Residues Determining the Binding Specificity of Uncompetitive Inhibitors to Tissue-Nonspecific Alkaline Phosphatase

Residues Determining the Binding Specificity of Uncompetitive Inhibitorsto Tissue-Nonspecific Alkaline Phosphatase

Alexey Kozlenkov,1 Marie Helene Le Du,2 Philippe Cuniasse,2 Tor Ny,3 Marc F Hoylaerts,4 and Jose Luis Millan1,5

ABSTRACT: Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnor-malities. Here, we used mutagenesis, kinetic analysis, and computer modeling to identify the residuesimportant for the binding of known ALP inhibitors to the TNALP active site. These data will enable drugdesign efforts aimed at developing improved specific TNALP inhibitors for therapeutic use.

Introduction: We have shown previously that the genetic ablation of tissue-nonspecific alkaline phosphatase(TNALP) function leads to amelioration of soft-tissue ossification in mouse models of osteoarthritis and ankylosis(i.e., Enpp1�/� and ank/ank mutant mice). We surmise that the pharmacologic inhibition of TNALP activityrepresents a viable therapeutic approach for these diseases. As a first step toward developing suitable TNALPtherapeutics, we have now clarified the residues involved in binding well-known uncompetitive inhibitors to theTNALP active site.Materials and Methods: We compared the modeled 3D structure of TNALP with the 3D structure of humanplacental alkaline phosphatase (PLALP) and identified the residues that differ between these isozymes within a 12Å radius of the active site, because these isozymes differ significantly in inhibitor specificity. We then usedsite-directed mutagenesis to substitute TNALP residues to their respective homolog in PLALP. In addition, wemutagenized most of these residues in TNALP to Ala and the corresponding residues in PLALP to their TNALPhomolog. All mutants were characterized for their sensitivity toward the uncompetitive inhibitors L-homoarginine(L-hArg), levamisole, theophylline, and L-phenylalanine.Results and Conclusions: We found that the identity of residue 108 in TNALP largely determines the specificity ofinhibition by L-hArg. The conserved Tyr-371 is also necessary for binding of L-hArg. In contrast, the binding oflevamisole to TNALP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. The maindeterminant of sensitivity to theophylline is His-434. Thus, we have clarified the location of the binding sites for allthree TNALP inhibitors, and we have also been able to exchange inhibitor specificities between TNALP and PLALP.These data will enable drug design efforts aimed at developing improved, selective, and drug-like TNALP inhibitorsfor therapeutic use.J Bone Miner Res 2004;19:1862–1872. Published online on June 28, 2004; doi: 10.1359/JBMR.040608

Key words: alkaline phosphatase, enzyme kinetics, site-directed mutagenesis, computer modeling, soft tissueossification, therapeutic inhibitors, drug design

INTRODUCTION

ALKALINE PHOSPHATASES (E.C.3.1.3.1; (ALPs) aredimeric enzymes present in most organisms.(1) They

catalyze the hydrolysis of phosphomonoesters with releaseof inorganic phosphate and alcohol.(2) In humans, three ofthe four isozymes are tissue-specific, the intestinal (IALP),placental (PLALP), and germ cell (GCALP) ALPs, whereasthe fourth ALP is tissue-nonspecific (TNALP) and is ex-

pressed in bone, liver, and kidney. The first three isozymesare �90% homologous to each other at the protein level,with PLALP and GCALP differing only by 12 amino acidsubstitutions. In contrast, TNALP is only 50% homologousto the three tissue-specific human isozymes.(3)

The clearest evidence that ALPs are important in vivo hasbeen provided by studies of human hypophosphatasia wherea deficiency in the TNALP isozyme, caused by deactivatingmutations in the TNALP gene,(4–6) is associated with defec-tive bone mineralization in the form of rickets and osteo-malacia.(7) The severity and expressivity of hypophosphata-sia depends on the nature of the TNALP mutation.(8,9) The

Dr Ny owns stock in Omnio AB and has filed patents related towound healing and arthritis. All other authors have no conflict ofinterest.

1Department of Medical Biosciences, Medical Genetics, Umeå University, Umeå, Sweden; 2Department d’Ingenierie et d’Etude desProteines, Gif-sur-Yvette, France; 3Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden; 4Center forMolecular and Vascular Biology, University of Leuven—Campus Gasthuisberg, Leuven, Belgium; 5The Burnham Institute, La Jolla,California, USA.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 19, Number 11, 2004Published online on June 28, 2004; doi: 10.1359/JBMR.040608© 2004 American Society for Bone and Mineral Research

1862

mapping of hypophosphatasia mutations to specific 3D lo-cations on the TNALP molecule has provided clues as to thestructural significance of these areas for enzyme structureand function.(10) In bone, TNALP is confined to the cellsurface of osteoblasts and growth plate chondrocytes, in-cluding the membranes of their shed matrix vesicles(MVs).(11–14) In fact, by an unknown mechanism, MVs aremarkedly enriched in TNALP compared with both wholecells and the plasma membrane.(15) It has been proposedthat the role of TNALP in the bone matrix is to generatethe inorganic phosphate needed for hydroxyapatitecrystallization.(16–18) However, TNALP has also been hy-pothesized to hydrolyze the mineralization inhibitor PPi tofacilitate mineral precipitation and growth.(19–21) Electronmicroscopy observations of MVs derived obtained fromhypophosphatasia patients(22) or from TNALP knockoutmice(23) reveal that they contain apatite-like mineral crystalsbut that extravesicular crystal propagation is retarded. Thisgrowth retardation could be because of either the lack ofTNALP’s pyrophosphatase function or the lack of inorganicphosphate generation. Our recent studies have providedcompelling proof that a major role for TNALP in bonetissue is to hydrolyze PPi to maintain a proper concentrationof this mineralization inhibitor ensuring normal bonemineralization.(24–26) We showed that crossbreeding theEnpp1�/� and the ank/ank mutant mice to TNALP knock-out mice caused normalization of extracellular PPi levelsand resulted in the correction of the soft-tissue ossificationabnormalities.(25,27) The increase in extracellular PPi con-centrations led, in turn, to an increased expression of os-teopontin. Correction of the soft tissue ossification wasattributed to the combined upregulation of two inhibitors ofcalcification (i.e., PPi and osteopontin). Importantly, thesestudies have pointed to TNALP as a potentially usefultherapeutic target for the treatment of soft tissue ossificationabnormalities including ankylosis, osteoarthritis, and arte-rial calcification.

The fact that deletion of the TNALP gene results in theelevation of PPi and osteopontin concentrations in bonematrix and suppresses the soft tissue ossification in bothEnpp1�/� and ank/ank mutant mice suggests that the chem-ical ablation of TNALP function may be a promisingtherapeutic strategy for soft tissue ossification. Since theearly 1960s, several specific inhibitors of ALP isozymeshave been uncovered. They include L-amino acids, suchas L-phenylalanine, L-tryptophan, L-leucine, and L-homoarginine,(28–30) as well as some nonrelated com-pounds, such as levamisole(31) and theophylline (Fig. 1).(32)

The inhibition is of a rare uncompetitive type,(33) but theexact binding site and precise mechanism of inhibition haveonly been elucidated for the binding of L-phenylalanine andL-leucine to the PLALP active site.(34–37) No information isavailable about the binding sites of the three commonlyused inhibitors of TNALP, that is, L-homoarginine (L-hArg), levamisole, and theophylline. Furthermore, theseinhibitors are not entirely specific for TNALP(28,38) andhave low binding affinity, requiring the in vivo administra-tion of very high concentrations to achieve biologicaleffects.(39–44)

In this study, we compared the modeled 3D structure ofTNALP with the structure of PLALP and pinpointed theresidues that differ between the two isozymes in the activesite area. We then clarified the location of the binding sitesfor the three TNALP inhibitors studied, and we also ex-changed inhibitor specificities between TNALP andPLALP. These data will help in drug design efforts tosynthesize improved, selective, and drug-like TNALP in-hibitors suitable for therapeutic use for the treatment of softtissue ossification.

