Site-directed Mutagenesis of Prostatic Acid Phosphatase

TKE JOURNAL OF B1aLoerc,u CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, h e

Vol. 269.No. 36, Issue of September 9, pp. 22642-22646, 1994 Printed in U.S.A.

Site-directed Mutagenesisof Prostatic Acid Phosphatase CAT&X”ICALLY I ~ P O R T ASPARTIC ~ T ACID258, SUBSTRATE SPECIFICITY, AND ~ L I ~ ~ E R I ~ A T I O ~ * (Received forpub~ication,March 9, 1994, and in revised form, May 7,1994)

Katja S. PorvariS, AnnakaisaM. HerralaS, Riitta M.KurkelaS, Piiivi A. TaavitsainenS, Ylva LindqvistO, Gunter SchneiderB,and PirkkoT.Vlhkoh From the $Biocenter Oulu and Department of Clinical Chemistry, University of Outs, Kajaanintie 50, FIH-90220 Udu, Finland and the 8Department of Molecular Biolom. Swedish University of Agricultural Sciences, Box 590, Biomedical Center, $-75224 Upisala, Sweden

At the active site of rat prostatic acid phosphatase brane proteins has been reported to be important for the fer(rPAP), residueAspm is a suitable candidate to as act an tilizing capacity of human sperm cells (11). It remains to be acidhase catalyst during phosphoester hydrolysis. It ascertained, whether hPAP is involved in the regulation of was changed to Am, Ser, and Ala by site-directed mu- fertility through its phosphotyrosine phosphatase activity (4, tagenesis. All these mutants were inactive, indicating 12-14). There have been several suggestions as to the natural that Aspzm may as acta proton donor in catalysis. TyrlZ3 substrate of PAP. It has been observed that the epidermal and ArglZ7 residues, located at the entrance of the active growth factor receptor is d e p h o s p h o ~ l a ~byd hPAP in uitro site surface in rPAP, are likely to be responsible for the (15).Another possible substrate is phosphocreatine, a high ensubstrate specificityof the enzyme. The corresponding ergy compound present in theseminal fluid (16). Prostatic 83residues in lysosomal acid phosphatase (LAP) are Lys kDa cytosolic protein, which is dephosphorylated at serine/ and Gly. In order to clarify the roles of the TyrlZand s threonine residues, is a further possible substrate of PAP (17). Argl” residues, lysosomal type rPAPmutants(Y123K, Moreover, it is known that PAP hydrolyzes phosphocholine(18R127G and Y123K,R127G) were generated. Sensitivity of Y123K,Rl27G to tartrate inhibition was similar to that 211, which is found in thereproductive system of several species. The three-dimensional structure of recombinant rPAP has observed in the case of LAP, indicating that these residues might be responsible for differencesin substrate been determined by us, using protein crystallographic methods specificity between the enzymes of prostatic and lysoso-(22, 23). The enzyme subunit consists of two domains, an a J @ mal origin. However, unlike humanW , the lysosomal domain and a smaller a domain. The topology of the former domain resembles the structure of phosphoglycerate mutase typemutantshydrolyzedthesuggestedPAP-specific (24). The dimer is formed through 2-fold interactions between substrates, phosphocreatine and phosphocholine, showsubunits. Thecritical roles of histidine and arginine residues in ing thatT y r t Z s and ArglZ7 are not the only residues contributing to the substrate specificity of rPAP. The resi- the activity of WAP have been demonstrated previously (25be important in the 27). Recently, some of the conserved histidine and arginine dues TrplOe andappearedto dimerization of rPAP.O ~ g o m e ~ a t i o mutants n ~ 1 0 6 ~residues , were changed by site-directed mutagenesis in order to Hll2D and W l ~ ~ , H l l 2 D existed ) in a monomeric form clarify the enzymatic mechanism of ~ s c ~ e r i ccoZi ~ i acid a phoswithout catalytic activity or a tartrate binding ability. phatase (28). A complete elimination of Escherichia