purification, enzymatic properties, and active site environment

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Pnnted in U.S.A. Vol. 256, No. 20, Issue of October 25, pp. 10664-10670, 1981

Purification, Enzymatic Properties, and Active Site Environment of a Novel Manganese(II1)-containing Acid Phosphatase*

(Received for publication, March 9, 1981)

Yukio SugiuraS, Hideo Kawabe, Hisashi Tanaka, Sadaki F’ujimoto, and Akira Ohara From the Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan and Kyoto College of Pharmacy, Kyoto 607, Japan

A new manganese-containing acid phosphatase has been isolated and crystallized from sweet potato tubers. The pure enzyme contains one atom of manganese per M. = 110,000 polypeptide and shows phosphatase activity toward various phosphate substrates. The pH optimum of the enzyme was 5.8 and the enzyme activity was inhibited by Cu2+, Zn2+, H8+, A s O ~ ~ - , and Moo4’-. This stable metalloenzyme is red-violet in color with an intense absorption band at 515 nm ( E = 2460). Our electronic, circular dichroism, and electron spin resonance findings strongly indicate that the Mn-valence state of the native enzyme is trivalent. When the Mn- enzyme is excited by the 5145 A line of Ar+ laser, prominent Raman lines at 1230, 1298, 1508, and 1620 cm” were detected. This Raman spectrum can probably be interpreted in terms of internal vibration of a coordinated tyrosine phenolate anion. The tryptophan- modified enzyme showed a positive Raman band at 370 cm”, which is preferentially assigned to a Mn(III)-S streching mode. The modification of the Mn-enzyme by N-bromosuccinimide led to a large decrease in the fluorescence intensity at 335 nm which was dominated by its tryptophan residues within a considerable hydrophobic environment. The acid phosphatase activity was significantly decreased by the tryptophan modification. With respect to the active site donor sets, the Mn(II1)- containing acid phosphatase is distinctly different from the Zn(I1)-containing alkaline phosphatase. Of interest is also the appreciable similarity of some enzymatic and spectroscopic properties between the present enzyme and uteroferrin.

Acid phosphatases (EC 3.1.3.2) are a group of enzymes which catalyze the nonspecific hydrolysis of phosphate mon- oesters and oxygen exchange from water into inorganic phosphate in an acidic environment (1). The enzymes are widely distributed in mammalian body fluids and tissues, plants, and microorganisms. In particular, the human prostate is rich in this enzyme and its serum enzyme levels are a useful tool in the diagnosis and management of prostatic diseases (2). How- ever, the structural and molecular properties of acid phosphatase are remarkably obscure, compared with those of alkaline phosphatase. Recently, the iron-containing acid phosphatase from pig allantoic fluid was purified to homogeneity and the violet metalloenzyme showed an absorption maximum near

* This study was supported in part by a grant from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom all correspondence should be addressed at the Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan.

550 nm (E = 2000) (3). Campbell et al. (4) have suggested that the violet iron-containing acid phosphatases are widespread from the investigation of the pig allantoic fluid, beef spleen enzymes, and several other acid phosphatases. We also found a novel acid phosphatase containing manganese rather than iron from sweet potato (Kintoki) and communicated the metal-binding properties (5, 6). The stable metalloenzyme is violet in color with an intense absorption band at 515 nm (E = 2460). The presence of similar violet acid phosphatases has been reported in sweet potato (Kokei No. 14), soybean, and other plant sources (7, 8).

Although manganese is ubiquitous in nature with apparent levels comparable to those of copper, only a few metalloen- zymes have a tightly bound manganese. Typical examples include pyruvate carboxylase (9), superoxide dismutase (lo), and diamine oxidase (11). In addition, it is known that the manganese-containing superoxide dismutase isolated from bacterial sources resembles the iron-containing superoxide dismutase from bacterial and algal sources in subunit molecular weight, amino acid content, and cyanide insensitivity (12).

In this paper, the purification, enzymatic and spectroscopic properties, and Mn(II1)-active site environment of new manganese(II1)-containing acid phosphatase from sweet potato (Kintoki) have been characterized in detail and compared with those of zinc(I1)-containing alkaline phosphatase.

EXPERIMENTAL PROCEDURES

Materials-Sweet potato (Kintoki) tubers were purchased from Chiba agricultural union, Japan. Sephacril S-200, DEAE-Sephacel, and blue dextran were obtained from Pharmacia Fine Chemicals, and hydroxylapatite was prepared according to the procedure of Siegel- man et al. (13). AU other reagents were the highest quality available and deionized water was used throughout the experiments.