MATERIALS AND METHODS

Site-directed mutagenesis

Mutant variants of TNALP or PLALP were constructedusing either the QuikChange XL site-directed mutagenesiskit (Stratagene, San Diego, CA, USA) or by PCR using theoverlap extension approach as before.(37) TNALP-FLAG orPLALP-FLAG cDNA, cloned into pcDNA3 mammalianexpression vector (Invitrogen, Carlsbad, CA, USA), wereused as templates in the mutagenesis reactions. Final DNAconstructs were checked by automatic sequencing for thepresence of the desired mutation and absence of unwantedsecondary mutations.

The QuikChange XL kit was used to introduce the fol-lowing mutations: F108, (F108, Q109), A120, E434, G434,Q434, and (D433, E434) in TNALP, and E107, R428,H429, and (R428, H429) in PLALP. The following primers,together with the analogous reverse complementary prim-ers, were used according to the mutagenesis protocol fromthe manufacturer—in TNALP: F108, 5�-CTGTGTGGGGT-GAAGGCCAATTTCGGCACCGTGGGGGTAAGCGC-3�;(F108, Q109), 5�-GTGTGGGGTGAAGGCCAATTTCCA-GACCGTGGGGGTAAGCGCAGCC-3�; A120, AGCG-C A G C C A C T G A G C G T G C C C G G T G C A A C A C -C A C C C A G - 3 � ; E 4 3 4 , 5 � - C A G T C T G C T G T G -CCCCTGCGCGAGGAGACCCACGGCGGGGAG-3�;G434, 5�-CAGTCTGCTGTGCCCCTGCGCGGC-GAGACCCACGGCGGGGAG-3�; Q434, 5�-CAGTCT-G C T G T G C C C C T G C G C C A G G A G A C C C A C G -GCGGGGAG-3 � ; (D433 , E434) , 5 � -CAGTCT-

FIG. 1. Chemical structure of the uncompetitive inhibitors (A)L-homoarginine, (B) levamisole, and (C) theophylline.

1863UNCOMPETITIVE INHIBITION OF TNALP

GCTGAGCCCCTGGACGAGGAGACCCACGGCGG-GGAG-3� and in PLALP: E107, 5�-CCTGTGCGGGGT-CAAGGGCAACGAGCAGACCATTGGCTTGAGTGC;R428, 5�-CGGCAGCAGTCAGCAGTGCCCCTGCGCG-AGGAGACCCACGCAGGCG-3�; H429, 5�-CGGCAGCAG-TCAGCAGTGCCCCTGGACCACGAGAACCCACGC-AGGCG-3 � ; (R428, H429) , 5 � -CGGCAGCAG-TCAGCAGTGCCCCTGCGCCACGAGACCCAC-GCAGGCG-3�.

To create other mutants, a PCR mutagenesis approachbased on the overlap extension method was used. InTNALP, primers 5�-GAAGGCCAATGAGGCCACCGT-GGGGGTA-3� for A109,5�-GAAGGCCAATGAGCA-GACCGTGGGGGTA-3� forQ109,5�-AGCGCAGCCACT-GAGCGTGACCGGTGCAACACCACCCAG-3� for D120,5�-AGCGCAGCCACGGAGCGTAACCGGTGCAACAC-CACCCAG-3� for N120, 5�-GCCGCCTACGCCCACTCG-GCTGCCCGGGACTGGTACTCAGAC-3� for A166, 5�-GCCGCCTACGCCCACTCGGCTAACCGGGACTGG-T A C T C A G A C - 3 � f o r N 1 6 6 , 5 � - A C G C C C -ACTCGGCTGACCGGGCCTGGTACTCAGACAAC-GAG-3� for A168, 5�-ACGCCCACTCGGCTGACCGGA-A C T G G T A C T C A G A C A A C G A G - 3 � f o r N 1 6 8 ,5�-CTTCACATTTGGTGGAGCCACCCCCCGTGGC-AACTCT-3� for A371, 5�-CAGTCTGCTGTGCCCCTG-GCCCACGAGACCCACGGCGGGGAG-3� for A433, 5�-C A G T C T G C T G T G C C C C T G G A C C A C G A G A C -C C A C G G C G G G G A G - 3 � f o r D 4 3 3 , 5 � - C A G -TCTGCTGTGCCCCTGCGCGCCGAGACCCACGGC-GGGGAG-3� for A434, and 5�-CTGCTGTGCCCCTGC-GCTCCGAGACCACGGCGGGGAGG-3� for S434 wereused, and their reverse complementary analog were initiallyused together with flanking primers 5�-CTGGCCATTGGC-ACCTGCCTTACTAACTCCTTAGTGCCAGAG-3� and5�-GTGGTCAATTCTGCCTCCTTCCACCAGCAAGAA-GAAGCC-3� (for mutants in the region 108–166) or 5�-CTCCTGACCCTTGACCCCCACAATGTGGACTAC-C T A T T G G G T - 3 � a n d 5 � - G T G C T G G A A T T C -GGCTTCTGCAGTTACTTGTCATCGTCGTCC-3� (formutants in the region 371–434) in two PCR reactions foreach mutant. The PCR products were then combined in anoverlap extension PCR reaction with the flanking primers.These PCR products were next ligated into pCRII/TOPOvector (Invitrogen), and sequenced to confirm the muta-tions. The resulting intermediate constructs were digestedwith PflMI, DraIII and EcoRI, or Eco91I and Eco81I andligated together with the corresponding fragments of thetemplate plasmid TNALP-FLAG/pcDNA3, digested withthe same enzymes.

In PLALP, primers 5�-CAAGGGCAACTTCGGGACCAT-TGGCTTGAGTGC-3� for G108, 5�-TGCGGGGTCAAG-GGCAACGAAGGTACCATTGGCTTGAGT-3� for (E107,G108), 5�-GCAGCCGCCCGCTTTAGCCAGTGCAACAC-GAC-3� for S119, and 5�-ACCAGTTGCGGTCCACCG-TGTGGGCGT-3� for D165 were used in a similar approachusing flanking primers 5�-ATGGGCGGTAGGCGTGTA-CGGTGGGAGG-3� (forward) and 5�-CCGGTCTCGATG-CGACCACCCTCCAC-3� (reverse). The overlap extensionPCR products were ligated into pCRII/TOPO vector, digestedwith PflMI, and ligated together with the corresponding main

fragments of the PLALP-FLAG/pcDNA3 plasmid. The mu-tants (E107, G429)PLALP and (G108, G429)PLALP weremade with similar ligation reactions, but using the main frag-ments of the plasmid (G429)PLALP-FLAG/pcDNA3(37) in-stead of the wildtype PLALP-FLAG/pcDNA3.

