Full text of DOI:10.1359/jbmr.2002.17.8.1383

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 8, 2002
© 2002 American Society for Bone and Mineral Research
Kinetic Characterization of Hypophosphatasia Mutations
With Physiological Substrates
SONIA DI MAURO,1,2 THOMAS MANES,1 LOVISA HESSLE,1,3 ALEXEY KOZLENKOV,3
2
4
1
˜
´
´
JOAO MARTINS PIZAURO JR., MARC F. HOYLAERTS, and JOSE LUIS MILLAN
ABSTRACT
We have analyzed 16 missense mutations of the tissue-nonspecific AP (TNAP) gene found in patients with
hypophosphatasia. These mutations span the phenotypic spectrum of the disease, from the lethal perinatal/
infantile forms to the less severe adult and odontohypophosphatasia. Site-directed mutagenesis was used to
introduce a sequence tag into the TNAP cDNA and eliminate the glycosylphosphatidylinositol (GPI)-anchor
recognition sequence to produce a secreted epitope-tagged TNAP (setTNAP). The properties of GPI-anchored
TNAP (gpiTNAP) and setTNAP were found comparable. After introducing each single hypophosphatasia
mutation, the setTNAP and mutant TNAP cDNAs were expressed in COS-1 cells and the recombinant flagged
enzymes were affinity purified. We characterized the kinetic behavior, inhibition, and heat stability properties
of each mutant using the artificial substrate p-nitrophenylphosphate (pNPP) at pH 9.8. We also determined the
ability of the mutants to metabolize two natural substrates of TNAP, that is, pyridoxal-5؅-phosphate (PLP) and
inorganic pyrophosphate (PPi), at physiological pH. Six of the mutant enzymes were completely devoid of
catalytic activity (R54C, R54P, A94T, R206W, G317D, and V365I), and 10 others (A16V, A115V, A160T,
A162T, E174K, E174G, D277A, E281K, D361V, and G439R) showed various levels of residual activity. The
A160T substitution was found to decrease the catalytic efficiency of the mutant enzyme toward pNPP to retain
normal activity toward PPi and to display increased activity toward PLP. The A162T substitution caused a
considerable reduction in the pNPPase, PPiase, and PLPase activities of the mutant enzyme. The D277A
mutant was found to maintain high catalytic efficiency toward pNPP as substrate but not against PLP or PPi.
Three mutations ( E174G, E174K, and E281K) were found to retain normal or slightly subnormal catalytic
efficiency toward pNPP and PPi but not against PLP. Because abnormalities in PLP metabolism have been
shown to cause epileptic seizures in mice null for the TNAP gene, these kinetic data help explain the variable
expressivity of epileptic seizures in hypophosphatasia patients. (J Bone Miner Res 2002;17:1383–1391)
Key words: genetic disease, missense mutations, catalytic efficiency, natural substrates, alkaline phosphatase
Robison(2) within ossifying bone and cartilage and he pro-
posed that this catalytic action had a role in hydrolyzing
INTRODUCTION
P (orthophosphoric-monoester phosphohydrolase, alka-
line optimum EC 3.1.3.1) is present in most species
monophosphate esters thus increasing the concentration of
inorganic phosphate. The clearest evidence that APs are
important in bone mineralization has been provided by
studies of human hypophosphatasia where a deficiency in
the tissue-nonspecific AP (TNAP) isozyme is associated
A
from bacteria to man.(1) The enzyme was discovered by
The authors have no conflict of interest.
1The Burnham Institute, La Jolla, California, USA.
2Faculdade de Cieˆncias Agra´rias e Veterina´rias/UNESP, Jaboticabal, Sa˜o Paulo, Brazil.
3Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
4Department of Medical Genetics, Umeå University, Umeå, Sweden.
1383

1384
DI MAURO ET AL.
with defective bone mineralization.(3) The clinical severity zures and apnea, which are prominent in some cases of
in hypophosphatasia patients varies widely.(4) The different hypophosphatasia.
syndromes, listed from the most severe to the mildest, are
perinatal, infantile, childhood, adult, and odonto- and
MATERIALS AND METHODS
Wild-type and TNAP mutant cDNAs
pseudohypophosphatasia. The phenotypic abnormalities
range from complete absence of bone mineralization and
stillbirth to spontaneous fractures and loss of decidual teeth
in adult life. The severe forms of hypophosphatasia usually
are inherited as autosomal recessive traits with parents of
such patients showing subnormal levels of serum AP activ-
ity. Variable expressivity and incomplete penetrance are
common features.(4) For the milder forms, that is, adult and
odontohypophosphatasia, an autosomal dominant pattern of
inheritance has been documented.(4) The molecular basis for
these complex patterns of inheritance is not understood
completely.
