Chemical modification of arginine residues of porcine muscle acylphosphatase

234

Biochimica et Biophysica Acta 870 (1986) 234-241 Elsevier

BBA32488

Chemical modification of arginine residues of porcine m u s c l e acylphosphatase T a k i k o T a m u r a *, Y u s u k e M i z u n o a n d H i r o y u k i S h i o k a w a Section of Biochemistry, Institute of lrnmunological Science, Hokkaido Unioersity, Kita-ku, Sapporo, Hokkaido 060 (Japan) (Received August 14th, 1985) (Revised manuscript received November 11th, 1985)

Key words: Acylphosphatase inactivation; Arginine residue; Chemical modification; Phenylglyoxal; Phosphate-binding site; (Porcine muscle)

Acylphosphatase (acylphosphate phosphohydrolase, EC 3.6.1.7) from porcine skeletal muscle is inactivated by phenylglyoxal following pseudo-first-order kinetics. The dependence of the apparent first-order rate constant for inactivation on the phenylglyoxai concentration shows that the inactivation is also first order with respect to the reagent concentration. Among the competitive inhibitors for the enzyme examined, inorganic phosphate and ATP almost completely, and CI- partially, protect the enzyme against the inactivation. The dissociation constants for inorganic phosphate and ATP determined from protection experiments by these inhihitors agree well with those from inhibition experiments by them. These results support the idea that the modification occurs at the phosphate-binding site. The amino-acid analysis reveals the lack of reaction at residues other than arginine. Circular dichroism spectra of the modified enzymes show that the inactivation seems not to be due to denaturation of the enzyme resulting from the modification of the non-essential arginine residues. The relationship between the loss of the enzyme activity and the number of arginine residues modified in the presence and absence of ATP shows that one arginine residue is possibly responsible for the inactivation of acylphosphatase.

Introduction Acylphosphatase (acylphosphate phosphohydrolase, EC 3.6.1.7) hydrolytically cleaves the carboxyl-phosphate bond of acylphosphates, such as 1,3-diphosphoglycerate, carbamoyl phosphate, acetyl phosphate and synthetic acylphosphates. The enzyme is widely distributed in bacteria, plants and animal tissues, and has been purified from various species. The molecular weight of the enzyme is about 10 000 and the primary structures of the enzymes from some species have been reported [1-4]. However, no information is available on amino-acid residues which participate in the bind-

* To whom correspondence should be addressed.

ing of substrates or in catalysis of the enzyme, except for lysine residue, which was reported to form a Schiff base with pyridoxal phosphate [5]. Recent modification studies of arginine residues have revealed that these can serve as positively charged recognition sites for negatively cha~ged phosphate or carboxylate moieties of substrates or cofactors [6]. The substrates of acylphosphatase contain negatively charged phosphate moieties and the enzyme is competitively inhibited with a variety of anions such as SO2- and phosphate compounds [7]. Therefore, positively charged lysine a n d / o r arginine are the most likely candidates for the functional amino-acid residues of acylphosphatase. Phenylglyoxal has been shown as a specific modifier for arginine residues under mild conditions [8]. Hence, we performed studies on the

0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

235 modification of porcine muscle acylphosphatase by this reagent and we now present evidence of the participation of possibly one arginine residue essential for the enzyme activity. Materials and Methods