Expression and purification of enzymes

The mutant constructs were transfected into COS-7 cellsfor transient expression. Either the DEAE-dextran mediatedmethod or a commercial transfection reagent SuperFect(Quiagen) was used for the transfections. The conditionedmedia with secreted proteins were collected after 3 and 6days, concentrated if needed by ultrafiltration on YM-50Centricon columns (Amicon, Beverly, MA, USA), and pu-rified by affinity chromatography with anti-FLAG M2 an-tibody gel (Sigma, St Louis, MO, USA) according to theprotocol from the manufacturer.

Enzyme kinetics experiments

Relative specific activities of the mutants were measuredas described previously.(37) In brief, samples of the enzymeswere added to microtiter plates coated with M2 anti-FLAGantibody, and saturating activities with the substrate pNPPwere measured in 1 M DEA/HCl buffer, pH 9.8, containing1 mM MgCl2 and 20 �M ZnCl2. The determinations of Km

and the inhibition studies were done in the same buffer, withvarying concentrations of pNPP and/or inhibitors. Levami-sole (L-tetramisole), L-homoarginine, L-phenylalanine (allfrom Sigma) and theophylline (Fluka) were used as reagentsin the inhibition studies. Ki values for the uncompetitiveinhibitors were obtained from the inhibition studies using 20mM pNPP (saturating substrate concentration) as well as at1 mM pNPP. The results of enzyme kinetics studies wereanalyzed by nonlinear regression using software Prism 3.02(GraphPad Software). Variations in Ki within a factor of 2were not considered functionally relevant.

Molecular modeling

The 3D model of TNALP was constructed with thePLALP structure as a template using software MODELLER3.0 as described previously.(10,45) The further analysis of theTNALP structure and its comparison with that of PLALPwas carried out with SwissPDB viewer,(46) RasMol, andTURBO-FRODO. Molecular models of the interaction ofthe ligands with TNALP were achieved using a simulatedannealing-based protocol(47) with the program X-PLOR(version 3.1).(48) The parameter set used for these calcula-tions is the CHARMM version 22.(49) For the ligands,geometrical and nonbonded parameters were derived fromab initio quantum calculations with the program GAUSS-IAN98.(50) During the simulated annealing-based protocol,the ligand was driven to the catalytic site of the enzymeusing a single distance restraint between one atom of theligand and the phosphorus atom of the P-Ser 93 residueusing the noe function of X-PLOR. For each ligand, thisprotocol was reiterated 30 times to give a corresponding setof solutions.

1864 KOZLENKOV ET AL.

RESULTS

Computer modeling and mutagenesis

We constructed the 3D model of TNALP based on theknown structure of PLALP,(51) using the software MOD-ELLER 3.0. The two structures were superimposed, and theamino acid differences in the active site area (the 12 Åregion around the catalytic Zn1 ion) were pinpointed (Fig.2). The differences between human TNALP and chickenTNALP were also mapped, because it has been reported thatchicken TNALP is less susceptible to inhibition by levami-sole than human TNALP.(52) In total, six positions withamino acid differences were found, which could be clus-tered into two groups. The first group, using the TNALP

numbers, includes residues 433 and 434, and the secondgroup includes residues 108, 109, 120, and 166 (Table 1). Inaddition, residues Asp168 and Tyr371 in TNALP were alsoinvestigated as possible sites of interaction with inhibitormolecules, as suggested by our previous data.(37)

Based on this analysis, we embarked on a mutagenesisstudy, constructing the following mutants: F108, A108,Q109, (F108, Q109), A120, D120, N120, A166, N166,A168, N168, A371, A433, D433, A434, Q434, G434, E434,S434, and (D433, E434) in TNALP and E107, G108, (E107,G108), S119, R428, H429, (R428, H429), (E107, G429),and (G108, G429) in PLALP. The (G429)PLALP mutantwas constructed previously.(37) Mutations were introducedinto the TNALP or PLALP cDNA cloned into the pcDNA3expression vector. In all mutants, the COOH-terminal signalfor the attachment of the GPI-anchor was substituted with ahydrophilic FLAG peptide as described before.(9) Resultingproteins, expressed in COS-7 cells, were secreted to themedium and could be purified by anti-FLAG affinity chro-matography. The GPI-anchored membrane-bound TNALPwas also studied and confirmed to have properties notsignificantly different from the FLAG-tagged TNALP (datanot shown). In addition to human TNALP, we also ex-pressed the GPI-anchored form of the chicken TNALPisozyme and studied its inhibition properties.

All of the mutant proteins retained significant activitywhen expressed in COS-7 cells, as can be observed fromtheir kinetic parameters kcat and Km values presented inTable 2, which summarizes all the kinetic data discussed inthis paper. The most noticeable decrease in kcat was found inthe (A109), (Q109), (F108, Q109), (N168), (A371), (A433),and (D433) mutants of TNALP, whereas the largest increasein Km was found in the (E434) and (D433, E434) TNALPmutants.

Inhibition by L-amino acids

L-Homoarginine (L-hArg), a homolog of arginine, is aspecific uncompetitive inhibitor of TNALP.(53) On theother hand, L-phenylalanine (L-Phe) is a well-knownselective inhibitor of PLALP.(28) In agreement with pre-vious data, we found that both human and chickenTNALP are inhibited by L-hArg with a Ki of about 2 mM,at least 30 times more efficiently than PLALP. L-Pheinhibited PLALP 25 times better than TNALP. To clarifywhich amino acid residues in TNALP and PLALP wereresponsible for these marked differences in inhibitionselectivity, we produced a large number of mutated vari-ants of both isozymes, concentrating on those residuesthat are different between TNALP and PLALP in theactive site area. The results are presented in Table 2 andalso shown graphically in Fig. 3 to facilitate the identi-fication of the changes. Among the TNALP mutationsinvestigated, the biggest changes in L-hArg inhibitionwere found for substitutions Phe-108 and Ala-371, whichdecreased inhibition by 35- and 22-fold, respectively, in(F108)TNALP and (A371)TNALP. The mutants(A108)TNALP, (F108, Q109)TNALP, (E434)TNALP,and (D433, E434)TNALP displayed a lesser, but stillnoticeable, negative change in inhibition. Apart fromthese changes, a large number of substitutions at different

FIG. 2. Active site region of (A) human TNALP and (B) humanPLALP. The highlighted residues are those that differ between TNALPand PLALP, with the exception of Y371. The residues are coloredaccording to their chemical properties: blue � basic, red � acidic,violet � aromatic, yellow � aliphatic, and green � uncharged polar.The zinc atom is shown in orange, the magnesium is in green, and thephosphate moiety is shown in yellow and red. The figures were pro-duced with the use of TURBO-FRODO.


positions (Ala-120, Asn-120, Asp-120, Ala-166, Asn-166, Ala-168, Asn-168, Gln-109, Asp-433, Ala-433, Ala-434, Ser-434, Gly-434, Gln-434, and Ser-434) had noeffect on the inhibition.