Enzyme Assay-Enzymatic activity was estimated by the method of Neil and Horner (14) with a minor modification. Phosphatase activity was normally assayed by incubating samples of the protein and 1 mM p-nitrophenyl phosphate in 0.1 M acetate buffer (pH 5.8). The total reaction volume was 3.2 ml and the incubation temperature 37 “C. At the end of the incubation (10 min), 1 ml of 0.5 N NaOH was added and the extinction was read at 410 nm; p-nitrophenol was used as a standard. The velocity of kinetic studies was easily calculated by this assay method. The reaction rate was linear over 15 min, meaning the enzyme was completely active over this time period and no significant product inhibition or substrate depletion occurred. Indeed, the Mn-enzyme was stable at room temperature during a few days. Phosphatase activity toward other substrates was determined by measuring the release of orthophosphate. The experimental conditions were as follows: incubation time, 10 min; temperature, 37 “C; buffer, acetate buffer (pH 5.8); and total volume, 1.6 ml. The reaction was stopped by the addition of 3.5 ml of 0.6 N H2S04.

Biochemical Methods-Protein concentrations were estimated by both the Lowry procedure (15) and ultraviolet absorption method (E:P = 18.7 at 280 nm). Polyacrylamide gel-disc electrophoresis was performed in a discontinuous system according to the method of Reisfeld et al. (16). The buffer used was diethylbarbituric acid/Tris

10664

Novel Mn(IIJkontaining Acid Phosphatase 10665

system (pH 7.6) and current was applied from a constant voltage power supply at 180 V for 70 min. Sodium dodecyl sulfate-acrylamide gel-disc electrophoresis was carried out in the presence of 7.5% acrylamide gels by the method of Weber and Osborn (17). The gels were stained with 0.25% Coomassie brilliant blue and destained in 7 8 acetic acid. Isoelectric points were determined by isoelectric focusing according to the procedure of Vesterberg and Svensson (18). The apparatus of an LKB Instrument was used and the pH range from 4 to 6 was adjusted with ampholine.

Physicochemical Methods-Electronic absorption and circular dichroism spectra were measured with a Hitachi 330 spectrophotometer and a JASCO 5-20 spectropolarimeter, respectively. Fluorescence spectral measurements were carried out with a Shimadzu RF-500 spectrofluorometer. Resonance Raman spectra were recorded on a JEOL-400 Raman spectrometer. X-band electron spin resonance measurements were performed using a JES-FE-3X spectrometer op- erating with a 100-kHz magnetic field modulation. Atomic absorption determination was carried out with a Shimadzu AA-630-01 type apparatus.

Enzyme Purification-Sweet potato (Kintoki) tubers were crushed in a juicer and compressed with a hand press to obtain the juice. Cold acetone was added gradually to the sweet potato juice until a final concentration of 258, v/v. The mixture was centrifuged and more cold acetone was added to the supernatant until a final concentration of 45% was reached. The precipitate collected by centrifugation was suspended in 0.01 M phosphate buffer (pH 7.0) containing 0.01 M EDTA (buffer 1) and it was dialyzed overnight against the same buffer. The dialyzed solution was brought to 35% saturation by the addition of solid ammonium sulfate (23 g/100 ml of solution). After 30 min, the precipitate was removed by centrifugation and the supernatant was brought to 70% saturation with ammonium sulfate. The precipitate, containing most of the phosphatase activity, was collected by centrifugation after 60 min and redissolved in buffer 1. To the solution, ammonium sulfate was added up to 35% saturation and then the pH of the solution was lowered to 4.0 by the addition of 2 N HCI. After stirring for 5 min, the precipitate was removed by centrifugation. The resulting supernatant was adjusted to pH 6.0 with 0.1 M KOH and brought to 80% saturation by the addition of solid ammonium sulfate. After 60 min, brown-colored precipitate was collected by centrifugation and redissolved in buffer 1. The enzyme-containing solution was applied to a column of Sephacril S-200 equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 1.0 mM EDTA (buffer 2). The collected fraction, containing phosphatase activity, was applied to a column of DEAE-Sephacel equilibrated with phosphate buffer 2. The obtained red-violet-colored fraction was dialyzed against 2.5 mM phosphate buffer (buffer 3) and then was applied to a column of hydroxylapatite equilibrated with buffer 3. The column was developed with a linear gradient of increasing phosphate concentration at pH 7.0. After the red-violet solution of acid phosphatase was dialyzed against 0.01 M phosphate buffer (pH 6.0) containing 0.1 mM EDTA (buffer 4), a DEAE-Sephacel (pre-equilibrated with buffer 2) chromatography was again carried out. Elution was made with a linear gradient of increasing NaCl concentration (0-0.6 M) at pH 6.0. The enzyme solution was collected and concentrated in a collodion bag under reduced pressure. The homogeneity of the enzyme preparation was examined by polyacrylamide gel electrophoresis at pH 7.6. It gave a single protein band (see Fig. 1). The enzyme was also homo- geneous as judged by electrophoresis on cellulose acetate strips a t various pH values. The typical purification scheme is summarized in Table I. In addition, crystallization readily occurred when sucrose (40%) was added and the solution was allowed to stand at 4 "C. Lozenge-shaped and violet-colored crystals grew within 1 week on the sides of the test tubes (see Fig. 2).