The first identified hypophosphatasia mutation was a mis-
sense mutation in exon 6, A162T.(5) Subsequently, com-
pound heterozygosity and new mutations, (i.e., R54C,
R54P, E174K, Q190P, Y246H, D277A, D361V, and
Y419H) were reported.(6,7) Other mutations found since
include G317D(8); F310L and G439R(9); E281K, A160T,
and a frame shift mutation at position 328 and another at
position 503.(10) Mornet et al.(11) reported 16 new missense
mutations in European patients (i.e., S-1F, A23V, R58S,
G103R, G112R, N153D, R167W, R206W, W253X,
E274K, S428P, R433C, G456S, G474R and two splice
mutations in intron 6 and 9, respectively). Interestingly, in a
recent historical vignette, the mutations present in the
original hypophosphatasia patient studied by Rathbun in
1948,(3) were identified also.(12) That 3-week-old boy was a
compound heterozygote for the A97T and D277A muta-
tions.
To date, a total of 65 distinct mutations have been de-
scribed for the human TNAP gene.(13) Mutagenesis studies
and structural predictions(14,15) have increased our under-
standing of the correlation between genotype and pheno-
type. Interestingly, defective trafficking of several hy-
pophosphatasia mutations (i.e., R54C,(16) A162T,(17) and
G317D(18)) through the Golgi has been established as a
mechanism that contributes to the variable expressivity of
this disease.
In this report we provide a kinetic characterization of 16
different hypophosphatasia mutations that span the spec-
trum of severity of the disease. The mutant enzymes were
produced in a soluble secreted form to enable us to charac-
terize their kinetic properties independently of how effi-
ciently these mutant enzymes may be exported to the cell
membrane. This strategy also allowed us to increase the
yield of purified recombinant TNAP and obtain the quanti-
ties necessary to perform the kinetic studies using physio-
The TNAP cDNA (ATCC no. 59635; American Type
Culture Collection, Manassas, VA, USA) was used as tem-
plate for the construction of the wild-type and mutated
TNAP expression vectors. To facilitate isolation of the
recombinant enzymes, an FLAG epitope (5Ј-TTA CTT
GTC ATC GTC GTC CTT GTA GTC-3Ј) was introduced
after the Leu489, followed by a termination codon to elim-
inate the glycosylphosphatidylinositol anchoring signal.
This secreted epitope-tagged TNAP (setTNAP) was used as
a template for the introduction of all hypophosphatasia
mutations.
Site-directed mutagenesis was performed as described by
Tomic.(19) The sequence of the primers used to generate the
mutations are shown in the following with amino acid
changes underlined: A16V, G TAC TGG CGA GAC CAA
GTC CAA GAG ACA CTG; R54Cc, CC AGG GTT GTG
GTG GAG CTG ACC CTT GAG GAT GCA GGC AGC
CGT C; R54Pc, CC AGG GTT GTG GTG GAG CTG ACC
CTT GAG GAT GGG GGC AGC CGT C; A94TC, G CTC
TCC GGT GGC GGT GCC GGT ACT GTC AGG G;
A115Vc, CGT CTC ACG CTC AGT GGC TAC GCT TAC
CCC CAC; A160Tc, CG TCT CTC AGC CGA GTG GGC
GTA GGT GGC GCT GG; A162Tc, CG TCT CTC AGC
CGA GTG GGT GTA GGC GGC; E174Kc, CG TCT CAA
GGC CTC AGG GGG CAT CTT GTT GTC TGA G;
E174Gc, CG TCT CAA GGC CTC AGG GGG CAT CCC
GTT GTC TGA G; R206W, ATT GAC GTG ATC ATG
GGG GGT GGC TGG AAA TAC ATG T; D277Ac, CG
TCT CTA CTG CAT GGC CCC CGG CTC G; E281K, C
GTC TCG CAG TAC AAG CTG AAC AGG AAC AA;
G317Dc, C GTC TCG CCC GTG GTC AAT TCT GTC
TCC TTC CAC C; D361V, CG TCT CTC ACT GCG GTC
CAT TCC CAC GTC; V365I, CG TCT CTC ACT GCG
GAC CAT TCC CAC ATC TTC ACA TTT GG; G439Rc,
AT GGG GCC CTT GGA GAA GAC GGC CAC GTC
CTC CCG GCC GTG GGT C.