eo€; acid phosphatase activity was observed when andHisx7 (corresponding to k g l l and His” in rPAP) residues were replaced. Prostatic acid phosphatase (PAP, EC 3.1.3.2)’ (1, 2) is a se- Argo,k g 7 ,and His303 AI$^, and His257in rPAP) were cretory enzyme (3,4, 5, 6) which hydrolyzes a wide variety of also catalytically important, because the corresponding mutant phosphomonoesters under acidic conditions. Similar to lysoso- enzymes had very low activities (below 0.4%) compared with mal acid phosphatase (LAP) (7, 81, it belongs to the group of the wild type enzyme. These results arein agreement with the high molecular weight tartrate-inhibitable acid phosphatases. x-ray structure of rPAP, since all the analyzed residues are at Until now, the physiological function of neither PAP nor LAP the active site (22). Based on the three-dimensional structure has been elucidated. However, it has been suggested that hu- analysis, we will here further characterize the active site of man prostatic acid phosphatase (hPAP) may be involved in the rPAP and particularly the possible role of Aspz5’ in the enzyprogression of tumor or metastases in prostatic cancer (9, 10). matic mechanism, using site-directed mutagenesis. The subOn the other hand, tyrosine phosphorylation of sperm mem- strate specificity and oligomerization of PAP will also be assessed in this work. * !t‘his work was supported by the Research Council for Medicine of E ~ E R i M PROCED~ES E ~ ~ the Academy of Finland, the Technology Development Center of Finland (TEKES), andthe Medical Research Council, Sweden. The Department PCR Mut~nesis~ite-directed mutagenesis was achieved with the of Clinical Chemistry, University of Oulu, isa WHO Collaborating Cen- overlap extension method (29). Mutations in rPAP eDNA (GenBank ter for research in reproduction supported by the Ministries of Educa- accession number M32397) were generated by PCR, using complemention, Health, and Social Affairs, Finland. The ofcosts publication of this tary mutagenic primer pairs containing the mismatched codons. The article were defrayed in part by the payment of page charges. This of mutant cDNAs were amplifiedby PCR, using complete coding regions article must thereforebe hereby marked“advertisement”in accordance the primers forN and C terminus. The 5’-end oligomer (S’GATGACTCwith 18 U.S.C. Section 1734 solely to indicatethis fact. contained XhoI and 9 whomcorrespondenceshould be addressed Biocenter and Dept. GAGAGATCTAC~TGAGAGCTGTCCCTCT~3~) of Clinical Chemistry, University of Oulu, Kajaanintie 50, FIN-90220 BglII restriction sites, whereas the 3‘-end oligomer (5”GCTGAGGTACC C C C ~ G ~ A C A G C G A C G C ~ ~ T G G ~contained G - 3 ‘ ) SmuI and Oulu,Finland. %I.: 358-81-3154413; Fax: 358-81-3155631. KpnI sites in addition to the rPAP sequences. The mutant cDNAs were Theabbreviationsusedare:PAP,prostaticacidphosphatase;h, by using XhoI and KpnI restrichuman; r,rat; AcNPV, Autogrupha culifornica nuclear polyhedrosisvi- cloned into the pSP72 vector (Promega) rus; J A P , lysosomal acid phosphatase; PCR, polymerase chain reaction; tion enzymes (New England Biolabs). The codon changesof the generated Asp mutants (D258N, D258S, and D258A), the lysosomal type rPAP PAGE, polyacrylamide gel electrophoresis.

22642

Catalysis, Specificity, and Oligomerization of Acid Phosphatase

J - " * - "

22643

4 "

1 2 3 4 5 6 7 8 9 101112 FIG.1. Immunoblot analysis of wild type and mutant rPAPs overproduced by baculovirus expression system in insect cells. Rabbit polyclonal antibody raised against peptidefrom the C terminus of rPAP was used in immunostainingof blotted SDS-PAGE gel. Lanes: 1, pure rPAP; 2, D258N; 3, D258S; 4 , D258A; 5 , Y123K, 6, R127G, 7, Y123YR127G; 8, W106E; 9, H112D; 10, W106E,H112D; 11, wild type rPAP; and 12, blank medium.