RESULTS AND DISCUSSION

Amino Acid Composition, Molecular Weight, and Man- ganese Content-Duplicate samples for amino acid analysis were hydrolyzed in uacuo for 24 and 48 h at 110 "C in 6 M HCl containing 0.1% phenol. The analyses were performed with a Hitachi 835 amino acid analyzer. Tryptophan content was determined by the spectrophotometric method of Edel- hoch (19). The result is shown in Table 11. The enzyme is considerably rich in the acidic amino acids aspartic and glutamic acids. Although the present result indicates that the enzyme has three half-cystine residues (1) three cysteine or 2) one cysteine and one cystine residues), the cysteine determi-

1

FIG. 1. Polyacrylamide gel electrophoresis of the purified acid phosphatase. Electrophoresis was carried out in a 7.5% gel a t pH 7.6 and stained with Coomassie brilliant blue.

TABLE I Purification of Mn(ZZZ)-containing acidphosphatase from sweet

potato (Kintoki) Total Total Specific Recov- Purification step and fraction orotein activitv activitv erv

mc?? units units/mg 90 1. Crude juice 3,059,700 292,480 0.096 100 2. Acetone fractionation 565,840 253,964 0.448 87 3. Ammonium sulfate frac- 34,710 132,660 3.82 45

4. Acid treatment 19,020 120,009 6.30 41 5. Sephacril S-200 chroma- 6,832 93,940 13.75 32

6. DEAE-Sephacel chro- 1,159 45,750 39.4 16

7. Hydroxylapatite chro- 604 30,225 50.0 10

8. DEAE-Sephacel chro- 514 26,900 52.4 9

tionation

tography

matography

matography

matography

nation with the Ellman reagent (20) showed that no free sulfhydryl groups were detected for the native enzyme, but 1 mol of -SH/mol of enzyme was determined for the denaturated enzyme obtained by the treatment of 6 M guanidine hydrochloride.

The molecular weight calculated from the present amino acid composition was in good agreement with the value observed by the gel chromatographic technique (21). Indeed, the molecular weight of the enzyme was estimated to be 110,OOO by Sephadex G-200 column chromatography, in which p-am- ylase, aldolase, serum albumin (bovine), ovalbumin, and chy- motrypsinogen A were used as standards. In polyacrylamide

10666 Novel Mn(II&containing Acid Phosphatase

FIG. 2. Crystals of the purified Mn(III)-containing acid phosphatase. These crystals are approximately 0.2 mm in length and are reddish violet in color.

TABLE I1 Amino acid composition of Mn(ZZ4-containing acidphosphatase

from sweet potato (Kintoki) Amino acid Amino acid content

Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Proline Tryptophan Glucosamine

mol/mol enzyme 124.0 (124) 47.9 (48) 65.9 (66) 94.3 (94) 79.9 (80) 85.7 (86) 54.2 ( 5 4 ) 2.7 (3) 7.1 (7)

37.8 (38) 51.1 (51) 61.5 (62) 41.3 (41) 41.8 (42) 22.1 (22) 37.2 (37) 57.3 (57) 23.5 (24) 13.2 (13)

Total residues (936)

gel-disc electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate with P-mercaptoethanol (22), the present enzyme showed a single protein band correspond- ing to a molecular weight of about 55,000. Thus, we may conclude that the Mn-containing acid phosphatase is com- posed of two subunits of M, = 55,000.