All TNAP cDNAs were subcloned into the pcDNA 3.1
expression vector (HindIII/SpeI) downstream from the
SV40 early promoter. All constructs were verified by re-
striction digestion and DNA sequencing.
Homology modeling of the TNAP molecule
We used the program MODELLER(20) to construct a
logical substrates at neutral pH. We have characterized homology model of dimeric TNAP based on a recently
these mutants for their hydrolyzing and transferase activity published structure of human placental AP (PLAP).(21) The
using the synthetic substrate p-nitrophenylphosphate (pNPP) relatively high level of sequence similarity (55% identical
and the two natural TNAP substrates (i.e., pyridoxal-5Ј- residues) between amino acid sequences of TNAP and
phosphate [PLP] and inorganic pyrophosphate [PPi]) that PLAP simplified the task of preparing the initial alignment.
are associated with the clinical manifestations of the dis- Five different models of TNAP were produced and the one
ease.(4) Our data indicate that the preferential use of PPi with the lowest MODELLER objective function was used
versus PLP as substrate by some missense mutations can for further examination. Metals and phosphate ions were
help explain the variable expressivity of the epileptic sei- included during the calculation to preserve the geometry of

HYPOPHOSPHATASIA MUTATIONS
1385
their conserved ligands. The only areas where the models leased in the reaction medium was determined by the pro-
differed significantly were a three-amino acid insertion after cedure of Heinonen and Lahti.(24) All determinations were
Ser241 and the C-terminal fragment after Ser485, for which carried out in duplicate and the initial velocities were con-
no template in the structure of PLAP was available.
stant for at least 60 minutes provided that Ͻ5% of substrate
was hydrolyzed. Controls without added enzyme were in-
cluded in each experiment to measure the nonenzymatic
hydrolysis of the substrate. Comparisons between mean
Tissue culture and enzyme purification
The expression constructs were transfected into COS-1 values were done using the Tukey test at 5% probability
cells by the diethylaminoethyl (DEAE) dextran (100 mg/ml level.
in Tris-buffered saline [TBS]) procedure and a 10%
DMSO/2% fetal calf serum shock to increase the transfec-
tion efficiency. The medium (25 ml/150-mm plate) was
Protein determination and Western blots
The enzyme concentration of each purified sample was
determined by the Quantigold (Diversified Biotech, Boston,
MA, USA) kit using bovine serum albumin (BSA) as a
standard and independently by a densitometric analysis of
Western blots. For Western blots, electrophoresed proteins
were transferred to polyvinylidene difluoride (PVDF) mem-
branes (Amersham Pharmacia Biotech, Buckinghamshire,
UK) followed by blocking in 25 mM of Tris-HCl, pH 7.4,
containing 137 mM of NaCl (TBS) also containing 1%
Tween 20 and 2% BSA. Subsequently, the membranes were
incubated with 1 ␮g/ml of M2 anti-FLAG antibody (Sigma)
in TBS for 60 minutes. After three washes in TBS, the
membranes were treated with a second antibody using the
Vectastain ABC kit (Vector Laboratories, Burlingame, CA,
USA) following the manufacturer’s instructions.
collected 72, 120, and 168 h after transfection. Each se-
creted FLAG-tagged mutant enzyme was purified using an
anti-FLAG M2 monoclonal antibody affinity column
(Sigma, St. Louis, MO, USA) as per the manufacturer’s
instructions. GPI-anchored wild-type and mutant enzymes
were extracted 72 h after transfections using a 1:1 mixture
of n-butanol and 50 mM of sodium acetate buffer, pH 5.5,
containing 100 mM of NaCl, 20 ␮M of ZnCl2, 2 mM of
MgCl2, and 0.5% thimerosal.
Kinetic measurements
For the kinetic studies, the affinity-purified enzymes were
diluted in TBS to the same optical density at 280 nm.
