Kinetic Studies on
-Site Amyloid Precursor Protein-cleaving
Enzyme (BACE)
CONFIRMATION OF AN ISO-MECHANISM*
Larisa
Toulokhonova
,
William J.
Metzler§,
Mark R.
Witmer¶,
Robert A.
Copeland
, and
Jovita
Marcinkeviciene
From the
Departments of Chemical Enzymology,
Bristol-Myers Squibb Pharmaceutical Company, Wilmington,
Delaware 19880-0400, § Macromolecular Structure and
¶ Biopharmaceutical Science and Technologies,
Princeton, New Jersey 08543-4000
Received for publication, October 11, 2002, and in revised form, November 11, 2002
 |
ABSTRACT |
The steady-state kinetic mechanism
of
-amyloid precursor protein-cleaving enzyme (BACE)-catalyzed
proteolytic cleavage was evaluated using product and statine- (Stat(V))
or hydroxyethylene-containing (OM99-2) peptide inhibition data, solvent
kinetic isotope effects, and proton NMR spectroscopy. The
noncompetitive inhibition pattern observed for both cleavage products,
together with the independence of Stat(V) inhibition on substrate
concentration, suggests a uni-bi-iso kinetic mechanism. According to
this mechanism, the enzyme undergoes multiple conformation changes
during the catalytic cycle. If any of these steps are rate-limiting to
turnover, an enzyme form preceding the rate-limiting conformational
change should accumulate. An insignificant solvent kinetic isotope
effect (SKIE) on kcat/Km, a
large inverse solvent kinetic isotope effect on
kcat, and the absence of any SKIE on the
inhibition onset by Stat(V) during catalysis together indicate that the
rate-limiting iso-step occurs after formation of a tetrahedral
intermediate. A moderately short and strong hydrogen bond (at
13.0 ppm and
of 0.6) has been observed by NMR spectroscopy in the
enzyme-hydroxyethylene peptide (OM99-2) complex that presumably mimics
the tetrahedral intermediate of catalysis. Collapse of this
intermediate, involving multiple steps and interconversion of enzyme
forms, has been suggested to impose a rate limitation, which is
manifested in a significant SKIE on kcat.
Multiple enzyme forms and their distribution during catalysis were
evaluated by measuring the SKIE on the noncompetitive (mixed)
inhibition constants for the C-terminal reaction product. Large, normal
SKIE values were observed for these inhibition constants, suggesting
that both kinetic and thermodynamic components contribute to the
Kii and Kis expressions, as has
been suggested for other iso-mechanism featuring enzymes. We propose that a conformational change related to the reprotonation of aspartates during or after the bond-breaking event is the rate-limiting segment in
the catalytic reaction of
-amyloid precursor protein-cleaving enzyme, and ligands binding to other than the ground-state forms of the
enzyme might provide inhibitors of greater pharmacological relevance.
 |
INTRODUCTION |
Extracellular amyloid deposits in brain, a characteristic feature
of Alzheimer's disease, is a result of proteolytic cleavage of
membrane-bound amyloid precursor protein by two enzymes,
-secretase and
-secretase. The second cleavage activity (
-secretase) is strongly associated with the presenilin multisubunit complexes (1),
whereas
-secretase (BACE)1
has been identified as a novel transmembrane aspartyl protease (2-4).
Although aspartyl proteases have been studied for more than 4 decades,
new aspects of catalysis and inhibition continue to emerge. A
substantial number of these enzymes have been identified as useful
targets for chemotherapeutic intervention in human diseases (5-8), yet
there has been limited success in identifying clinically relevant
inhibitors; hence, it is important to explore alternative drug design
approaches. An understanding of the catalytic mechanism of the target
enzyme is a powerful tool in the search for new inhibitors, and this
has motivated us to study BACE more carefully in an attempt to find
determinants that could lead the way to more successful drug design.