Materials. Phenylglyoxal monohydrate was purchased from Aldrich and recrystallized from water. [7-14C]Phenylglyoxal (15.1 m C i / m m o l ) was obtained from Amersham, ATP and dithiothreitol from Sigma, Sephadex G-25 and G-50(fine) from Pharmacia. Benzoyl phosphate was synthesized as described by Camici et al. [9]. Porcine acylphosphatase was prepared by the method of Mizuno et al. [10]. The enzyme thus obtained was largely monomer owing to the addition of 2mercaptoethanol. The dimer of the enzyme was obtained as follows. The enzyme solution was passed through a Sephadex G-25 column to remove 2-mercaptoethanol and exchange 0.1 M sodium acetate buffer (pH 5.3) for 0.1 M Tris-HC1 buffer (pH 8.0). The eluate was bubbled with oxygen gas for 1 h at room temperature and left to stand overnight at 4°C. After concentration of the enzyme by precipitation with 90% saturation of ammonium sulfate, the dimer was separated from the monomer by Sephadex G-50 column chromatography. The monomer was obtained from the original enzyme preparation by the chromatography on a Sephadex G-50 column equilibrated with 0.1 M sodium acetate buffer (pH 5.3) containing 1 mM dithiothrietol. The monomer obtained was immediately used for the inactivation experiments. Enzyme activity and protein concentration. Acylphosphatase activity was assayed spectrophotometrically according to the method of Ramponi et al. [11] with the following modification. Microcells of 0.5 cm light-path were used. The sample cell contained the reaction mixture (0.25 ml) consisting of 0.1 M sodium acetate buffer (pH 5.3), 5 mM benzoyl phosphate, 0.05% Tween 20 and an appropriate amount of the enzyme. The reference cell contained the same constituents as the sample cell except that the enzyme was removed and benzoyl phosphate was 4 mM. The decrease in absorbance at 283 nm of the reaction mixture was recorded by Cary Model 17 spectrophotometer for 30 s at 25°C. The enzyme activity in the presence

of Tween 20 was the same as in the presence of bovine serum albumin, which was used by Mizuno et al. [10]. The concentration of porcine muscle acylphosphatase was estimated from the absorbance at 280 nm by the use of the absorption coefficient, A (1%) = 14.2 [101.

Modification of acylphosphatase by phenylglyoxal. Modification of acylphosphatase by phenylglyoxal was carried out in the dark at 30°C in 0.1 M N-ethylmorpholine acetate buffer (pH 8.0) unless otherwise indicated. Acylphosphatase dimer was used for the modification, unless otherwise stated, for the reason described under 'Discussion'. All data and concentration of the enzyme described below are expressed for the monomer. Competitive inhibitors were added to the reaction mixture, when needed. The reactions were initiated by adding an aliquot of a freshly prepared phenylglyoxal solution in water. Aliquots (2-5 /xl) were removed from the reaction mixture and diluted 80-fold with a cold diluting solution consisting of 0.1 M sodium acetate buffer (pH 5.3) and 0.05% Tween 20, and their enzyme activity was immediately measured. In order to prevent activity loss arising from low protein concentration, the containers for dilution had been treated as follows: they had been filled with 1% Tween 20, incubated for 2 h at 37°C, rinsed with distilled water, and dried. The modification of the monomer was performed in the reaction mixture containing 1 mM dithiothreitol. The enzyme incubated without phenylglyoxal was used as the control. The concentrations of phenylglyoxal and competitive inhibitors in the final assay medium were low enough for them not to interfere with the activity measurements. Amino-acid analysis. The modification reaction of acylphosphatase was carried out as described above. Aliquots (usually 50 /~1) were withdrawn from the reaction mixture and mixed with 1 M acetic acid (one-tenth of the volume of the aliquots) in the containers kept in an ice bath. The p H of the mixtures was 4.9. These were assayed for the enzyme activity and the arginine content. Aliquots (2 /~1) were used for the enzyme activity. The activity of the modified enzyme treated in this way did not change for at least 1 h. For the amino-acid analyses, aliquots (usually 50 ~tl) were withdrawn

236 from the mixtures and separated from the unreacted phenylglyoxal by a column centrifugation technique [12] in which a column of Sephadex G-50 (fine) was equilibrated with 1 M acetic acid. After evaporation of acetic acid solution from the eluates, the residues were hydrolyzed in 6 M HC1 for 22 h in evacuated, sealed tubes at l l 0 ° C in the presence of 0.05% mercaptoacetic acid [13]. Amino-acid analyses were performed on a Hitachi amino-acid analyzer (model 835). [14C]Phenylglyoxal incorporation. Modification reaction was performed in a manner similar to that described above. The enzyme (36.5 ~tM) was incubated in the dark with 6 m M [7-14C]phenylglyoxal (3.44-106 cpm//~mol) in 0.1 M N-ethylmorpholine buffer (pH 8.0) at 30°C in the absence or presence of 2.5 m M ATP. Aliquots (50/~1) from the reaction mixture were processed by the column centrifugation procedure [12]. Aliquots (2 #1) of the eluate were used for the enzyme activity. The remaining eluates were mixed with 10 ml of toluene-based scintillator and their radioactivities were measured with an Aloka liquid scintillation spectrometer (model LSC-900). The amount of the enzyme recovered after the column centrifugation was calculated from the total activity of the enzyme before and after the column centrifugation. Circular dichroism measurements. The native enzyme and enzymes modified by phenylglyoxal for 30 min or 60 rain in the absence or presence of 25 mM ATP were passed through a Sephadex G-25 column equilibrated with 0.1 M cacodylate buffer (pH 5.3). The eluates were used for CD measurements. The CD was measured at 4°C in a cell of 0.1 cm light-path with a JASCO J-500A spectropolarimeter and the baselines were corrected with a JASCO DP-500 data processor. The data are expressed in terms of mean residue ellipticity. The contents of a-helix, fl-structure and random coil were calculated by the method of Chen et al. [14]. Results