Determination of manganese was carried out by the atomic absorption method. Sample solutions were extensively dialyzed against 5 mM Tris-HC1 buffer containing 0.5 mM EDTA. The enzyme contained manganese which corresponds to one atom of Mn per enzyme molecule. The tetrameric superoxide dismutase from Themus aquaticus is also known to contain two atoms of manganese (23). The isoelectric point of the present Mn-enzyme was pH 5.18 and the specific extinction coefficientEy,, (287 nm) was 18.7. In addition, the pH optimum of the Mn-containing acid phosphatase was 5.8.

Substrate Specificity and Inhibitors-The phosphatase activity of the Mn-containing enzyme toward various phosphate substrates is listed in Table 111. Although there was appreciable activity toward fructose 1,6-diphosphate, P-glycophos- phate, 3'-AMP, NADP', and pyrophosphate, the compounds such as 5'-AMP, 2'-AMP, 5'-CMP, and NAD' were hydrolyzed only poorly, suggesting that these are not natural substrates for the enzyme.

Table IV summarizes the inhibitive effect of various substances on the Mn-containing acid phosphatase activity. Sev- eral transition metals, Cu2+, Zn", and Hg'+, were potent inhibitors for the present Mn-containing enzyme. Fluoride, arsenate, and particularly molybdate strongly inhibited the phosphatase activity. On the other hand, Na+, K', Ca"', Cr"+, Co2+, C1-, Br-, and I- had very little effect. The effect of L- (+)-tartaric acid, an inhibitor of prostate acid phosphatase (I), was also small. Levamisole, which is a typical inhibitor for alkaline phosphatase (24), had no effect on the enzymatic activity of the Mn-containing acid phosphatase. In contrast, N-bromosuccinimide markedly affected this enzyme activity, suggesting significant contributions of the tryptophan residues toward the acid phosphatase reaction.

Table V shows the inhibition mode and the Ki values of some potent inhibitors. The competitive inhibition by MOO^^-, AS():-, and Poi'- suggests that these anions presumably act as the substrate analogue. Although both Hg2+ and p-chloro-

TABLE I11 Substrate specificity of Mn(ZZZ))-containing acidphosphatase from

sweet potato (Kintoki) The assay mixture contained 320 pmol of sodium acetate buffer

(pH 5.8), 3 pmol of substrate, and 0.02 pg of native enzyme in a total volume of 1.6 ml. After 10 min at 37 "C, a 3.5-ml solution of 0.6 N H~SOI was added to 0.5 ml of the sample. Orthophosphate was determined colorimetrically. The enzyme activity was expressed as a per cent of that of the native enzyme for p-nitrophenyl phosphate (=loo). The K,,, value determined with p-nitrophenyl phosphate was 1.7 x 1 0 - ~ M.

Compound Relative rate of hydrolysis

%

p-Nitrophenyl phosphate 100 Glucose 6-phosphate 33 Fructose 6-phosphate 29 Fructose 1.6-diphosphate 69 Ribose 5-phosphate 25 a-Glycerophosphate 48 /&Glycerophosphate 69 5'-ATP 38 5'-ADP 15 5"AMP 9 3"AMP 60 2'-AMP 6 5'-CMP 3 NADP' 68 Pyridoxal phosphate 38 FMN NAD'

35 0

Diphenyl phosphate 0 Pyrophosphate 93

Novel Mn(III)-containing Acid Phosphatase 10667

TABLE IV Inhibitive effect of various substances for acidphosphatase activity

Substance (1 mM) activitv

Substance (1 mM)

Relative activitv

%

Na' 100 K+ 100 Mg2+ 111 Ca2+ 100 CrZ+ 100 Cr3+ 100 Mn2+ 81 Fe2+ 58 Fe3+ 38 GO2+ 98 CU2+ 2 Ag+ 88 Zn2+ Cd2+

21 92

Hg2+ 0

F- c1- Br- I-

As02- Moo4'- p-Chloromercuribenzoic acid Benzoic acid L-(+)-Tartaric acid D-(-)-Tartaric acid Bromosuccinimide Levamisole Acetylacetone Pyridine

~ 0 ~ 3 -

% 50 94

100 100 80 28 0

65 90 92 90 0

100 89 57

TABLE V Effect of inhibitors on the acid phosphate activity

Inhibitor Mode of inhibition K,

Hgz+ M004'- AsOd3- F- p-Chloromercuribenzoic

acid Pod3- Acetylacetone

M

Noncompetitive 1.9 x Competitive 1.1 x Competitive 5.0 X lo-' Noncompetitive 3.0 X 10-~ Competitive 7.1 X