Enzymatic reactions were carried out using pNPP in 1 M of
diethanolamine (DEA) buffer (pH 9.8) containing 1 mM of
MgCl2 and 20 ␮M of ZnCl2. Enzyme reactions also were
performed at pH 7.5 in 50 mM of Tris buffer, containing 1
mM of MgCl2 and 20 ␮M of ZnCl2. The inhibition prop-
erties of the mutant enzymes toward L-homoarginine and
levamisole were measured in microtiter plates by adding 2
RESULTS
Hypophosphatasia mutations
We introduced an eight-amino acid FLAG tag sequence
␮l of enzyme to a mixture of 100 ␮l of 10 mM of pNPP in and a premature stop codon at position 489 into the wild-
DEA, pH 9.8, supplemented with 1 mM of MgCl2, 20 ␮M type TNAP cDNA to facilitate the expression, recovery, and
of ZnCl2, and 100 ␮l of inhibitor solution (ranging in purification of the large amounts of recombinant enzyme
concentration from 0 to 10 mM for L-homoarginine and 0 to from COS-1 cells needed for the subsequent characteriza-
100 ␮M for levamisole). For heat stability studies, the tions. To ascertain how these modifications might affect the
GPI-anchored TNAP and all setTNAP mutants were diluted properties of the resulting enzyme, we compared the
in TBS containing 10% casein, 1 mM of MgCl2, and 20 ␮M setTNAP and the wild-type GPI-anchored TNAP (gpiTNAP)
of ZnCl2 and incubated in a thermocycler for 10 minutes at in terms of affinity for substrate, inhibition properties, and
a temperature ranging from 45 to 62.5°C at 2.5°C intervals heat stability. We observed a small, but statistically signif-
as previously described.(22) Heat stability was determined icant increase in Km for setTNAP (0.45 Ϯ 0.04 mM) com-
also by inactivation at 56°C up to 25 minutes. Ten- pared with gpiTNAP (0.34 Ϯ 0.10 mM; p Ͻ 0.05) while the
microliter samples were removed every 5 minutes and pi- inhibition constants toward the uncompetitive inhibitor
petted into a well of a microtiter plate kept on ice. Residual L-homoarginine (3.6 mM vs. 4.0 mM) were comparable.
activities then were measured in triplicate. Michaelis- Both setTNAP and gpiTNAP showed 50% inactivation at
Menten constants (Km and Vmax) were determined graphi- 56°C. The setTNAP showed a 15-minute half-life compared
cally from Woolf plots.
with a 13-minute half-life for gpiTNAP when subjected to a
time-dependent inactivation at 56°C.
Using setTNAP as a template, we constructed 16 TNAP
mutant cDNAs corresponding to a wide spectrum of re-
PLP and pyrophosphatase activity
PLP and pyrophosphatase activities were assayed discon- ported hypophosphatasia mutations (Table 1) and expressed
tinuously at 37°C. Standard assay conditions were 50 mM them in COS-1 cells. All mutant cDNAs were expressed in
of Tris buffer, pH 7.5, containing 1 mM of MgCl2 and 20 the cells and recombinant protein could be recovered from
␮M of ZnCl2 and increasing concentrations of substrate (0.1 the culture medium as shown by Western blot analysis (Fig.
␮M–10 mM) in a final volume of 0.08 ml. The reaction was 1). The small differences in electrophoretic migration of the
initiated by the addition of the enzyme and interrupted with mutant TNAPs are reduced, but not eliminated, by neur-
the addition of 0.02 ml of 50% trichloroacetic acid at aminidase treatment. The mutant enzymes were affinity-
appropriate times as described.(23) Inorganic phosphate re- purified and characterized. The purified TNAPs also were

1386
DI MAURO ET AL.
TABLE 1. HYPOPHOSPHATASIA MUTATIONS STUDIED
Mutations
Type
Inheritance
Compound heterozygote with
Reference
A16V
R54C
R54P
A94T
P/I
P/I
P/I
Odonto
Adult
Adult
P/I
I/Adult
Odonto
Lethal
I/Adult
P/I
Lethal
Adult/Odonto
I
R
R
R
D
D
R
R
R?
D?
D?
R
R
R
D?