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Fig. 1. Time-courseof inactivation of acylpbosphataseby phenylglyoxal. The enzyme (36.5 /LM) was incubated with no (D), 2.0 (zx),4.0 (A), 5.0 (©) and 6.0 (e) mM phenylglyoxalin 0.1 M N-ethylmorpholine buffer (pH 8.0) at 30°C. At the times indicated, aliquots were removed for measurements of the residual enzyme activity.

in a pseudo-first-order loss of the enzyme activity (Fig. 1). After the incubation of the enzyme for 60 min with 6 m M phenylglyoxal, about 14% of the original activity was observed. Prolonged incubation with phenylglyoxal resulted in precipitation of the enzyme. The apparent first-order rate constants for the inactivation were linearly related to the phenylglyoxal concentrations (data not shown), and the slope of the plot gave a value of 5.50 M - 1 - m i n -1 for the second-order rate constant. The monomer enzyme gave the same kinetics as the dimer (data not shown). The reversibility of the modified enzyme was examined. The activity of the enzyme (36.5 /~M) modified with 6 m M phenylglyoxal in 0.1 M N-ethylmorpholine buffer (pH 8.0) at 30°C for 1 h was not recovered at all by the incubation of the modified enzyme in 0.1 M Tris-acetate buffer (pH 8.0) at 25°C for 3 h.

Modification of acylphosphatase Acylphosphatase was not satisfactorily inactivated by phenylglyoxal at p H 8.0 in 50 mM borate buffer, but was inactivated in 0.1 M N-ethylmorpholine buffer. Incubation of acylphosphatase with phenylglyoxal in the latter buffer resulted

A mino-acid residues modified by phenylglyoxal Amino-acid residues modified by phenylglyoxal were examined by amino-acid analysis. Table I shows the result of amino-acid analysis on the enzyme modified with 6 m M phenylglyoxal at p H

237 TABLE I AMINO-ACID COMPOSITIONS OF THE CONTROL A N D PHENYLGLYOXAL-TREATED ENZYMES The enzyme (36.5 /~M) was incubated for 60 min with 6 mM phenylglyoxal and the modified enzyme was processed for amino-acid analysis as described under Materials and Methods. The number of residues are calculated on the basis of 8 Asx residues per tool enzyme monomer. In parentheses the aminoacid composition of porcine acylphosphatase obtained from its amino-acid sequence [3] is shown. The results shown are mean values of three different experiments. The mean deviation of the value for each amino acid was within + 4%. Amino acid

Enzyme control

Phenylglyoxaltreated enzyme

Asx Thr Ser Glx Pro Gly Ala Val Met lie Leu Tyr Phe Lys His Arg

8.00 6.57 11.53 10.41 2.79 8.34 2.07 8.73 1.98 4.60 3.10 3.91 3.66 9.13 0.00 6.93

8.00 6.58 11.23 10.52 3.03 8.06 2.09 8.77 1.92 4.71 3.09 3.98 4.09 9.21 0.00 3.09

(8) (7) (13) (10) (3) (8) (2) (10) (2) (5) (3) (4) (4) (9) (0) (7)

8.0 and 30°C for 60 rain, of which the residual activity was about 14% of the original activity. The arginine content decreased from 6.93 residues of the control to 3.09 residues of the modified enzyme. No significant change was observed in the content of other amino acids. Table II summarizes the numbers of unmodified arginine residues at various reaction times in the absence or presence of ATP (2.5 mM) determined by amino-acid analysis. The last column in Table II represents the number of arginine residues protected by ATP from the modification. At 60 rain of the reaction time, 3.91 and 2.19 arginine residues were modified in the absence and presence of ATP, respectively. Therefore, 1.72 of 3.91 arginine residues modified in the absence of ATP escaped from the modification in the presence of ATP. Using Pi instead of ATP as a protecting reagent, similar results were obtained.