In contrast, several mutations made in the context ofPLALP improved the inhibition by L-hArg, most signifi-cantly in (E107)PLALP, but also in the (H429)PLALP,

(R428, H429)PLALP, (G429)PLALP, and (E107,G108)PLALP mutants. The double mutant (E107,G429)PLALP was inhibited by L-hArg as strongly as thewildtype TNALP enzyme. When the inhibition by L-Phewas tested, a reversed effect was found for mutations(F108)TNALP and (E107)PLALP, which now significantlyimproved or decreased the inhibition, respectively, com-

TABLE 1. ACTIVE SITE RESIDUES STUDIED

Enzymes Homologous amino acid residues

Human TNALP E108 G109 S120 D166 D168 Y371 R433 H434Chicken TNALP E108 G109 D120 N166 D168 Y371 R433 Q434Human GCALP F107 Q108 N119 N165 N167 Y367 D428 G429Human PLALP F107 Q108 N119 N165 N167 Y367 D428 E429

TABLE 2. KINETIC PARAMETERS AND INHIBITION CONSTANTS (�SD) OF WILDTYPE AND MUTANT TNALP AND PLALP ISOZYMES

Mutants kcat (103 � s�1) Km (mM)

Ki for various inhibitors

L-hArg(mM)

L-Phe(mM)

Levamisole(�M)

Theophylline(20)* (�M)

Theophylline(1)* (�M)

hTNALP 2.1 � 0.2 0.36 � 0.02 1.4 � 0.1 19 � 6 16 � 1 25 � 2 82 � 11chTNALP ND 0.64 � 0.03 1.9 � 0.2 11 � 0.9 136 � 20 111 � 8 393 � 150hPLALP 0.46 0.38 � 0.02 59 � 15 0.75 � 0.05 563 � 56 947 � 120 932 � 90hTNALP mutants

A108 4.3 � 0.1 0.28 � 0.03 14 � 1 27 � 4 20 � 1 15 � 1 31 � 3F108 1.8 � 0.1 0.19 � 0.01 48 � 11 3.7 � 0.5 19 � 2 5.6 � 0.3 16 � 2A109 0.39 � 0.02 0.27 � 0.02 1.3 � 0.2 20 � 4 16 � 1 29 � 2 104 � 10Q109 0.33 � 0.05 0.30 � 0.02 1.6 � 0.1 4.0 � 1.1 50 � 4 40 � 12 317 � 33(F108, Q109) 0.46 � 0.03 0.19 � 0.03 15 � 2 2.1 � 0.4 36 � 3 10 � 0.4 6 � 1A120 2.0 � 0.8 0.31 � 0.02 1.4 � 0.1 24 � 2 14 � 1 19 � 1 103 � 6D120 1.2 � 0.1 0.37 � 0.03 1.4 � 0.1 34 � 4 13 � 1 24 � 2 104 � 10N120 2.9 � 0.1 0.35 � 0.01 1.3 � 0.1 23 � 8 14 � 0.5 22 � 1 83 � 10A166 1.6 � 0.1 0.28 � 0.02 1.6 � 0.3 24 � 4 26 � 1 33 � 2 67 � 4N166 3.4 � 0.1 0.39 � 0.05 1.8 � 0.1 32 � 5 15 � 1 30 � 2 87 � 4A168 1.4 � 0.3 0.29 � 0.03 1.5 � 0.3 30 � 4 18 � 2 25 � 1 58 � 6N168 0.59 � 0.01 0.28 � 0.03 1.8 � 0.1 42 � 6 20 � 1 33 � 2 66 � 6A371 0.46 � 0.03 0.29 � 0.02 31 � 3 65 � 24 122 � 6 52 � 3 118 � 11A433 0.68 � 0.05 0.41 � 0.01 1.6 � 0.2 31 � 3 18 � 2 52 � 4 134 � 8D433 0.98 � 0.05 0.52 � 0.02 1.5 � 0.2 23 � 5 18 � 1 51 � 2 218 � 28A434 2.8 � 0.1 0.69 � 0.08 1.5 � 0.1 21 � 6 133 � 7 121 � 9 219 � 20Q434 2.5 � 0.1 0.79 � 0.10 2.0 � 0.1 9.8 � 1 268 � 53 126 � 27 374 � 20G434 1.6 � 0.1 0.50 � 0.04 1.6 � 0.2 9.6 � 1 311 � 38 158 � 23 176 � 20S434 2.3 � 0.1 0.66 � 0.03 1.6 � 0.1 13 � 2 163 � 17 186 � 10 248 � 18E434 2.3 � 0.1 2.0 � 0.1 6.8 � 0.5 43 � 8 731 � 70 1240 � 80 2350 � 310(D433, E434) 1.4 � 0.1 3.1 � 0.9 13 � 1.6 206 � 111 1430 � 120 2400 � 250 5200 � 750

hPLALP mutantsE107 0.43 � 0.02 0.49 � 0.03 5.7 � 0.9 3.8 � 0.4 1050 � 40 4200 � 260 5800 � 670G108 0.57 � 0.02 0.30 � 0.01 60 � 9 2.4 � 0.3 458 � 30 540 � 85 650 � 95(E107, G108) 0.45 � 0.02 0.38 � 0.04 12 � 1.6 23 � 2 1150 � 60 6400 � 500 9700 � 1300S119 0.6 � 0.1 0.32 � 0.05 61 � 13 0.67 � 0.08 630 � 90 710 � 70 920 � 90D165 0.44 � 0.03 0.33 � 0.04 59 � 25 0.54 � 0.04 680 � 140 690 � 40 1080 � 100R428 1.4 � 0.2 0.6 � 0.03 36 � 4 0.54 � 0.05 460 � 40 330 � 40 630 � 50G429 0.36 � 0.05 0.09 � 0.01 12 � 2 0.09 � 0.01 474 � 56 200 � 40 216 � 7H429 0.95 � 0.04 0.14 � 0.01 10.7 � 1.5 0.31 � 0.03 50 � 3 34 � 1 115 � 8(R428, H429) 1.4 � 0.1 0.09 � 0.006 14 � 1 0.35 � 0.04 34 � 1 19 � 1 76 � 5(E107, G429) 0.10 � 0.01 0.11 � 0.01 1.5 � 0.2 1.0 � 0.16 535 � 80 161 � 54 443 � 70(G108, G429) 0.44 � 0.02 0.08 � 0.01 18 � 2 0.49 � 0.06 480 � 60 158 � 15 189 � 13

*Because of the occurrence of substrate inhibition, apparent Ki values were also calculated at [pNPP] � 1 mM. For those mutants with elevated Km, thisis a suboptimal substrate concentration, but they are not subject to substrate inhibition in the presence of theophylline (shown in Fig. 5 for (Q434)TNALP).


pared with the wildtype enzymes. A similar effect wasfound for reciprocal substitutions (Q109)TNALP and(G108)PLALP.

On the contrary, the effects of substitutions at position434 in TNALP, particularly (E434), on the L-Phe inhibitionwere parallel to those observed for L-hArg, and mutation(H429) in PLALP decreased Ki for both inhibitors, so thisresidue cannot explain the selectivity of inhibition. Also, thesubstitution Tyr-371 in TNALP reduced L-Phe inhibitionsignificantly, as it did for L-hArg inhibition. Apart for asmall decrease in inhibition found in (N168)TNALP, allother mutations had no effect on inhibition.