R
R
Y419H
D277A
Q190P
none
none
F310L
none
D361V/D277A/E274K
503-fr. Shift
None
R54C/E174K
503-fr. Shift
none
E174K
F310L
F310L
Henthorn et al.(7)
Henthorn et al.(7)
Henthorn et al.(7)
Goseki-Sone et al.(29)
Watanabe et al.(28)
Goseki-Sone et al.(29)
Weiss et al.(5)
A115V
A160T
A162T
E174K
E174G
R206W
D277A
E281K
G317D
D361V
V365I
G439R
Henthorn et al.(7)
Goseki-Sone et al.(29)
Mornet et al.(11)
Henthorn et al.(7)
Orimo et al.(10)
Greenberg et al.(8)
Henthron et al.(7)
Goseki-Sone et al.(29)
Ozono et al.(9)
P/I
Included are the type of hypophosphatasia in which the mutation was found, the mode of inheritance, with what other mutation it was
found in compound heterozygotes (if applicable), and the original reference that identified the mutation.
P, perinatal hypophosphatasia; I, infantile hypophosphatasia; Odonto, odontohypophosphatasia; Adult, adult hypophosphatasia; R,
recessive inheritance; D, dominant inheritance.
FIG. 1. Western blot analysis of the setTNAP and secreted hypophosphatasia mutant enzymes. All mutant enzymes were expressed and secreted
appropriately into the culture medium. The setTNAP and a commercial secreted and flagged-bacterial AP (BAP) were used as positive controls.
electrophoresed on nondenaturing gels and stained by Coo-
massie blue as well as for AP activity (not shown). This
analysis revealed that although all mutant enzymes were
isolated from the medium as dimers, only some mutants
retained catalytic activity. In fact, six mutant TNAPs were
completely inactive (i.e., R54C, R54P, A94T, R206W,
G317R, and V365I). However, 10 of the 16 mutants dis-
played various amounts of residual activity (i.e., A16V,
A115V, A160T, A162T, E174K, E174G, D277A, E281K,
D361V, and G439R). Figure 2 shows a model representa-
tion of those amino acid substitutions that lead to complete
inactivation of the TNAP activity, as well as of those
Kinetic parameters under optimal in vitro conditions
Table 2 shows the kinetic parameters and heat stability
properties of the 10 active mutant TNAPs using the artificial
substrate pNPP at pH 9.8. The kcat value for the wild-type
setTNAP was 551. The mutations introduced major changes
in affinity for the substrate as well as in the catalytic rate
constants.
The Km values for the D277A, D361V, and G439R mu-
tants were increased 2.2-, 344-, and 253-fold compared with
setTNAP; the E281K mutant on the contrary showed a 50%
reduction in Km, reflecting a higher affinity for pNPP. The
mutations that result in mutants with residual enzyme ac- A16V and A115V mutations result in reduced Km values in
tivity. The model of TNAP was constructed based on the association with a reduction in kcat, yielding a catalytic
crystal structure of human PLAP.(21)
efficiency of 10% of the control value. The D361V and

HYPOPHOSPHATASIA MUTATIONS
1387
FIG. 2. Location of the investi-
gated hypophosphatasia mutants
on
the
modeled
three-
dimensional structure of TNAP.
(A) Amino acid substitutions
leading to complete loss of func-
tion (R54C, R54P, A94T,
R206W, G317R, and V365I) are
shown in space-filling representa-
tion colored in red. Amino acid
substitutions leading to various
degrees of TNAP activity loss are
represented as space-filling resi-
dues with different colors: or-
ange, very low activity remaining
(A115T, D361V, and G439R);
yellow, moderate activity remain-
ing (E174G, E174K, D277A, and
E281K); and green, activity com-
parable or greater than with wild-
type TNAP (A160T). The muta-
tions are shown as space-filling
residues only on the panel A sub-
unit, which is displayed in back-
bone representation and colored
in white. The panel B subunit is
displayed as ribbons emphasizing
regions of secondary structure
and is colored in magenta. The
metal ions (Zn1, Zn2, Mg, and
Ca) are identified in the panel B
subunit for reference.