Incorporation of [ H C]phenylglyoxal There are many reports that phenylglyoxal reacts with arginine residues in a 2 : 1 stoichiometry [6,8], but 1 : 1 stoichiometry [15], and the reversion of 2:1 adduct to 1:1 complex have also been reported [16]. Hence, the incorporation of [14C]phenylglyoxal to the enzyme was examined at various reaction times in the absence and presence

TABLE II N U M B E R O F A R G I N I N E RESIDUES MODIFIED BY PHENYLGLYOXAL AS D E T E R M I N E D FROM AMINO-ACID ANALYSIS Modification reactions and amino-acid analyses were performed as described under Materials and Methods. Mean values of three different experiments are shown. The numbers of arginine found were calculated by assuming that the native enzyme contained 8 Asx residues. The number of modified arginine residues was calculated by subtraction of number of arginine found from 7, which is the content of arginine residues in porcine muscle acylphosphatase. Reaction time

(min) 5 10 15 20 30 45 60

Number of arginines - ATP

+ ATP

modified

found

modified

found

modified

( - A T P ) - ( + ATP)

6.28 5.82 5.41 5.01 4.39 3.56 3.09

0.72 1.18 1.59 1.99 2.61 3.44 3.91

6.74 6.57 6.35 6.20 5.69 5.21 4.81

0.26 0.43 0.65 0.80 1.31 1.79 2.19

0.46 0.75 0.94 1.19 1.30 1.65 1.72

238 TABLE III NUMBER OF [14C]PHENYLGLYOXALS INCORPORATED Modification reactions and measurements of the radioactivity were performed as described under Materials and Methods. Reaction time (rnin)

Number of [14C]phenylglyoxalsincorporated

5 10 15 20 30 45 60

1.12 1.95 2.72 3.76 5.12 7.43 8.48

-

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0.55 1.02 1.47 1.93 2.84 4.18 4.88

0.57 0.93 1.25 1.83 2.28 3.25 3.60

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of ATP. The result is summarized in Table III. After 60 min reaction, 8.48 mol and 4.88 mol []4C]phenylglyoxal per mol acylphosphatase were incorporated into the enzyme in the absence and presence of ATP, respectively. The number of [14C]phenylglyoxals incorporated at each reaction time was between 1.6- and r2.4-fold, mostly about 2-fold, the number of modified arginine residues determined by amino-acid analysis. Therefore it is considered that in acylphosphatase two phenylglyoxals per one arginine residue reacted under the reaction conditions used. The number of arginine residues protected by ATP is shown in the last column. After 60 rain reaction, 3.60 mol phenylglyoxal per mol acylphosphatase were prevented from incorporation by the presence of ATP.

Relationship between inactivation and number of residues modified Fig. 2 shows the correlation of the residual enzyme activity with the number of arginine residues modified. The number of arginine residues modified was determined independently either by amino-acid analysis or by [14C]phenylglyoxal incorporation. The data obtained by the incorporation of [14C]phenylglyoxal fit a straight line up to about 90% loss of the original activity, and the intercepts of the line on the abscissa and on the ordinate are 2.0 residues and 100%, respectively. The data obtained by amino-acid analysis fit almost to a straight line up to 86% loss of the original activity, and the intercepts of the line on

1.0 2.0 AR~NINE RESIDUES MODIFIED Fig. 2. Correlation between residual enzyme activity and number of arginine residues modified. The number of residues modified was determined from amino-acid analysis (O) or from the incorporation of [14C]phenylglyoxal(O), assuming a stoichiometry of 2 mol phenylglyoxalper mol arginine.

the abscissa and on the ordinate are 2.3 residues and 100%, respectively.