1 2 3 4 5 6 7 8 9101112 FIG.2. Acid phosphatase activity staining of native PAGE. Lanes: 1, blank medium; 2, wild type rPAp, 3, D258N; 4, D258S; 5 , D258A,6,Y123K, 7, R127G8,Y123K,R127G9,W106E; 10,H112D; 11, W106E,H112D; and 12, purerPAP.

tants did not bind tothe tartrate column, W106E was further purified by hydrophobic interaction chromatography with alkyl superose HR5/5. It was bound to the hydrophobic interaction chromatography column mutants (Y123K, R127G and Y123K,R127G), and the oligomerization with 2 M ammonium sulfate in50 mM sodium acetate buffer (pH 6) and mutants (W106E, H112D, and W106E,H112D) are listed in Table I. eluted using linear gradientof salt-free buffer. Construction of the Recombinant Plasmid l'kansfer Vectors-After Molecular Weight Determination-The molecular weights of pure confirmation of theentire coding region sequence by the dideoxy rPAP and partlypurified mutant protein W106E were determined using method (30), the mutantcDNAs were inserted into the BglIIISmaI site a Superose 12 gel filtration column in a Smart system (Pharmacia). of the pVL1392 nonfusion transfer vector. The wild type cDNA was subcloned in the samevector a s described previously (4). RESULTS Generation of the Recombinant Baculouiruses a n d Protein ProducExpression of Mutant Enzymes in SP Cells-The baculovirus tion-Wild type and mutant rPAP AcNPVs were produced by using linearized AcNPV-DNA (Invitrogen) or BaculoGold-DNA(Pharmingen), expression system was used to overproduce wild type and muas reported by Summers and Smith (31). The generated viruses were tant rPAP enzymes in Sf9 cells. Media containing either the checked by PCR with recombinant baculovirus primers (Invitrogen). wild type or the mutant enzymes were examined by immunoRecombinant proteins were produced using Sf9 insect cells infected by different rPAP-AcNPVs a t a multiplicity of infection of 1 in T-flasks. blot analysis (Fig. 1).Two subunits very close to each other 10% FCS incomplete TNM-FH insect medium (Sigma) with antibiotics were detected from both the wild type and the mutants. The was used as a medium. For harvesting,the cells were centrifuged, and sizes of 46 and 48 kDa were reported for the differently glycothe supernatant wascollected and storeda t 4 "C for further character- sylated subunits of pure recombinant rPAP from silver-stained ization of recombinant proteins. SDS-PAGE (4). The mutant proteins, with the exception of Gel Electrophoresisand Immunoblot Analysis-Electrophoretic char- D258N, co-migrated with the wild type rPAP. The anomalous acterization of recombinant proteins was performed by using supernamigration of D258N is probably due to an additional N-glycotants harvested 3 days after the infection. The electrophoresis were carried outon a PhastSystem (Pharmacia) with PhastGel gradient me- sylation site (Am-Thr-Thr, residues 258-260), which was credia 10-15 for SDS-PAGE (32, 33) and 8-25 (34, 35) for native PAGE. ated at the mutation site. The activities of overproduced enThe activities of recombinant enzymes in native gels were located by zymes in concentrated media were checked by native PAGE, acid phosphataseactivitystaining,the gel beingincubatedwith using acid phosphatase activity staining (Fig. 2). The Asp mua-naphthyl phosphate(0.5mg/ml) and fast garnet 2-methyl-4-[(2-meth- tantsandthe oligomerization mutants were all inactive, ylphenyl)azolbenzeneamine salt (Sigma)(0.6-0.75 mg/ml) dissolved in whereas the lysosomal type rPAP mutants were active en0.1 M sodium acetate (pH 5.4). In Western blotting, the proteins were transferred on to nitrocellulose membranes with PhastTransfer (Phar- zymes. Concentrated mediumfrom noninfected Sf9 cells (blank macia) (36). Rabbit polyclonal antibodies raised against synthetic rPAP medium) did not show any acid phosphatase activity in native peptides (4) were used together witha ProtoBlot AP system (Promega) gel (Fig. 2). to detect the expressed proteins. Characterization of Aspartic Acid Mutants-Histidine 257 Production a n d Isolation of hZA-Baby hamster kidney cells (BHK- and asparticacid 258 are located close to thephosphate binding 21) transfected with humanLAP cDNA, kindly provided by Prof. Kurt von Figura, were used a s a source of hLAP enzyme. The cells were site of acid phosphatase (22h2 Theyare suitablecandidates to grown in minimalEagle's medium with Earle'ssalts supplemented with act as acid/base catalysts duringphosphoester hydrolysis. It is 5% fetal calf serum and antibiotics, including puromycin (5mg/ml) for known that replacement of histidine 303 (corresponding to selection. Fractions containingLAP were prepareda s described (37) for His257in therat enzyme) by alanine drastically reduces E. coli enzymatic activity assays. acid phosphatase activity (28). In order todetermine the cataEnzymatic Actiuity Assays-The media containing wild type or mu- lytic importance of aspartic acid 258, we generated D258N, tant enzymes collected 3 days after the infection were used in the activity measurements. Relative enzyme amounts ofthe denatured wild D258S, and D258A mutants. The acid phosphatase activity, type and mutants in the media were measured from the immunostained measured using p-nitrophenyl phosphate as a substrate, was slot blot by densitometric scanning (MolecularDynamics). Activity as- completely lost when Asp2" was changed to asparagine (Table says with p-nitrophenyl phosphate substrate were performed as de- I). Inactivity of this mutant may be due to a bulky glycosyl scribed (38),using 50 mM citrate bufferpH 5.4 or pH2.5-7 for pH profile group in theactive site. When Asp258was replaced by serine or determinations. Sensitivities to L-(+)-tartrate were analyzed by using alanine, thecorresponding mutant proteins exhibited activities various concentrationsof sodium L-(+)-tartrate(0-100 mM). Using phoswhich were only 0.9 and 0.3%,respectively, of that of the wild phocreatine or phosphocholine a s a substrate, the enzymatic assays were performed in 50 mM sodium citrate buffer (pH 5.41, incubating type enzyme. Aspartic acid mutants were also inactive when equal volumes of substrate and enzyme dilution for 15 min at 37 "C. The tested with a-naphthyl phosphate (Fig. 21, phosphocreatine, or enzymatic activity was related the to inorganic phosphate released(39). phosphocholine (results not shown). Especially D258S and One unit of activity was the amount of enzyme providing for the for- D258A mutant proteins reflect the important role of aspartic mation of 1nmol of productlmin. The kinetic data were correctedfor the acid in the enzymatic mechanism. nonenzymatic hydrolysis of substrates. Lineweaver-Burk plots were Substrate Specificity of Acid Phosphatases-Two polar resiused to determine theK, and V,, parameters. Protein concentrations dues, tyrosine 123and arginine127, are located at the entrance were measured with the Bio-Rad protein assay (40). Purification of Oligomerization Mutants-Mutant proteins W106E and W106E,H112D were purified as described (4). Because these muY. Lindqvist, G. Schneider, andP. Vihko, submitted for publication.