Inhibition by levamisole

Levamisole, the L-stereoisomer of tetramisole, is a potentuncompetitive inhibitor of TNALP. In fact, it is nearly 100times more effective than L-hArg. It shows marked prefer-ence toward TNALP. In contrast with the data obtained forL-hArg, we found that levamisole inhibition depends verymuch on the nature of residue 434 in TNALP. SubstitutionsH434E (as in PLALP), H434Q (as in chicken TNALP),H434S (as in the intestinal isozyme), H434G (as in GCAP),or H434A all lead to a significant decrease in inhibition. The(E434)TNALP mutant displayed a greatly increased Ki

value; in fact, it was inhibited about as weakly as PLALP.We also confirmed that wildtype chicken TNALP is muchless inhibited by levamisole (about 8.5-fold) than humanTNALP as previously reported.(52)

In contrast to the substitutions at His-434 in TNALP,mutations at positions 108 (Ala-108 and Phe-108), 120(Ala-120, Asp-120, and Asn-120), 166 (Ala-166 and Asn-166), 168 (Ala-168 and Asn-168), and 433 (Ala-433 andAsp-433) did not affect the inhibition by levamisole. A2-fold increase in Ki was observed in TNALP mutants

containing the substitution G109Q and in PLALP mutantscontaining the mutation F107E. However, no effect wasfound for the reciprocal mutations Q108G in PLALP andE108F in TNALP or in the (A108)TNALP mutant, suggest-ing that the positions 108 and 109 (TNALP numbering) playonly a minor role in levamisole inhibition. We also observeda 2-fold decrease in inhibition caused by the Asp-433 mu-tation in the double (D433, E434)TNALP mutant comparedwith the (E434)TNALP mutant and a similar effect in thereciprocal PLALP mutants. However, this effect was notseen in the single Ala-433 and Asp-433 substitutions, sug-gesting that in this case, residue 433 did not directly interactwith the inhibitor, but rather indirectly influenced the inter-action of residue 434 with levamisole. It is likely that theside chains of residues 433 and 434 in TNALP have highmobility, which makes it difficult to assess the exact natureof their interactions.

Besides the substitutions at His-434, the only mutationthat significantly influenced the inhibition by levamisolewas Ala-371, increasing the Ki value about eight times. Thissuggests that, together with His-434, Tyr-371 in wildtypeTNALP forms the binding area for levamisole. We per-formed a double inhibition experiment using both L-hArgand levamisole on wildtype TNALP (Fig. 4). The parallellines in the reciprocal plot of v versus inhibitor concentra-tion suggest that the two uncompetitive inhibitors can onlyact independently on the enzyme (i.e., no simultaneousbinding is possible and their effect is additive, although bothinhibitors are stabilized in the active site pocket on differentamino acid residues).

Inhibition by theophylline

Theophylline (a 1,3-dimethyl derivative of xanthine) isa potent inhibitor of TNALP. Double reciprocal plots of

FIG. 3. Inhibition constants of PLALP, TNALP, and their mutants. Bar chart representation of Ki as a function of the mutants for L-hArg, L-Phe,levamisole, and theophylline, measured at 20 mM pNPP. Native enzymes are identified as wildtype (wt), TNALP mutants (TNALP), and PLALPmutants (PLALP) and indicated by the amino acid substitution.


v versus substrate concentration (Fig. 5A) confirmed thatTNALP is inhibited by theophylline in an uncompetitivemanner, but in agreement with findings by Glogowski etal.,(54) we found evidence of substrate inhibition. This didnot preclude assessing Ki values during inhibition stud-ies, as shown from secondary replots of the y-interceptsversus the concentration of theophylline, enabling correctassessment of the Ki (Fig. 5A, inset). Moreover, at[pNPP] � 1 mM, substrate inhibition was virtually ab-sent, motivating us to study enzyme inhibition of TNALPand PLALP mutants both at 1 and 20 mM pNPP (Table2). In general, lower apparent Kis were found with 20mM pNPP, as expected. In agreement with publisheddata, we thus found that theophylline is a much betterinhibitor of TNALP than of PLALP. In addition, mu-tagenesis of TNALP, while reducing the affinity of the-ophylline for the resulting mutant, did not modify theuncompetitive nature of the inhibition, as shown for the(Q434)TNALP mutant (Fig. 5B). As with levamisoleinhibition, the main determinant of sensitivity to theoph-ylline is His-434. The H434E substitution (as in PLALP)causes a 50-fold reduction in inhibition, making the cor-responding TNALP mutant even less inhibited than wild-type PLALP. The reciprocal E429H mutation in PLALPimproves the apparent Ki for theophylline inhibition by30-fold, nearly to the level of wildtype TNALP. Thus,strikingly, only this single substitution can account for allthe differences in theophylline inhibition betweenTNALP and PLALP. Several other substitutions at posi-tion 434 have been investigated and displayed interme-diate apparent Ki values between those of TNALP (25�M measured at 20 mM pNPP) and PLALP (947 �Mmeasured at 20 mM pNPP). One such mutation is H434S(as in the intestinal isozymes). Earlier experimental data

showed that human intestinal ALP is inhibited by the-ophylline better than PLALP. In agreement with thesedata, we found that (S434)TNALP is inhibited noticeablybetter than (E434)TNALP, although worse than wildtypeTNALP. Similarly, chicken TNALP, which has Gln-434,has a Ki value of 111 �M, measured at 20 mM pNPP.

Despite the fact that His-434 is clearly the most importantresidue for theophylline inhibition, we found that the inhi-bition can also be influenced by the nature of residues 433,371, and 108. TNALP has Arg in position 433. Mutations

FIG. 4. Additive uncompetitive inhibition of TNALP. Plot of resid-ual ALP enzyme activity (1/v) as a function of the concentration oflevamisole, measured with 20 mM pNPP in the presence of increasingconcentrations of L-homoarginine (f, absent; �, 0.5 mM; E, 2.5 mM).

FIG. 5. Uncompetitive nature of TNALP inhibition by theophylline.Double-reciprocal plot of ALP enzyme activity (v) vs. substrate con-centration ([S]) during the inhibition of TNALP by increasing concen-trations of theophylline. (A) Inhibition of wildtype TNALP by increas-ing concentrations of theophylline (f, absent; Œ, 25 �M; �, 50 �M).The inset shows the replot of 1/V(max) vs. [Theophylline] (�M) thatenabled correct assessment of Ki. (B) Inhibition of (H434Q)TNALP byincreasing concentrations of theophylline (f, absent; Œ, 250 �M; �,500 �M; F, 750 �M). The inset shows the replot of 1/V(max) vs.[Theophylline] (�M) that enabled correct assessment of Ki.


R433A and R433D (as in PLALP) decreased theophyllineinhibition two times. This effect was also noticeable in(D433, E434)TNALP and in the reciprocal (R433,H434)PLALP double mutant. The Y371A substitution onlyshowed a 2-fold decrease in inhibition. Interestingly, the-ophylline inhibition in TNALP could be further improvedby introducing Phe-108 (as in PLALP) instead of Glu inTNALP.

DISCUSSION

In PLALP, the best-studied mammalian ALP isozyme,two residues have been implicated in the binding of uncom-petitive inhibitors to the active site. The first is Glu-429(Gly-429 in GCAP), which determines the selectivity to-ward L-Leu between these two isozymes.(34,35,55) The sec-ond is Tyr-367, which we have shown to also have asignificant effect on the inhibition by L-Phe and L-Leu.(37)

As was suggested by computer modeling, this residue, per-fectly conserved in all mammalian ALPs, constitutes part ofthe hydrophobic binding site for the side chain of theinhibitor. However, nothing is currently known about theresidues that determine the specificity in inhibition in theTNALP molecule.