Protection against inactivation by phenylglyoxal Some competitive inhibitors for the enzyme, that is, inorganic phosphate, ATP and CI-, were tested to determine whether they protect the enzyme from inactivation by phenylglyoxal or not. The enzyme (36.5 #m) was incubated with 6 mM phenylglyoxal in 0.1 M N-ethylmorpholine buffer (pH 8.0) for 60 min at 30°C in the absence or presence of one of the above inhibitors. The K~ values for these inhibitors, where K i is defined as the dissociation constant for inhibitor, were 0.20 mM, 22 /~M and 26 m M for Pi, ATP and CI-, respectively (unpublished data). At 125-times the K~ of each inhibitor, inorganic phosphate provided complete protection for 60 min of reaction, whereas A T P provided almost complete protection up to 30 min of reaction but had allowed about 10% decrease of the original activity at 60 min. NaCI provided little protection at 2 M (77-times Ki), but at 0.65 M (25-times K i) it caused a decrease in the rate constant from 0.033 to 0.013 m i n - ] .

239

To elucidate whether the active site is actually attacked by phenylglyoxai, the concentration dependencies of protecting effects of these inhibitors for the inactivation were examined. The rate of inactivation of the enzyme by phenylglyoxal in the presence of a competitive inhibitor may be treated in terms of the following equation, if the reagent reacts only with the free enzyme and not with the enzyme-inhibitor complex: ko 1+ []

k.pp

Kp

where kapp is the observed pseudo-first-order rate constant; k 0 is the rate constant without the inhibitor; K p is the dissociation constant of the enzyme-inhibitor complex. Then, Kp is obtained from a plot of 1 / k a p p v e r s u s concentration of inhibitor. Such a plot for inorganic phosphate is shown in Fig. 3 and a K 0 of 0.19 mM is obtained from the slope. This value is in fair agreement with a K~ of 0.20 mM obtained from the inhibition experiments of the enzyme. The effect on the inactivation with various concentrations of ATP was also examined. In a plot of fractions of the activity remaining versus reaction time on semilog paper, the experimental points nearly fitted to a straight line up to 30 min of reaction and then to a

curve whose slope increased slightly in negative sense with time. The kap p values were obtained from reaction times up to 30 min. The plot of 1/kap p versus concentrations of ATP gave a Kp of 15 #M, which agreed with a K i of 22/~M obtained from the direct inhibition experiments. The protcting effect on the inactivation with various concentrations of NaC1 is shown in Fig. 4. Full protection from the inactivation was not afforded with 0.65 M NaC1 (25-times the gi), where more than 96% of the enzyme was considered to exist as the enzyme-inhibitor complex, as calculated from the K i of 26 mM. The kapp at this concentration of NaC1 was 0.013 min - t and k 0 was 0.033 min -1. As shown in Fig. 4, at 13, 26 and 52 mM of NaC1, the experimental points fell on the theoretical curves calculated on the assumption that the enzyme-inhibitor complex (the concentration being calculated with K i = 26 mM) reacts with the reagent at a rate constant of 0.013 min -1 and the free enzyme at 0.033 min-1. Thus, the binding of CI- at the active site produces a decrease in the

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Pi (raM) Fig. 3. Effect of inorganic phosphate on inactivation of acylphosphatase by phenylglyoxal. The enzyme (36.5/~M) was incubated with 6 mM phenylglyoxal in 0.1 M N-ethylmorpholine buffer (pH 8.0) for 60 min at 30°C in the absence or presence of inorganic phosphate. At specified times, aliquots of the reaction mixture were removed for measurements of the enzyme activity.

Fig. 4. Time-course of inactivation of acylphosphatase by phenylgloxal in the presence of NaC1. The inactivation reaction was carried out as described in Fig. 3 except for the presence of various concentrations of NaCI instead of inorganic phosphate: none (e), 13 (Q), 26 (&), 52 (zx) and 650 (O) raM. The broken lines are calculated from the equation , A / A o = ([Ef]e -°'°33t +[EI]e-°°13t)AEt], where A / A o is the fraction of activity remaining, and [Et], [Et] and [EI] are the concentrations of total enzyme, free enzyme and enzyme-inhibitor complex, respectively. [Ef] and [El] are calculated with Ki(NaCI)= 26 mM.

240 rate constant of the inactivation reaction, but does not prevent the inactivation reaction.