Competitive 2.3 X Competitive 1.2 x

mercuribenzoate are typical "SH reagents, their inhibition modes were different. The competitive fluoride inhibition has been observed in human prostate acid phosphatase (25). In the present Mn-containing acid phosphatase, however, fluoride ion inhibited noncompetitively. Acetylacetone, a Mn(II1)- chelating agent, was also a competitive inhibitor for the present enzyme. The Yonetani-Theorell plot (26) in the multiple inhibition experiment by POZ- and p-chloromercuribenzoate gave infinity as the interaction constant of these two inhibitors, indicating that the two compounds compete for the same site of the Mn-enzyme.

Electron Spin Resonance Features-Fig. 3 exhibits the X- band ESR spectra at 293 K for the native Mn-enzyme (A) and the denaturated one (B). No ESR signals were detected with the native violet enzyme at 293 and 77 K. In contrast, the acid- and heat-treated colorless enzyme showed characteristic six-line ESR patterns due to the aquated Mn(I1) ion (55Mn, I = 5/2) around g = 2.0. The result is reasonable if it is assumed that the violet chromophore represents an ESR-silent form of the metal, Mn(II1). In the case of S = 2 or S = 1, if the zero field splitting is sufficiently large, normal laboratory fields may not be large enough to bring the transition (between S, = 0 and S, = +1) within the energy range of the applied X- band microwave radiation. Indeed, similar ESR behavior has been observed in the Mn-containing superoxide dismutase and Fee et al. (27) reported that the absence of an observable ESR signal is quite characteristic of a Mn(II1) integral spin system with zero field splitting of 1-2 cm". An alternative interpretation for the loss of ESR signal is that Mn(I1) is tightly bound to protein and thereby immobilizes within its ligand field. In such a case, however, the intensely visible band would not appear.

Electronic and Circular Dichroism Spectra-The violet Mn-enzyme gave an absorption maximum at 515 nm (E = 2460) and CD extremum at 55 nm ( A E -0.53), respectively (see

Fig. 4). The ratio of AE to E is 2.1 X for the characteristic visible band. In a rough approximation, the following equation can be utilized to estimate Kuhn's anisotropic factor (28), y = IAE/EI, where AE and E are C D ( E L - ER) and optical absorption in terms of extinction coefficients, respectively. The ratio is typically s10-' for magnetically allowed and electrically forbidden transition of the d-d type. Therefore, the intense 515 nm band is assigned to an electrically allowed charge-transfer band from the ligand to the metal, which is expected in Mn(III)(d) rather than Mn(II)(d5). Indeed, the native enzyme showed the band at 1160 nm in the near- infrared region which can be assigned to a transition 'A + 5B of high spin Mn(II1) (29). The extinction coefficient of the Mn-enzyme is significantly larger than that of the Escherichia coli Mn-superoxide dismutase (X,,, = 473 nm and E = 400) (30) and is close to that of Mn(II1)-transferrin (X,,, = 430 nm and E = 4400) (31) due to charge-transfer interaction with the protein tyrosine residues.

As seen in Fig. 5, the enzyme activity is reduced in parallel with a decrease in the 515 nm absorption attributed to the Mn ion directly coordinated with some amino acid residues.

FIG. 3. ESR spectra of the native enzyme (A) and the denatured enzyme (B) at 20 "C. B was obtained by mixing A (0.5 mM; 0.2 ml) in water with HCl (1.0 M; 0.05 ml) and then heating to 100 "C for 3 min.

3000 \ c3.0

- i

1 I 1 I I J -2.0 200 300 400 5 00 600 700

Wavelength, m

FIG. 4. Electronic and circular dichroism spectra of Mn(II1)- containing acid phosphatase at pH 6.8.

10668

0.X

2 0.1s Ln ri Ln

+ 0 u c

D h :: 0.18 e 4

0.17

Novel Mn(II4-containing Acid Phosphatase

70 80 90 100

Relative activity,

FIG. 5. Correlation between enzymatic activity and absorbance at 515 nm in Mn(II1)-containing acid phosphatase. Every 10 min after the treatment of native acid phosphatase (0.8 mM) with 6 M guanidine HC1, the absorbance at 515 nm and the enzymatic activity for p-nitrophenyl phosphate were determined.

