalytic center of the porcine enzyme. The same residue, His-36, had been suggested from a structural point of view [4].

Two cysteine residues are found in mammalian muscle adenylate kinase [3,9,24]. Blocking them can result in decrease or complete loss of activity [10,25]. Although these experiments show that at least one of the two cysteines is in the active center region, they do not distinguish between them. Again, comparison with the only cysteine in the carp enzyme resolves the ambiguity: The amino acid composition of the cysteine-containing peptide B (Table 4) fits the amino acid sequence around Cys-25 of the pig enzyme and is different from the sequence around the other cysteine. In addition, for the enzyme from rabbit one of the two cysteine peptides sequenced [26] is identical to the Cys-25 peptide from the porcine enzyme and the composition matches the present peptide B from the carp enzyme. Since the carp enzyme is completely inactivated by blocking its cysteine with 5,5'-dithio- bis(2-nitrobenzoic acid) or 7-chloro-4-nitrobenzo-2-

oxa- 1,3-diazole and since this cysteine is effectively protected by diadenosine pentaphosphate [27] it is probably in the active center region. Therefore, we conclude that in the rabbit and pig enzymes the homologous Cys-25 is in the active center region as is also supported by proximity of Cys-25 to the essential His-36 as established in the three-dimensional structure [4].

A comparison of the tryptic fingerprints of the carp and the porcine enzymes showed another peptide that seemed to be conserved. This peptide was isolated and analyzed (peptide C, Table4). Its amino acid composition fits the amino acid sequence around Asp-93 in the porcine enzyme except for one exchange between tyrosine and phenylalanine. In the three- dimensional structure this peptide is in the catalytic center region. Moreover, Asp-93 has been suggested as taking part in catalysis [4].

In the porcine enzyme the peptides corresponding to A, B, and C are located adjacent to each other in the deep cleft of the molecule [4]. In particular, the side chains of Cys-25, His-36 and Asp-93 are touching each other. The fact that this region was conserved whereas many other changes occurred during evolution is one indication that it is near to the active center.

The properties of the carp enzyme will facilitate the analysis of the catalytic mechanism because the crucial histidine and the crucial cysteine are single so that interpretations can be much more precise.

The technical assistance of Takiko Shimizu Tamura, Hokkaido University, Sapporo, Japan, in some early work and the helpful discussions and assistance of Heiner Schirmer, Tom Pinder, and Thomas Frohlich are gratefully acknowledged. The support in part by the National Institutes of Health, Grant HL03599 and the tenure of a Guggenheim fellowship during the early part of this work of one of us (L. N.) are also gratefully acknowledged.

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L. Noda, Department of Biochemistry, Dartmouth Medical School Hanover, New Hampshire, U.S.A. 03755

G. E. Schulz and I. von Zabern, Abteilung Riophysik, Max-Planck-Institut fur Medizinische Forschung, D-6900 Heidelberg, JahnstraBe 29, Federal Republic of Germany

Note Added in Proof' (January 6 , 1975). Z. Ooshiro, M. Fukushima, and S . Hayashi [Bull. Jup. Soc. Sci. Fish. 40, 291 - 298 and 299- 302 (1974)] report the isolation and properties of myokinase I and 11 from carp muscle. The isozymes were found in roughly equal quantities having molecular weights of 27000 and 22000 respectively. We have not observed a second isozyme of adenylate kinase in our purifications from the skeletal muscles of carp, pig and rabbit. The question of a second isozyme in carp muscle my possibly be due to unexplained differences in isolation procedures.


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Crystalline Adenylate Kinase from Carp Muscle