CD spectra of modified acylphosphatase CD spectra of the native enzyme, the enzyme incubated with phenylglycoxal for 30 rain, and that for 60 rain in the presence of ATP were similar. But CD spectrum of the enzyme incubated with phenylglyoxal for 60 min in the absence of ATP was slightly different from that of the native enzyme (data not shown). The contents of a-helix, fl-structure and random coil were 6.7%, 42.4% and 50.9%, respectively, for the native enzyme, while those were 6.5%, 35.3% and 58.2%, respectively, for the enzyme modified for 60 min in the absence of ATP. Discussion

Phenylglyoxal reacts with arginine residues in a highly selective manner, but it reacts with the a-amino groups of proteins and lysine residues, and possibly, in addition, with cysteine residues [8,17]. There is no possilJility for the a-amino group of porcine muscle acylphosphatase to be modified, for it is acetylated [3]. The enzyme contains one cysteine residue and partially forms the dimer even in the presence of 10 mM 2mercaptoethanol at acidic pH [10]. At alkaline pH, where the modification reaction is usually carried out, the dimer forms more easily than at acidic pH. Hence, the prepared dimer was used for the modification. The specific activities and their pH dependencies of the monomer and the dimer enzymes were similar (unpublished data). The pseudo-first-order loss of the enzyme activity induced by phenylglyoxal indicates that there is no cooperativity in the reaction of the reagent with arginine residues [18]. It is considered that the reaction of arginine with two phenylglyoxal molecules proceeds in two steps: arginine is attacked by the first molecule of the reagent (this is the possible rate-limiting step) followed by addition of the second molecule [8]. The kinetics of the inactivation of acylphosphatase was first order with respect to phenylglyoxal concentration. On the other hand, the stoichiometry for the reaction was 2 tool phenylglyoxal per mol arginine residue. Therefore it is considered that the binding of one molecule of

phenylglyoxal to each essential arginine residue is sufficient for the inactivation of the enzyme. In the plot of the activity remaining versus the number of arginine residues modified in acylphosphatase, the data nearly fit a straight line whose intercept on the abscissa is about two residues. When in such a plot a straight line results, its intercept on the abscissa gives the maximum number of essential residues [19]. Therefore, one or two arginine residues are probably essential in acylphosphatase. When two arginine residues are essential, the above plot does not show a straight line. Only when one of two arginine residues is essential and rate constants for the modification reaction of the two arginine residues are similar, does the above plot show a nearly straight line of which the intercept is about 2. Therefore it is likely that in acylphosphatase one arginine residue is essential but the other is not, and their rate constants for the modification reaction are similar. In a number of enzymes, arginine residues have been shown to serve as a cationic site in the binding of a negatively charged group of substrate or coenzyme. Porcine muscle acylphosphatase was competitively inhibited by various anions, such as inorganic phosphate, ATP and C1-. Among these inhibitors, inorganic phosphate and ATP at saturating level produced total and nearly total protection, respectively from the inactivation. In addition, the dissociation constants obtained from the protection experiments by these inhibitors agreed well with those obtained from the ordinary inhibition experiments. On the other hand, CIfailed to produce total protection. The concentration dependency of the protecting effect of the inhibitor showed that phenylglyoxal was able to attack the essential arginine residue of the enzymeC1- complex. These results support the concept that the essential arginine residue in acylphosphatase serves as a cationic site for the negatively charged phosphate moiety of the substrate. About 7% decrease in fl-structure was observed in the enzyme-modified four arginine residues. Thus, the modification of arginine residues caused a decrease in fl-structure. However there was little structural change in the enzyme modified for 30 rain and only about 7% decrease in fl-structure in the enzyme which had lost 86% of its original