In this study, we have therefore produced a number ofTNALP and PLALP mutants, the catalytic efficiency ofwhich was only mildly affected. Therefore, these mutantswere suitable to study uncompetitive inhibition byL-hArg, L-Phe, levamisole, and theophylline, becausedifferences in Ki measured for different ALP mutantsreflect affinity changes in the active site pocket for thedifferent inhibitors, rather than mutation-dependentchanges in kinetic parameters.(36) The first obvious con-clusion relates to the crucial role of TNALP residue 108in determining the selectivity of inhibition by aminoacids. A single substitution, that is, E108F in TNALP,completely reversed the pattern of TNALP inhibition,rendering it more than 10 times more sensitive to L-Phethan to L-hArg inhibition. The reciprocal mutation, thatis, F107E in PLALP, made the enzyme nearly equallysensitive to L-Phe and L-hArg. However the double(E108, G109)PLALP mutant displayed a 2-fold selectiv-ity toward L-hArg compared with L-Phe. The importanceof residue 108 in TNALP is further supported by analysisof the (A108)TNALP mutant, which displayed a 10-foldreduction in inhibition by L-hArg and a slight reductionin L-Phe inhibition. These data suggest that selectivitytoward L-amino acid inhibition, in both TNALP andPLALP, is largely determined by residues 108 and 109.Docking studies for L-Phe in the PLALP active site(37)

and of L-hArg in the TNALP active site (Fig. 6A) giveadditional support to this conclusion, because they showthe side chains of amino acid inhibitors contacting withthe pocket formed by residue 108 and its neighbors.Residue 108 lies within the same pocket as Tyr-371(Tyr-367 in PLALP), which, as we have recentlyshown,(37) is important for the efficient inhibition ofPLALP by L-Phe. If the suggested mode of binding ofamino acid side chains in the active site of TNALP orPLALP is correct, one should expect reduced inhibition

in the case of Ala-371 substitution in TNALP. The dataobtained for the Ala-371 proves that this is true. Whentested for either L-Phe or L-hArg inhibition,

FIG. 6. Calculated optimal docking of the inhibitors (A)L-homoarginine, (B) levamisole, and (C) theophylline into the modeledactive site of TNALP. The figures were produced with the use ofTURBO-FRODO.


(A371)TNALP is one of the least efficiently inhibitedmutants in this study.

In addition to residues 108, 109, and 371, another posi-tion that has a large effect on the inhibition is His-434(Glu-429 in PLALP). In PLALP, the important effect ofsubstituting Glu-429 has been clearly shown.(34,35,55) How-ever, here we show that changes at His-434 in TNALP donot significantly affect the inhibition by either L-Phe orL-hArg (Table 2; Fig. 3). There is some experimental un-certainty in the determination of the Ki, as reflected by theSD values shown in Table 2. For this reason, we have notinterpreted differences in Ki smaller than a factor of 2. Thus,we can state that mutagenesis of residue 434 in TNALPhardly affects inhibition by L-Phe or by L-hArg. Thesesubstitutions include H434Q (a substitution distinguishingchicken TNALP from human TNALP), H434S (as in theintestinal isozyme), H434G (as in GCAP), and H434A. Theonly exception was observed in the (E434)TNALP mutant.Here a noticeable increase in Ki for both L-Phe and L-hArgwas observed, and the inhibition was further reduced in the(D433, E434)TNALP double mutant. Interestingly, the sin-gle substitutions at residue 433 did not affect the inhibition.In the reciprocal (H429)PLALP and (R428, H429)PLALPmutants, a 2-fold improvement in inhibition was observedfor L-Phe, whereas the inhibition by L-hArg was increased4- to 5-fold. Thus, all tested substitutions at residue 434 inTNALP or 429 in PLALP had parallel effects on the inhi-bition by L-Phe and L-hArg.

Summarizing the data for L-Phe and L-hArg inhibition,we can conclude that the binding of amino acid inhibitorsoccurs in the area E108-G109-Y371-H434 in TNALP(equivalent to F107-Q108-Y367-E429 in PLALP). Thespecificity of L-hArg inhibition in TNALP is most likelydetermined by electrostatic interaction of the positivelycharged side chain of the inhibitor with residue Glu-108 inthe active site. The conserved Tyr-371 is also necessary forthe binding, most likely by providing the hydrophobic areafor the nonpolar alkyl part of the inhibitor side chain. InPLALP, the residues Phe-107 and Gln-108, together withTyr-367, provide a hydrophobic pocket that accommodatesthe phenyl ring of phenylalanine. Finally, residue 434 inTNALP (429 in PLALP) modulates the inhibition. Here thenegatively charged glutamate side chain is clearly unfavor-able for the inhibition by either L-hArg or L-Phe. However,other substitutions at 434 show nearly the same level ofinhibition as in wildtype TNALP. We conclude that residue434 interacts with the carboxyl part of amino acid inhibitors,and the electrostatic effect is most important, while thespecific nature of the side chain at 434 plays minor role.

Levamisole, the L-stereoisomer of tetramisole, is a well-known potent uncompetitive inhibitor of TNALP,(31) and ithad been reported that chicken TNALP is about eight timesless sensitive to levamisole than human TNALP.(52)

Chicken TNALP has only a few substitutions in the activesite area compared with human TNALP, one of them beingglutamine at position 434. Our results showed that chickenTNALP is inhibited comparably, although slightly betterthan, the (Q434)TNALP variant of TNALP. The fact thatsuch different amino acids as Gly or Glu at this position allshowed a marked decrease in inhibition suggests that the

effect is not caused by steric hindrance, but rather dependson the particular presence of the amino acid His at thisposition. A possible explanation for the role of His-434 is astacking interaction between the flat hydrophobic groups ofthe protein and the ligand. The reciprocal E429H mutationin PLALP improved the inhibition by levamisole about 10times, further confirming the crucial role of this residue. Weconclude that the binding area for levamisole partially over-laps with the binding pocket for amino acid inhibitors, butis more spatially restricted than the latter. A more importantdifference between the two inhibitors is the nature of theirinteractions with the active site. In contrast with the inhibi-tion by amino acids, the selectivity of levamisole toward theTNALP isozyme is nearly fully explained by the substitu-tion at residue 434 (Table 2; Fig. 6B).