G439R mutants had the highest Km with the lowest kcat the stability of the mutants. Because D361V and G439R
values and consequently had the lowest catalytic efficiency. mutants had very low activity, they could not be tested in
The change of a glutamic acid to glycine or lysine in E174K the heat stability studies. The enzyme resulting from the
and E174G had little impact on Km and kcat, despite the D277A substitution at 56°C loses activity with a half-life of
major change of charge in the E174 mutant. The catalytic 5 minutes. Both D277A, a mutation present in infantile and
efficiency decreased to 50% for the E174G mutant. The adult hypophosphatasia, and A16V, present in perinatal and
A160T and A162T mutants had the same value for Km, but infantile hypophosphatasia, progressively lost their activity
A160T showed an increase of 100% in kcat, resulting in an above 47.5°C; at 57.5°C the D277A mutant was almost
enzyme with an almost threefold increased catalytic effi- inactive after 15 minutes and the A16V mutant retained
ciency compared with setTNAP.
20% of its initial activity. Interestingly, whereas the E174G
TNAP loses its activity very rapidly at 56°C.(25) As mutant had a heat stability comparable with that of
shown in Table 2, most mutations had very little impact on setTNAP, the E174K mutant appeared to be more stable

1388
DI MAURO ET AL.
TABLE 2. KINETIC PARAMETERS AND STABILITY PROPERTIES OF SETTNAP AND HYPOPHOSPHATASIA MUTATIONS
Half-life
56°C (min)
50%
Inactivation (°C)
L-homoarginine
Levamisole
Ki (␮M)
kcat
k
cat/Km ϫ 103
MUTANT
Km (mM)
Ki (mM)
(sϪ1
)
(MϪ1sϪ1
)
setTNAP
A16V
0.45 Ϯ .04
0.34 Ϯ .07
0.32 Ϯ .04
0.34 Ϯ .04
0.34 Ϯ .06
0.47 Ϯ .07
0.34 Ϯ .07
1.0 Ϯ .09
15.0
12.2
12.8
12.2
12.0
13.0
Ͼ25.0
5.0
56.0
55.0
55.0
56.0
55.5
55.5
57.0
53.0
56.0
ND
4.0
4.8
5.6
3.8
5.6
3.0
4.3
6.0
3.8
ND
ND
51.0
ND
ND
55.0
30.0
68.0
100.0
64.0
61.0
ND
551
40
36
1083
331
294
452
350
156
8
1224
116
114
3187
973
627
1329
350
650
Ͻ0.05
0.05
A115V
A160T
A162T
E174G
E174K
D277A
E281K
D361V
G439R
0.24 Ϯ .07
Ͼ155 Ϯ 23
114 Ϯ 12
20.0
ND
ND
ND
ND
6
Measurements were done at pH 9.8 using pNPP as substrate.
(Table 2). After 15 minutes at 56°C (half-life for setTNAP) simultaneously causing a raise in kcat and Km. For A162T
the E174K mutant retained 75% of its activity, making it the and D277A we were not able, using this methodology, to
most stable enzyme tested. Also, the E281K mutant ap- detect any activity when PLP was the substrate. In contrast,
peared to have gained stability with respect to setTNAP, setTNAP showed comparable catalytic efficiency for both
with a half-life of 20 minutes.
PPi and PLP as substrates.
The setTNAP and all active mutant enzymes were tested
for their residual activity in the presence of the uncompeti-
tive inhibitors L-levamisole or L-homoarginine. As shown
on Table 2, the Ki value was only mildly affected for most
mutated enzymes studied, using either inhibitor. The largest
variation, a twofold increase in Ki for levamisole, was
observed with the E174K mutant. Thus, the amino acid
substitutions interfered less with enzyme inhibition than
with the actual catalytic process.
DISCUSSION
TNAP dysfunction has been associated with hypophos-
phatasia, a disease characterized by deficiency in bone min-
eralization.(4) In this study we characterized 16 mutations
found in patients with a different degree of severity of the
disease. To be able to perform our analysis with purified
enzyme, we tagged the 3Ј-terminal sequence of the TNAP
encoding cDNA with FLAG in front of the stop codon and
PLP and pyrophosphatase activity at physiological pH
To validate whether the shift in activity measured for the the sequence encoding the GPI anchor. This approach made
TNAP mutants under optimal conditions (i.e., in the pres- it possible to produce the large amounts of recombinant
ence of a transphosphorylating alcohol at pH 9.8) reflects TNAP needed for the kinetic experiments at physiological
residual activities at more physiological pH, activity mea- pH, where the requirements for recombinant enzyme in-
surements were done at pH 7.5. As shown in Table 3, the creased 20- to 100-fold for most mutants. The kinetic pa-
relative activities of those six mutants that had retained rameters determined for released setTNAP and membrane-
sufficiently high activity at pH 9.8 were differentially af- bound TNAP were comparable, indicating that this
fected at pH 7.5.