Oligomerization of Acid Phosphatase

Catalysis, Specificity, and

22644

TABLEI Specific activities of wild type and r n u t ~ rPAPs ~t enzyme Relative Codon Protein

101

acid

Wild type D258N D258S D258A Y123K R127G Y123K,B127G

Amino changed

changed

Asp258+ Asn Aspz6' + Ser Aspz6' + Ma T y P 3 "-* Lys Arg"" + Gly 143 TyrlZ3 Lys and ArglZ7 Gly Trplffi + Glu His"' "-t Asp3.61 T r p 1 0 6 -+ Glu and 0.12 His''' Asp -j

W106E H112D W106E,H112D

GAC + AAC GAC AGC GAC -+ GCC TAC AAG 100 AGG -+ GGG TAC -> AAG and AGG --* GGG TGG -+ GAA CAC -+ GAC TGG GAA and CAC + GAC --f

--f

-

--f

--f

(I

Specific

amount

activity"

Specific activity

unitsirelative enzyme amount

%

1.00 0.54 0.65 1.00 0.86 0.67 178 0.84

142 0.15 1.26 0.36 142

100

0.58 0.72 0.48

3.23

0.1 0.9 0.3 125 2.3 2.5 0.1

Specific activity was determined with 5 RIM p-nitrophenyl phosphate as a substrate.