While our mutagenesis study has emphasized that His-434 is the main determinant of theophylline binding toTNALP, it is not possible to establish the exact orientationof the theophylline molecule in the active site. Data in theliterature have documented the effect of several substitu-tions in the xanthine structure.(56) For example, substitutionsat N7, C8, and N9 lead to compounds with no inhibitoryactivity (see Fig. 1 for numbering). One can conclude thatN7 or N9 is involved in important interactions with theenzyme, probably through Zn1 ion in the active site. Incontrast, several substitutions at N1 and N3 in theophyllinegave active compounds with altered selectivities towardbovine TNALP or intestinal isozymes. For example, a neg-atively charged 1-carboxymethyl derivative of theophyllinehas a greatly decreased activity against the TNALP isozymebut enhanced activity against the intestinal isozyme, thusbeing a selective inhibitor of the latter. Interestingly, bovineintestinal isozymes have a positively charged residue (Argor Lys) at the position homologous to Gly-109 in TNALPand a noncharged tyrosine at the position homologous tonegatively charged Glu-108 in TNALP. These data, to-gether with the positive effect of the Phe-108 substitution ontheophylline inhibition in our study, suggest that the bindingarea for the large ring of theophylline might lie near TNALPresidues 108/109 and that the nature of the substitutions atN1 (or N3) in theophylline should be complementary to thenature of residues 108/109 for effective inhibition to takeplace. Mutations at residue 434 lead to a wide range oftheophylline inhibition levels, suggestive of close interac-tions between residue 434 and the inhibitor (Fig. 6C). Thereason for the special significance of His-434 for the inhi-bition by theophylline (and levamisole) is not fully clear. Asalready discussed, a similar flattened structure of both in-hibitors may help form favorable stacking interactions withHis-434. In the PLALP structure, residues Asp-428 andGlu-429 have high side chain mobility, as judged by theB-factor values. The same should hold true for the homol-ogous residues Arg-433 and His-434 in TNALP. At thesame time, the increased mobility of the side chains of theseresidues may lead to a better adjustment during the bindingof the inhibitor molecules. While there is a consensus aboutthe uncompetitive nature of TNALP inhibition in the case ofL-amino acids and levamisole, inhibition by theophyllinehas been found to be either uncompetitive or noncompeti-tive.(32,38) We found theophylline inhibition of wildtype


TNALP to be uncompetitive, but also found, in agreementwith Glogowski et al.,(54) evidence of substrate inhibition athigh substrate concentrations, complicating a correct assess-ment of the Ki values for theophylline inhibition. ApparentKi values reported in Table 2 have therefore been deter-mined both at high (20 mM) and low (1 mM) substrateconcentrations.

In conclusion, by using four established uncompetitiveinhibitors of ALPs, this study identified all amino acidresidues, explaining the selectivity of inhibition of humanTNALP by these inhibitors. Through drug design ap-proaches, these findings will pave the way for the develop-ment of more specific and safe TNALP inhibitors for ther-apeutic use to treat pathological soft tissue mineralizationdisorders.

ACKNOWLEDGMENTS

This work was supported in part by National Institutes ofHealth Grants DE 12889 and AR 47908. AK was supportedby a stipend from Umeå University.

REFERENCES

1. McComb RB, Bowers GN, Posen S 1979 Alkaline Phosphatases.Plenum Press, New York, NY, USA.

2. Schwartz JH, Lipmann F 1961 Phosphate incorporation into alka-line phosphatase of E. coli. Proc Natl Acad Sci USA 47:1996–2005.

3. Harris H 1989 The human alkaline phosphatases: What we knowand what we don’t know. Clin Chim Acta 186:137–150.

4. Weiss MJ, Cole DEC, Ray K, Whyte MP, Lafferty MA, MulivorRA, Harris H 1988 A missense mutation in the human liver/bone/kidney alkaline phosphatase gene causing a form of lethal hy-pophosphatasia. Proc Natl Acad Sci USA 85:7666–7669.

5. Henthorn PS, Whyte MP 1992 Missense mutations of the tissue-nonspecific alkaline phosphatase gene in hypophosphatasia. ClinChem 38:2501–2505.

6. Mornet E 2000 Hypophosphatasia: The mutations in the tissue-nonspecific alkaline phosphatase gene. Hum Mutat 15:309–315.

7. Whyte MP 2001 Alkaline phosphatase. In: Scriver CR, BeaudetAL, Sly WS, Valle D, Childs B, Kinzler KW, Vogelstein B (eds.)The Metabolic and Molecular Bases of Inherited Disease.MacGraw-Hill, New York, NY, USA, pp. 5313–5329.

8. Zurutuza L, Muller F, Gibrat JF, Taillandier A, Simon-Bouy B,Serre JL, Mornet A 1999 Correlations of genotype and phenotypein hypophosphatasia. Hum Mol Genet 8:1039–1046.

9. Di Mauro S, Manes T, Hessle H, Kozlenkov A, Pizauro JM,Hoylaerts MF, Millan JL 2002 Kinetic characterization of hy-pophosphatasia mutations with physiological substrates. J BoneMiner Res 17:1383–1391.

10. Mornet E, Stura E, Lia-Baldini AS, Stigbrand T, Menez A, Le DuMH 2001 Structural evidence for a functional role of human tissue:On-specific alkaline phosphatase in bone mineralization. J BiolChem 276:31171–31178.

11. Anderson HC 1969 Vesicles associated with calcification in thematrix of epiphyseal cartilage. J Cell Biol 41:59–72.

12. Ali SY, Sajdera SW, Anderson HC 1970 Isolation and character-ization of calcifying matrix vesicles from epiphyseal cartilage.Proc Natl Acad Sci USA 67:1513–1520.

13. Ali SY, Anderson HC, Sajdera SW 1971 Enzymic and electron-microscopic analysis of extracellular matrix vesicles associatedwith calcification in cartilage. Biochem J 122:56P.

14. Bernard GW 1978 Ultrastructural localization of alkaline phospha-tase in initial membranous osteogenesis. Clin Orthop 135:218–225.

15. Morris DC, Masuhara K, Takaoka K, Ono K, Anderson HC 1992Immunolocalization of alkaline phosphatase in osteoblasts andmatrix vesicles of human fetal bone. Bone Miner 19:287–298.

16. Robison R 1923 The possible significance of hexosephosphoricesters in ossification. Biochem J 17:286–293.

17. Majeska RJ, Wuthier RE 1975 Studies on matrix vesicles isolatedfrom chick epiphyseal cartilage. Association of pyrophosphataseand ATPase activities with alkaline phosphatase. Biochim BiophysActa 391:51–60.

18. Fallon MD, Whyte MP, Teitelbaum SL 1980 Stereospecific inhi-bition of alkaline phosphatase by L-tetramisole prevents in vitrocartilage calcification. Lab Invest 43:489–494.

19. Moss DW, Eaton RH, Smith JK, Whitby LG 1967 Association ofinorganic pyrophosphatase activity with human alkaline phospha-tase preparations. Biochem J 102:53–57.

20. Meyer JL 1984 Studies on matrix vesicles isolated from chickepiphyseal cartilage. Association of pyrophosphatase and ATPaseactivities with alkaline phosphatase. Arch Biochem Biophys 231:1–8.

21. Rezende A, Pizauro J, Ciancaglini P, Leone F 1994 Phosphodies-terase activity is a novel property of alkaline phosphatase fromosseous plate. Biochem J 301:517–522.

22. Anderson HC, Hsu HH, Morris DC, Fedde KN, Whyte PW 1997Matrix vesicle in osteomalacic hypophosphatasia bone containapatite-like mineral crystals. Am J Pathol 151:1555–1561.

23. Anderson HC, Sipe JE, Hessle L, Dhamayamraju R, Atti E, Ca-macho NP, Millan JL 2004 Impaired calcification around matrixvesicles of growth plate and bone in alkaline phosphatase-deficientmice. Am J Pathol 164:841–847.

24. Johnson KA, Hessle L, Wennberg C, Mauro S, Narisawa S, God-ing J, Sano K, Millan JL, Terkeltaub R 2000 Tissue-nonspecificalkaline phosphatase (TNALP) and plasma cell membraneglycoprotein-1 (PC-1) act as selective and mutual antagonists ofmineralizing activity by murine osteoblasts. Am J Physiol RegulIntegr Comp Physiol 279:R1365–R1377.