approach would not confound the kinetic properties of the
When measured with pNPP, the lowest kcat was found in mutant enzymes. This strategy also enabled us to character-
the case of the E174K mutation and the highest Km was ize the behavior of some missense mutations known to
found for the A160T mutation. To further know whether the traffic abnormally through the Golgi.
mutants would be active with physiological substrates, spe-
All mutant TNAP were produced efficiently and secreted
cific activity measurements also were done with two natural into the medium where they were recovered and purified.
substrates (PPi and PLP) and their Km and kcat and catalytic The purified recombinant TNAPs displayed small differ-
efficiency were determined as reported in Table 3. In com- ences in migration on SDS-PAGE, which are in part ex-
parison with setTNAP, the Km value with PPi and PLP was plained by differences in the sialic acid content, because
altered for all the mutant enzymes tested. When measured neuraminidase treatment reduced some of the heterogeneity.
with PPi, the A162T and D277A mutants showed a consid- However, conformational changes resulting from the point
erable drop in Km, indicative of facilitated substrate inter- mutations also are likely to contribute to this differential
actions. However, both mutants showed a strong reduction migration. The recombinant TNAPs were subjected also to
in kcat, that is, leading to weak catalytic efficiencies. When electrophoresis on nondenaturing gels. This analysis con-
PLP was the substrate, the mutants E174K and E281K firmed that all mutants were produced and secreted as
showed the largest drop in kcat (Table 3). The A160T dimeric molecules even when the point mutation resulted in
mutation maintained a comparable catalytic efficiency by the complete abolishment of enzymatic activity.

HYPOPHOSPHATASIA MUTATIONS
1389
The six mutations that produced inactive enzymes were
found in patients with perinatal and infantile forms of hy-
pophosphatasia alone or in compound heterozygosity. R54C
and R54P are mutations present in patients with the perina-
tal form of hypophosphatasia.(6) R54C has been shown to
remain trapped inside transfected cells.(16) Our data using a
soluble epitope-tagged mutant enzyme indicate that the lack
of activity is not only caused by inappropriate trafficking
but that this mutation effectively abolishes catalytic activity.
The same is true for the G317D mutation.(18) Figure 2 shows
that three inactivating mutations (V365I, R54C, and R54P)
are close to the enzyme dimer interface and it is possible
that these mutations severely destabilize dimer formation.
Two mutations (G317D and A94T) are positioned close to
the active site residues that are involved directly in the Zn2ϩ
and Mg2ϩ coordination.(21) Surprisingly, the R206W muta-
tions are distant from the active site pocket, while still
capable of causing loss of activity. However, this residue is
close to a fourth metal site, which as reported recently, may
be occupied by calcium ions in TNAP.(15) D361V and
G439R are mutations located in the Zn2ϩ coordination site
and were the least active mutants, that is, around 100-fold
less active than setTNAP. D361V has been found in dom-
inantly inherited hypophosphatasia and this mutation was
shown to inhibit the activity of the product of the remaining
wild-type allele.(26) Previously, we have shown that mam-
malian APs behave as allosteric enzymes and that the het-
erodimers display properties that are not the weighted av-
erage of the properties of the homodimers.(27) So the kinetic
properties of the D361V mutant and the fact that it forms
heterodimers can explain its role in causing dominantly
inherited hypophosphatasia. The same explanation may
hold true for the A115V mutation, which was reported as
dominant and found in a patient with adult hypophosphata-
sia.(28)
␮
One mutant (A160T)(29) had an increased kcat when mea-
sured with pNPP at pH 9.8 and with PLP, but not when
measured with PPi and pNPP at pH 7.5. In fact, an increase
in Km was observed for both PLP and pNPP under these
conditions. This mutation was found in a patient compound
heterozygote with an F310L who had only mild symptoms
of adult hypophosphatasia. The A162T(5) mutation dis-
played normal catalytic properties under optimal pH and
buffer conditions. This is in contrast with results from Weiss
et al.,(5) who reported complete inactivation when this mu-
tant was expressed in NIH3T3 cells. However, Shibata et
al.,(17) who expressed A162T in COS-1 cells, found that
most of the A162T enzyme remained inside the cells pro-
viding an explanation for the Weiss et al.(5) report of com-
plete inactivation. Our experimental approach, using set
enzymes enabled us to bypass this intracellular trafficking
block and obtain data that indicate that the active site of the
A162T mutant is largely unaffected by this substitution
using pNPP as substrate at pH 9.8. Nevertheless, the A162T
mutation showed considerable reduction in enzyme activity,
using PPi and PLP as substrates at physiological pH. So the
combination of reduced activity at physiological pH and
deficient transport explains the severity of this mutation,
which has been found in cases of perinatal hypophos-
phatasia.