1600 1400 1200 c m"

FIG. 6. Resonance Raman spectrum of Mn-containing acid phosphatase. The sample concentration was 0.5 mM and the spectrum was measured at pH 6.8 and 4 "C. Instrumental conditions were as follows: excitation, 514.5 nm line of Art laser; power, 20 milliwatts at a sample point; time constant, 16 s; slit width, 200 pm; scan speed, 10 cm"/min.

The result strongly indicates that the Mn ion present in the enzyme plays an essential role in the catalytic hydrolysis reaction by the Mn-containing acid phosphatase. Indeed, the Mn-removed apoenzyme was catalytically inactive.

Resonance Raman Characteristics-Fig. 6 shows the resonance Raman spectrum excited by the 5145 8, line of argon ion laser for the native Mn-containing acid phosphatase. The native enzyme exhibited prominent Raman lines at 1230,1298, 1508, and 1620 cm". Repeated experiments established that these Raman bands are indeed reproducible. These appear to be in resonance with the visible band and are probably due to internal vibrations of the coordinated amino acid residue toward Mn ion. The most striking feature of the resonance Raman spectrum of the Mn-containing acid phosphatase is the remarkable similarity of it to the spectra reported for Mn(II1)-ovotransferrin (1173, 1264, 1501, and 1603 cm") (31), Fe(II1)-transferrin (1174, 1288, 1508, and 1613 cm") (321, protocatechuate-3,4-dioxygenase (1177, 1265, 1505, and 1605 cm") (33), uteroferrin (1173, 1293, 1504, and 1607 cm") (34), andp-cresol-Fe(II1) complex (1180,1222,1488, and 1618 cm-l) (33). In these Mn(II1) and Fe(II1) complexes,the four characteristic Raman lines have been assigned to the vibration of

the coordinated phenolate anion. On the basis of the chemical similarity between Mn(II1) and Fe(II1) ions, the present Ra- man spectrum of the Mn-enzyme is interpreted in terms of the internal vibration of a coordinated tyrosine phenolate anion. Indeed, the amino acid composition revealed that the violet enzyme has a considerable abundance of the tyrosine residues.

Fig. 7 compares resonance Raman spectra for the N-bromosuccinimide-treated enzyme (A) and the native enzyme (B) in the region between 300 and 400 cm", obtained with 5145 A excitation. The Raman bands of the native enzyme are obscured by the fluorescent background. On the other hand, the tryptophan-modified enzyme showed a positive band at 370 cm". The typical Raman line at 370 cm" is preferentially assigned to a Mn(II1)-S streching mode, although other metal- ligand streching modes such as Mn-O(pheno1ate) have not been excluded conclusively. Such a Mn(II1)-S streching mode at approximately 370 cm" has been reported in the infrared spectrum of the tris(njA"diethy1 dithiocarbamato)Mn(III) complex (35). In addition, symmetric streching vibrations of sulfhydryl sulfur to Fe(II1) bonds have been assigned at 315- 365 cm-l in iron-sulfur proteins (36) and synthetic iron-sulfur clusters (37). The resonance Raman data are consistent with the sulfhydryl reagent effects. Therefore, the characteristic 515 nm absorption in the present Mn-enzyme is attributed to a charge-transfer transition of tyrosine ligand ( plr) + Mn(d) and cysteine (u or T) + Mn(d) on the basis of the enhancement of tyrosine ligand modes and Mn-S vibration in the resonance Raman spectra.

400 350 300 c m"

FIG. 7. Resonance Raman spectra of the tryptophan-modified enzyme (A) and the native enzyme (8). The sample concentration was 0.5 mM and the spectra were measured at pH 5.8 and 20 "C (A) and at pH 6.8 and 4 "C ( B ) . Instrumental conditions were as follows: excitation, 514.5 nm line of Ar+ laser; power, 40 milliwatts (A) and 20 milliwatts ( B ) at a sample point; time constant, 8 s (A) and 16 s (B) ; slit width, 250 pm (A) and 200 pm ( B ) ; scan speed, 10 cm"/min.