1.2 &

0.8

1 A

0.41

2.5

/ /

3

3.5

4

4.5

5

5.5

6

6.5

7

., PH

FIG.3. pH profiles of wild type and mutantrPAPs and W . e, Y123K; 0,R127G, x, Y123K,R127G wild type rPAP, 0, h W , and A, blank medium.

of the active site surface in rPAP (22, 23). They are only conserved in PAPS, whereas the corresponding residues in L A P S are lysine and glycine. In order to determine the importance of these polar residues in the substratespecificity betweenthese two types of acid phosphatases, mutant proteins Y123K, R127G, and Y123K,R127G were generated. These lysosomal type rPAP mutants were active enzymes, as was to be expected (Fig. 2). The Y123K mutant exhibited a pH profile similar to that of the wild type enzyme having the optimum near pH 5.4 (Fig. 3). The R127G and Y123K,R127G mutant enzymes, however, showed a wider pH optimum with maxima at pH 5.4 and pH 4.5, respectively. The catalytic activity of the hLAP enzyme, overproduced in BKK-21 cells, was highest at pH 3. Differences in sensitivity to L-(+)-tartratehave been reported for LAP and PAP (3). The tartrate inhibitions of the Y123K and R127G mutant enzymes were analogous to that of wild type rPAP (Fig. 4). The double mutant Y123K,R127G, however, was also been observed in the case more sensitive to tartrate, as has of LAP. The activity of this mutantenzyme was only 22% of the noninhibited enzyme activity, compared with the corresponding value of 78% for the wild type enzyme, when 1 nm L-(+)tartrate was added. The remaining activity of recombinant hLAP was 10%when 1 mM inhibitor was present. The specific activities of the Y123K, R127G, and Y123K,

01

0.1

10

20

40

60

L-(+)-tartrate concentration(mM)

80

100

.,

FIG.4. Tartrate inhibition curves of wild type and mutant rPAPs and W.Activities with tartrate were compared with noninhibited activity of an enzyme. e,Y123K, 0, R127G; x, Y123K,R127G, wild type rPAP; and 0, hW.

R127G mutants were close to that of the wild type enzyme (Table I). The kinetic parameters for these mutants (Table 111, determined by using p-nitrophenyl phosphate as a substrate, were similar to each other. The K,,, values (0.21-0.23 mM) of the lysosomal type mutant enzymes were almost identical with the previously reported K,,, values of hLAP (0.2 mM) (41) and rLAP (0.242 m ~ (3). ) The corresponding value for the wild type and purified rPAP enzyme was 0.16 mM, which is close to the 0.123 m~ reported for rPAP (3) and the 0.18mM for hFAP (42). We also studied the enzymatic properties of these mutants, using the suggested PAP-specific substrates, phosphocreatine (16) and phosphocholine (18-21). However, the lysosomal type mutants and the wild type rPAP enzyme did not show any remarkable changes in thekinetic parameters in the case of phosphocreatine. The K, and V,, values of these mutants and the wild type enzyme were alsoquite similar when phosphocholinewas used. On the contrary, recombinant hLAP enzyme did not hydrolyze either phosphocreatine or phosphocholineat observable levels. Oligomerization of Prostatic Acid Phosphatase-There are a number of hydrophobic contacts between the two subunits of rPAP. Furthermore, the residues Gln37and His67,T y r f i 5 and Aspv8,and Asp76and His*lZsupport the dimeric structure by possible hydrogen bonds betweenthe side chain atoms. !ikpl*' is