241

activity. Therefore, it seems unlikely that the inactivation is due mainly to denaturation of the enzyme resulting from the modification of the non-essential arginine residues. Porcine muscle acylphosphatase contains seven arginine residues, at positions 4, 16, 23, 31, 74, 77 and 97 [3]. In comparison of the primary structure of porcine muscle acylphosphatase with those of other three muscle acylphosphatases, two arginine residues in the porcine enzyme are substituted in the others, that is, Arg-4 for Gly and Arg-31 for Lys [1-4]. Therefore, Arg-4 can not be the essential arginine residue and Arg-31 is not likely to be the essential arginine residue. Some acylphosphate-hydrolyzing enzymes are known other than acylphosphatase [20,21]. Among these, a consensus sequence of (Ser or Thr)-X-Thr-Thr in oxidized glyceraldehyde-3-phosphate dehydrogenase and Ca 2+-dependent ATPase is possibly related to their hydrolytic activity for acylphosphates. There is a consensus sequence of (Ser or Thr)-(X, XX or XXX)-(Lys or Arg) for the phosphate-binding site in a number of phosphorylated proteins and enzymes which bind phosphate compounds [22,23]. In addition, in glyceraldehyde-3-phosphate dehydrogenase there are two sequences of (Arg or Lys)-X-X-Arg which have been implicated as possible binding sites for phosphates of NAD + [24]. In the amino-acid sequence of porcine acylphosphatase there was a sequence containing the above sequences, that is, Ser-Pro-Ser-Ser-ArgIle-Asp-Arg (from 70 to 77). Accordingly, Arg (74) or (77) is a possible arginine residue responsible for the inactivation of acylphosphatase by phenylglyoxal.

Acknowledgements We wish to thank Dr. F. Morita of the Department of Chemistry, Faculty of Science, Hokkaido University, for measurements of CD spectra and for helpful advice.

References 1 Cappugi, G., Manao, G., Camici, G. and Ramponi, G. (1980) J. Biol. Chem. 255, 6868-6874 2 Camici, G., Manao, G., Cappugi, G., Berti, A., Stefani, M., Liguri, G. and Ramponi, G. (1983) Eur. J. Biochem. 137, 269-277 3 Mizuno, Y., Kizaki, T., Takasawa, T. and Shiokawa, H. (1985) J. Biochem. (Tokyo) 97, 1135-1142 4 Kizaki, T., Takasawa, T., Mizuno, Y. and Shiokawa, H. (1985) J. Biochem. (Tokyo) 97, 1155-1161 5 Ramponi, G., Manao, G., Camici, G. and White, G.F. (1975) Biochim. Biophys. Acta 391, 486-493 6 Riordan, J.F. (1979) Mol. Cell. Biochem. 26, 71-92 7 Ramponi, G. (1975) Methods Enzymol. 42, 409-426 8 Takahashi, K. (1968) J. Biol. Chem. 243, 6171-6179 9 Camici, G., Manao, G., Cappugi, G. and Ramponi, G. (1976) Experientia, 32, 535-536 10 Mizuno, Y., Takasawa, T. and Shiokawa, H. (1984) J. Biochem. (Tokyo) 96, 313-320 11 Ramponi, G., Treves, C. and Guerritore, A. (1966) Experientia 22, 705-706 12 Penefsky, H.S. (1979) Methods Enzymol. 56, 527-530 13 Patthy, L. and Smith, E.L. (1975) J. Biol. Chem. 250, 557-564 14 Chen, Y., Yang, J.T. and Chan, K.H. (1974) Biochemistry 13, 3350-3359 15 Borders, C.L., Jr. and Riordan, J.F. (1975) Biochemistry 14, 4699-4704 16 Viale, A.M., Andreo, C.S. and Vallejos, R.H. (1982) Biochim. Biophys. Acta 682, 135-144 17 Takahashi, K. (1977) J. Biochem. (Tokyo) 81, 403-414 18 Ray, W.J. and Koshiand, D.E., Jr. (1961) J. Biol. Chem. 236, 1973-1979 19 Stevens, E. and Colman, R.F. (1980) Bull. Math. Biol. 42, 239-255 20 Ehring, R. and Colowick, S.P. (1969) J. Biol. Chem. 244, 4589-4599 21 Rega, A.F. and Garrahan, P.J. (1976) in The Enzymes of Biological Membranes (Martonosi, A., ed.), Vol. 3, pp. 303-314, Plenum Press, New York 22 Houghton, J.E., Bencini, D.A., O'Donovan, G.A. and Wild, J.R. (1984) Proc. Natl. Acad. Sci. USA 81, 4864-4868 23 Schulz, G.E. and Schirmer, R.H. (eds.) (1979) Principles of Protein Structure, pp. 224-226, Springer, New York 24 Harris, J.I. and Waters, M. (1976) in The Enzymes (Boyer, P.D., ed.), Vol. 13, pp. 1-49, Academic Press, New York