1390
DI MAURO ET AL.
The E174K(6) and E174G(29) mutations were detected in thesda, MD, USA). Sonia Di Mauro was supported by a
a patient with the infantile form of the disease and in cases fellowship from the Conselho Nacional de Desenvolvi-
of adult and odontohypophosphatasia in compound het- miento Cient´ıfico e Tecnolo´gico (CNPq), Sa˜o Paulo, Brasil.
erozygosity. Our results show a significantly reduced activ-
ity for E174K and E174G when tested with PLP. Mutation
E281K(10) was found in a patient with a severe form of
hypophosphatasia. A substantial loss of activity was found
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ization and respiratory distress and died at 5 months of age.
Mutation D277A produces an unstable enzyme with low kcat
values for all substrates analyzed (Tables 2 and 3) but
particularly undetectable activity toward PLP. D277A was
found in different compound heterozygous patients.(6) Re-
cently, in an interesting historical vignette, Mumm et al.(12)
identified this mutation in the original hypophosphatasia
case reported by Rathbun in 1948.(3) This severely affected
boy that appeared well at birth developed epileptic seizures
and cried as though in pain. These symptoms (i.e., epileptic
seizures, high-pitched vocalizations, and apnea) are a prom-
inent feature of the mouse model of hypophosphatasia(30)
and these manifestations have been attributed to abnormal
PLP metabolism(31,32) resulting from a TNAP inactivation.
Thus, the inability of these mutant enzymes (E174G,
D277A, and E281K) to hydrolyze PLP clearly correlates
with the clinical presentation of the disease.
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In conclusion, our results contribute to the understanding of
the variable expressivity of hypophosphatasia by pointing to
the preferential use of PPi versus PLP displayed by some
mutations. Data obtained using mouse models of hypo-
phosphatasia, indicate that abnormalities in PLP metabo-
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prevalent in the TNAP knockout mice. However, the bone
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a
potent inhibitor of mineralization. Hypophosphatasia pa-
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amounts of phosphoethanolamine in the urine. However, the
metabolic pathway that leads to this phosphoethanolamine
increase currently is unknown and no clinical abnormality has
been correlated to this biochemical finding to date.
Patients that inherit mutations such as E174G, D277A, or
E281K, which are very inefficient in hydrolyzing PLP,
would be expected to manifest more severe seizures and
apnea than patients with other mutations. Furthermore,
these mutant enzymes often are found in compound het-
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other missense mutations. We know that heterodimers have
catalytic properties that are not the pounded average of
those of the corresponding homodimers because of the
allosteric properties of mammalian APs.(27) Thus, preferen-
tial substrate use by each monomer in combination with
heterodimer formation and even impaired intracellular traf-
ficking are all mechanisms that help explain the complex
variable expressivity often observed in hypophosphatasia.
ACKNOWLEDGMENTS
This work was supported by grants CA42595 and
DE12889 from the National Institutes of Health (NIH; Be-
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Y, Oda K 2000 Possible interference between tissue-non spe-

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Address reprint requests to:
Jose´ Luis Milla´n, Ph.D.
The Burnham Institute
10901 North Torrey Pines Road
La Jolla, CA 92037, USA
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pophosphatasia inhibits the activity of the wild-type enzyme. Received in original form October 30, 2001; in revised form
J Clin Endocrinol Metab 85:743–747. January 16, 2002; accepted February 6, 2002.