25. Hessle L, Johnsson KA, Anderson HC, Narisawa S, Sali A, GodingJW, Terkeltaub R, Millan JL 2002 Tissue-nonspecific alkalinephosphatase and plasma cell membrane glycoprotein-1 are centralantagonistic regulators of bone mineralization. Proc Natl Acad SciUSA 99:9445–9449.

26. Johnson K, Goding J, Van Etten D, Sali A, Hu SI, Farley D, KrugH, Hessle L, Millan JL, Terkeltaub R 2003 Linked deficiencies inextracellular inorganic pyrophosphate (PPi) and osteopontin ex-pression mediate pathologic ossification in PC-1 null mice. J BoneMiner Res 18:994–1004.

27. Harmey D, Hessle L, Narisawa S, Johnson KA, Terkeltaub RA,Millan JL 2004 Concerted regulation of inorganic pyrophosphateand osteopontin by Akp2. Enpp1 and Ank: An integrated model ofthe pathogenesis of mineralization disorders. Am J Pathol 164:1199–1209.

28. Fishman WH, Sie HG 1971 Organ-specific inhibition of humanalkaline phosphatase isoenzymes of liver, bone, intestine and pla-centa; L-phenylalanine, L-tryptophan and L-homoarginine. Enzy-mologia 41:140–167.

29. Fishman WH 1974 Perspectives on alkaline phosphatase isoen-zymes. Am J Med 56:617–650.

30. Doellgast GJ, Fishman WH 1977 Inhibition of human placental-type alkaline phosphatase variants by peptides containingL-leucine. Clin Chim Acta 75:449–454.

31. Van Belle H 1976 Alkaline phosphatase. I. Kinetics and inhibitionby levamisole of purified isoenzymes from humans. Clin Chem22:972–976.

32. Farley JR, Ivey JL, Baylink DJ 1980 Human skeletal alkalinephosphatase-kinetic studies including pH dependence and inhibi-tion by theophylline. J Biol Chem 255:4680–4686.

33. Cornish-Bowden A 1995 Fundamentals of Enzyme Kinetics. Port-land Press Ltd, London, UK.

34. Hummer C, Millan JL 1991 Gly429 is the major determinant ofuncompetitive inhibition of human germ cell alkaline phosphataseby L-Leucine. Biochem J 274:91–95.

35. Hoylaerts MF, Millan JL 1991 Site-directed mutagenesis andepitope-mapped monoclonal antibodies define a specific importantconformational difference between human placental and germ cellalkaline phosphatase. Eur J Biochem 202:605–616.

36. Hoylaerts MF, Manes T, Millan JL 1992 Molecular mechanism ofuncompetitive inhibition of human placental and germ cell alkalinephosphatase. Biochem J 286:23–30.

37. Kozlenkov A, Manes T, Hoylaerts MF, Millan JL 2002 Functionassignment to conserved residues in mammalian alkaline phospha-tases. J Biol Chem 277:22992–22999.


38. Ansari A, Salem FA 1982 Determination of human serum placen-tal alkaline phosphatase using theophylline inhibition. Clin ChimActa 118:135–139.

39. Witte RS, Cnaan A, Mansour EG, Barylak E, Harris JE, Schutt AJ2001 Comparison of 5-fluorouracil alone, 5-flourouracil with le-vamisole, and 5-flourouracil with hepatic irradiation in the treat-ment of patients with residual, nonmeasurable, intra-abdominalmetastasis after undergoing resection for colorectal carcinoma.Cancer 91:1020–1028.

40. Taal BG, van Tinteren H, Zoetmulder FA, NACCP group 2001Adjuvant 5FU plus levamisole in colonic or rectal cancer; im-proved survival in stage II and III. Br J Cancer 85:1437–1443.

41. Powe TA, Powers RD 1985 Periorchitis after tetramisole treatmentin bulls implaned with Setaria labiatopapillos. J Am Vet MedAssoc 186:588–589.

42. Bhopale GM, Bhatnagar BS 1984 Serum protein profile of miceduring infection of Ancylostoma caninum and after the adminis-tration of tetramisole and levamisole. J Hyg Epidemiol MicrobiolImmunol 28:455–459.

43. Eisenberg E, Shklar G 1977 Levamisole and hamster pouch car-cinogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod43:562–571.

44. McPherson ML, Prince SR, Atamer ER, Maxwell DB, Ross-ClunisH, Estep HL 1986 Theophylline-induced hypercalcemia. Ann In-tern Med 105:52–54.

45. Le Du MH, Millan JL 2002 Structural evidence of functionaldivergence in human alkaline phosphatases. J Biol Chem 277:49808–49814.

46. Guex N, Peitsch MC 1997 SWISS-MODEL and the Swiss-Pdb-Viewer: An environment for comparative protein modeling. Elec-trophoresis 18:2714–2723.

47. Nilges M, Clore M, Gronenborn AM 1988 Determination of three-dimensional structures of proteins from interproton distance databy dynamical simulated annealing from a random array of atoms.Circumventing problems associated with folding. FEBS Lett 239:129–136.

48. Brunger AT 1993 X-PLOR, version 3.1. Yale University Press,New Haven, CT, USA.

49. MacKerell AD, Bashford D, Bellott M, Dunbrack RL, EvanseckJD, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph-McCarthy D,

Kucnhir L, Kuczera K, Lau FTK, Mattos C, Michnick S, Ngo T,Nguyen DT, Prodhom B, Reiher WE, Roux B, Schlenkrick M,Smith JC, Stote R, Straub J, Watanabe M, Wiorkiewicz-Kuczera J,Yin D, Karplus M 1998 All-atom empirical potential for molecularmodeling and dynamics studies of proteins. J Phys Chem 102:3586–3616.

50. Frisch M Available online at http://www.gaussian.com.51. Le Du MH, Stigbrand T, Taussig MJ, Menez A, Stura EA 2001

Crystal structure of alkaline phosphatase from human placenta at1.8 A resolution. Implications for substrate specificity. J BiolChem 276:9158–9165.

52. Reynolds JJ, Dew GW 1977 Comparison of the inhibition of avianand mammalian bone alkaline phosphatases by levamisole andcompound R8231. Experientia 33:154–155.

53. Lin CW, Fishman WH 1972 L-Homoarginine: An organ-specific,uncompetitive inhibitor of human liver and bone alkaline phos-phohydrolases. J Biol Chem 247:3082–3087.

54. Glogowski J, Danforth DR, Ciereszko A 2002 Inhibition of alka-line phosphatase activity of boar semen by pentoxifylline, caffeine,and theophylline. J Androl 23:783–792.

55. Watanabe T, Wada N, Kim EE, Wyckoff HW, Chou JY 1991Mutation of a single amino acid converts germ cell alkaline phos-phatase to placental alkaline phosphatase. J Biol Chem 266:21174–21178.

56. Croce MA, Kramer GL, Garbers DL 1979 Inhibition of alkalinephosphatase by substituted xanthines. Biochem Pharmacol 28:1227–1231.

Address reprint requests to:Jose Luis Millan, PhDThe Burnham Institute

10901 North Torrey Pines RoadLa Jolla, CA 92037, USA

E-mail: [email protected]

Received in original form February 26, 2004; in revised form April1, 2004; accepted June 28, 2004.


Residues Determining the Binding Specificity of Uncompetitive Inhibitors to Tissue-Nonspecific Alkaline Phosphatase

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