Catalysis, Specificity, and Oligomerization

of Acid Phosphatase

22645

TABLE I1 Kinetic Darameters of wild tvoe and mutant rPMs Phosphocholine

Phosphocreatinephosphate p-Nitrophenyl Protein

122 103 104 115

Wild type

Y 123K R127G Y123K,R127G

K m

V,",

K m

vmax

mM

unitslrelative enzyme amount

mM

unitslrelative enzyme amount

0.16 0.22 0.21 0.23

318 347 426 318

0.32 0.47 0.41 0.53

1052 730 932 628

K", mM

"X ,

unitslrelative enzyme amount

5.26 3.70 3.57 3.70

also an important residue in oligomerization. It connects the two subunits by stacked side chains oriented across the 2-fold -155 kDa axis in the rPAP dimer (22). We have tried to prevent dimerization in the W106E, H112D, and W106E,H112D mutants. These mutants were inactive in native PAGE upon acid phos- 69 kDa phatase activity staining (Fig. 2). The specific activities of these mutants in the presence ofp-nitrophenyl phosphate wereminimal compared with the wild type enzyme (Table I). Different 1 2 3 4 5 amino acid changes led to identical molecular organizations of FIG. 5. Immunoblot analysis of pure rPAP and oligomerization the oligomerization mutants, as seen in theWestern blot (Fig. mutants in native PAGE. Lanes: 1, pure rPAP, 2, nonbinding fraction 5). Altogether three native bands were detected from the oli- of tartrate aftinity column, containingpartly purified W106E; 3, gomerization mutants. The expected monomers existed in two W106E;4, H112D;5, W106E,H112D. forms close to each other, similar to the subunits of pure rPAP in denaturingconditions (41, with molecular masses around 69 the catalytic importance of Asp258 bymutating it toAsn, Ser, kDa. The molecular structure of the biggest band of the oli- and Ala. All the Asp mutants were severely impaired in catagomerization mutants is unknown. Two native forms of pure lytic activity, although theinactivity of D258N was not necesrecombinant rPAP were recognized in the Western blot, with sary due to amino acid change in this mutant. Mutant proteins reported molecular masses of 100 kDafor the inactive and 155 D258S and D258Aindicate thatAspZ5' may be a proton donor a t kDa for the active dimericenzyme (4,22). Themolecular mass the rPAP active site. Recently, it has been proposed that correof 69 kDa therefore agrees nicely with the monomeric appear- sponding Asp304, rather than His3'', donates a proton to the ance of the oligomerization mutants. Theaffinity purification of substrate leaving group at the catalytic center of E. coli acid W106E (Fig. 5) and W106E,H112D (data not shown) indicated phosphatase (44). that theoligomerization mutants had lost their ability to bind Crystallographic studies of rPAP have indicated that theactartrate. More accurate molecular masses of the wild type and tive site of the enzyme is an easily accessible, being an open W106E mutant were determined by gel filtration chromatog- cleft with a phosphate binding pocket (22), which enables the raphy. Contrary to the results from native PAGE, only one enzyme to accept a large variety of substrates (45). At the protein peak was seen in thegel filtration of pure recombinant entrance of the active site, there aretwo polar residues, rPAP. This peak represented the active enzyme with a partition and ArglZ7,which are conserved only in prostatic acid phoscoefticient K,, of 0.288, corresponding to a size of 82 kDa. A phatases, the corresponding residues in lysosomal acid phosmolecular mass of 109 kDahas been reported for native dimeric phatases are Lys and Gly. It is therefore possible, that these hPAP in studiesusing the samemethod (38).The gel filtration residues mightbe responsible for enzymatic variations such as fraction of W106E gave aK,,of 0.363 and a deduced molecular substrate specificity and sensitivity to tartrate inhibition bemass of 36 kDa,thus representing an inactive monomer of the tween the enzymes of prostatic and lysosomal origin. Our reenzyme. Differences in the size estimations for the monomer sults support thisidea as far as tartrate inhibition and thepH and thedimer of rPAP are understandable due to the irregularprofile of the Y123K,R127G mutant enzyme are concerned. migration of globular proteins in native PAGE. However, in the case of phosphocreatine and phosphocholine, which have been suggested as PAP-specific substrates from DISCUSSION spermatic fluid, the differences in substrate specificity of PAP Using the three-dimensional structure of rat acid phospha- does not depend exclusively on the T y r ' 2 3 and Arg"' residues. tase (22)as a basis, we investigated the catalytic importance of The fact that recombinant hLAP enzyme did not accept either Aspz5' and the substrate specificity and oligomerization of rPAP of these "PAP-specific substrates" shows that there isfare additional residuelresidues which affect the substrate specificity by site-directed mutagenesis. The residuesArg", Hid2,ArgI5, which are partof a sequence between PAP and LAP. As it is known, PAP is a secretory enmotif RHGXRXP typical of acid phosphatases (431, together zyme found in spermatic fluid (3, 5, 6) and LAP functions in with ArgI9, Hisz5', and Asp258generate the centerof the active lysosomes (46, 47, 48) not competing for the same substrates. site of rPAP (22). The conserved residues Arg", Hid2,Arg", and Thus, the active sites of these enzymes can be very similar Arg", create a positively charged cluster optimal for phosphate with slight substrate specificity. It has been observed that binding. During substrate hydrolysis, Hid2 becomes phospho- subcellular location is one of the things controlling the subrylated (22). The location of the residuesHisz5' and Asp25K not strate specificity of protein tyrosine phosphatases. The numfar from the phosphatebinding site makes them qualified to act ber of potential substrates for specific protein tyrosine phosasacidhasecatalystsduring phosphoester hydrolysis. The phatases islimited by the different targeting of the enzymes in acidic pH optima of acid phosphatases together with the unfa- the cell (49). It can thus be concluded that replacement of Tyr123 vorable distance and orientation of the Hisz5' side chain suggest with lysineand Arglz7with glycine changes the enzymatic propthat Asp258is more suitable as a proton donor.2Areplacementof erties of PAP toward those of LAP. The observed change in the the corresponding His303 byAla causes a drastic drop in activity pH profile upon replacement of these residues may have physfor E. coli acid phosphatase (28). Here, we have demonstrated iological significance, since it could be the molecular basis for