Novel Mn(III)-containing Acid Phosphatase 10669

Fluorescence Spectra-As shown in Fig. 8, maximal fluorescence intensity for the native Mn(II1)-containing enzyme occurred at 335 nm when excited at 280 nm, while that for the 6 M guanidine hydrochloride-treated enzyme was observed at 350 nm. Even if the excitation wavelength is varied from 270 to 295 n m , no evidence for significant tyrosine energy transfer was detected. In adrenal iron-sulfur protein (adrenodoxin), an anomalous emission (331 nm) of the tyrosyl residue at position 82 of the native protein has been reported (38). However, the apoadrenodoxin exhibited a normal tyrosine emission at 304 nm. Tryptophan fluorescence maximum can be expected to vary from 370 to 315 nm as the solvent polarity decreases (39). Thus, the fluorescence in this Mn(II1)-containing enzyme is dominated by its tryptophan residues. The 15 nm blue shift from the denaturated enzyme and standard N-acetyltrypto- phan methyl ester strongly indicates that several of the 24 tryptophans of the native Mn-enzyme must be within a considerably hydrophobic environment. Indeed, oxidation of the tryptophans by N-bromosuccinimide led to a disproportion- ately large decrease in the fluorescence intensity. Although the Mn(II1)-containing acid phosphatase activity was significantly decreased by the tryptophan modification with N-bromosuccinimide, the Mn(II1) optical activity at the typical 515 nm band was unaffected N-bromosuccinimide modification. The tryptophan oxidation also led to specific reduction in the 330 nm CD band, whereas ellipticity in the 550 nm region did not change. One can infer that the 330 nm transition is dominated by a charge-transfer transition markedly influ- enced by tryptophan residues. Therefore, the effect of the N- bromosuccinimide modification on the tryptophan fluorescence appears to reflect environmental and conformational changes in the active site locus, rather than direct quenching or intersystem crossing effect due to contact interaction between the Mn ion and tryptophans. Certainly, it is difficult to imagine how a tryptophan indole can be an endogenous ligand to the Mn(II1). A similar profound effect of tryptophan residues on the catalytic activity and fluorescence property has been demonstrated in galactose oxidase which contains one Cu(I1) ion and 18 tryptophans per enzyme molecule (40).

Mn(III)-active Site Environment-The present optical and

""""" "" -"" ""

I

3 0 0 350 4 00

Wavelength, MI

FIG. 8. Fluorescence emission spectra of the native enzyme (-), 6 M guanidine-treated enzyme (------) and N-bromosuccinimide-treated enzyme (- - -) in acetate buffer (pH 5.8). The protein concentration was 5 X 1O"j M and the excitation was at 280 nm.

ESR spectral data c o n f i i Mn(II1) (S = 2) for the valence state of the metal bound to the manganese-containing acid phosphatase. High spin Mn(II1) is subject to a considerable Jahn-Teller distortion which results in increased axial bond lengths, compared with high spin Fe(II1) complexes with regular octahedral structures. Taking into account the optimal pH region of the enzymatic reaction, it is reasonable that alkaline phosphatase and acid phosphatase require Zn(I1) and Mn(II1) (or Fe(II1)) ions, respectively. It is speculated that this enzyme uses Mn(II1) ion to induce effective binding of phosphate substrate and structural stability of the enzyme in an acidic environment. The resonance Raman and chemical evidence suggest at least tyrosine phenolate and cysteine sulfhydryl groups as the Mn(II1)-active site ligands. Since the cysteine sulfhydryl ligand is considerably a-donating toward metal ion, tyrosine phenolate ligand may be required for T-

back donation. With respect to the active site donor sets, the Mn(II1)-containing acid phosphatase is distinctly different from the Zn(I1)-containing alkaline phosphatase which con- sists of at least three histidyl imidazole nitrogen donors (41). In addition, the indole ring of tryptophan residues is one component of an active site cluster of hydrophobic side chains which is critical to the conformation of the entire acitve site. The N-bromosuccinimide modification experiments demonstrated the significant influence of the tryptophan groups on the Mn-environment. Recently, 31P NMR results provided the first unequivocal evidence for direct metal-phosphate interaction in alkaline phosphatase (42). By "P NMR and spin label ESR using 4-hydroxy-2,2,6,6-tetramethyl-piperidinoxyl dihydrogen phosphate, we must determine Mn(II1)-phosphate interaction in the present acid phosphatase. Of interest is also the appreciable similarity of some enzymatic and physicochemical properties between the present Mn(II1)-enzyme and uterofenin, an iron (111)-containing glycoprotein (34,43,44).

Acknowledgments-We are grateful to Dr. T. Kitagawa for resonance Raman measurements and Dr. K. Hayashi for amino acid analysis.

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