22646

Catalysis, Specificity, Oligomerization and

the adaptationof LAP to function optimally at thelower pH in lysosomes. Theresiduesand His'" wereshown to be importantin the oligomerization of rPAP. The formation of dimeric enzyme was inhibited in the W106E, H112D, and W106E,H112D mutants. The results from both native PAGE and gel filtration chromatography suggested that the oligomerization mutants were in a monomeric form. These mutants were expected to be active monomers, because the active sites are far apart from each other in a dimer (22), indicating a lack of cooperation between these sites. However, the loss of catalytic activity and the tartratebinding ability suggestedthat theoverall folding of the oligomerization mutants was abnormal. It might be possible that the formation of a dimeric enzyme represents an activation mechanism, regulating the function of PAP. Acknowledgments-We thank Prof. Dr. Kurt von Figura and Dr. Christoph Peters for LAP overexpressing cells and Saini Sydanmaa for her expert technical assistance. REFERENCES 1. Roiko, K., Jiinne, 0. A., and Vihko, P. (1990) Gene (Amst.)89,223-229 2. Vlhko, P., Virkkunen, P., Henttu, P., Roiko, K., Solin, T., and Huhtala, M.-L. (1988)FEBS Lett. 236, 275-281 3. Vanha-Perttula, T.,Niemi, R., and Helminen, H. J. (1972) Inuest. Urol. 9, 345-352 4. Vihko, P., Kurkela, R., Porvari, K., Herrala, A,, Lindfors, A,, Lindqvist, Y., and Schneider, G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 799-803 5. Yam, L. T.,Li, C. Y., and Lam, K. W. (1980) in Male Accessory Sex Glands (Spring-Mills, E., and Hafez, E. S. E., eds) pp. 183-196, ElseviermorthHolland Publishing Co., Amsterdam 6. Ronnberg, L., Vihko, P., Sajanti, E., and Vihko, R. (1981) Int. J . Androl. 4, 372-378 7. Pohlmann, R., Krentler, C., Schmidt, B., Schroder, W., Lorkowski, G., Culley, J., Mersmann, G., Geier, C., Waheed, A,, Gottschalk, S., Grzeschik, K.-H., Hasilik, A., and von Figura, K. (1988) EMBO J. 7, 2343-2350 8. Geier, C., von Figura, K., and Pohlmann, R. (1989) Eur J . Biochem. 183, 611-616 9. Mukhopadhyay, N., Saha,A., Smith,W., Dowling, J., Hiserodt, J.,and Glew, R. (1989) Clin. Chim. Acta 182, 31-40 10. Ishibe, M., Rosier, R., and Puzas, E. (1991)J. Clin. Endocrinol. & Metab. 73, 785-792 11. Naz, R., Ahmad, K., and Kumar, R.(1991) J. Cell Sci. 99,157-165 12. Li, H. C., Chernoff, J., Chen, L. B., and Kirschonbaum, A. (1984) Eur: J. Biochem. 138,45-51 13. Lin, M.-F., and Clinton, G. M. (1986)Biochem. J . 235, 351-357 14. Chevalier, S., Landry, D., and Chapdeline, A. (1988) Prostate 12, 209-219 15. Lin, M . 3 , a n d Clinton, G. M. (1988) Mol. Cell. Biol. 8, 5477-5485

of Acid Phosphatase

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