The proposed chemical mechanism for aspartyl proteases involves
activation of the attacking water molecule by the general base
Asp-COO
with concomitant protonation of the substrate
carbonyl by a general acid Asp-COOH, yielding a tetrahedral
intermediate amide hydrate (9). Kinetic mechanism studies suggest that
pepsin and HIV protease undergo isomerization steps during catalysis
(10, 11), and some evidence was obtained that this iso-step might be
rate-limiting (12). Inhibitors binding to the form of the enzyme that
accumulates because of the slow interconversion between enzyme isomers
would be expected to display higher affinity and greater selectivity for the target enzyme.2 A
recent proposal for the mechanism suggests that aspartyl proteases undergo multiple conformation states, which differ in geometry, protonation state, and proton localization (13). The coplanar geometry
of the aspartyl groups in the protease active site (14) and the
pK values of the catalytic aspartates allow for the
possibility that aspartyl proteases hydrolyze peptide bonds using
cyclic proton transfers and reactant state hydrogen tunneling,
featuring a low barrier hydrogen bond (LBHB) (13). If so, ligands,
attracted and preserving this particular feature, would yield very
tight and specific inhibitors (15). With this in mind, we performed product and statine-based peptide inhibition analysis, proton NMR of
the free and inhibitor-bound enzyme, as well as solvent isotope effect
studies to address the kinetic mechanism catalyzed by BACE and to
dissect possible rate-limiting segments and dominating enzyme forms.
 |
EXPERIMENTAL PROCEDURES |
Cloning, Expression, and Purification of Human
-Secretase--
The cDNA for the catalytic domain of human BACE
(residues 1-460) was PCR-amplified and subcloned into the mammalian
expression vector pTV1.6, upstream of a thrombin cleavage site linked
to cDNA encoding human IgG1 heavy chain. This
construct, pTV1.6-BACE-T-IgG, was used to produce stably transfected
dihydrofolate reductase-deficient Chinese hamster ovary DG44 cells,
which were then scaled up using methotrexate for selection. Multiple
rounds of selection were used, with a final expression level of ~10
mg/liter based on quantitation by enzyme-linked immunosorbent assay.
BACE-T-IgG was scaled up to 35-80 liters of bioreactor production
runs, and the secreted fusion protein was purified as described below.
The clarified growth media harvested from a bioreactor run was
concentrated using tangential flow filtration fitted with a 30,000 molecular weight cut-off unit to a smaller volume,
typically from 35-80 down to 4 liters to facilitate purification. The
fusion protein was captured on rProtein
A-SepharoseTM column (5 × 20 cm, Amersham
Biosciences) at 4 °C. The column was washed with PBS, pH 7.1, until
base-line absorbance was observed, and BACE-T-IgG was eluted with 0.10 M citrate, pH 3.0, into tubes containing 0.5 volumes of 4 M Tris, pH 8. Fractions containing the fusion protein were
dialyzed extensively against PBS, pH 7.1, at 4 °C. Analysis by
SDS-PAGE and N-terminal sequencing (not shown) showed the presence of a
mixture of two forms, consistent with published results from Amgen. The
protein was sterile-filtered (0.2 µm) and stored at 4 °C for long
term storage with no significant loss of activity observed even after
~1 year.
BACE-T-IgG was treated with human
-thrombin (Enzyme Research
Laboratories) at a ratio of 1:500 in PBS, pH 7.1, at 37 °C for 2 h. Thrombin was removed by passing the sample over
benzamidine-Sephadex column (Amersham Biosciences), and IgG was
captured with rProtein A-SepharoseTM. Finally, the
protein sample was further purified by applying onto Superdex 200 (26/60) gel filtration column washed with PBS, pH 7.1, at room
temperature. Fractions containing BACE were combined, sterile-filtered
(0.22 µm), and stored at 4 °C. The protein was characterized by
SDS-PAGE, and a variety of biophysical techniques, including isothermal
titration calorimetry, to demonstrate that it was glycosylated,
monomeric, catalytically active, and fully competent to bind BACE
active site inhibitor OM99-2 (16).
Substrates and Inhibitors--
Two BACE substrates were employed
in this study: Ac-EIDL
MVLDWHDK-DNP-OH (synthesized in-house) and
MCA-EVNL
DAEF(K-DNP)-COOH (Biosource International), where MCA
is 7-methoxycoumarin. The first substrate (low Km)
was used in experiments where substrate saturation was needed. The
second substrate (high Km) was used in fluorescence
assays to perform experiments at substrate concentrations well below
the Km value. Product inhibition studies were
performed using Ac-EIDL-OH (corresponding to the N-terminal part of the
cleaved substrate, referred to as product P) and Ac-MVLDWHDK-OH
(corresponding to the C-terminal portion of the substrate, referred to
as product Q). The peptidomimetic inhibitor OM-99 and statine-valine
inhibitor (Stat(V)) (Fig. 1) were purchased from Bachem and Enzyme
Systems Inc., respectively. All substrate, product, and inhibitor stock
solutions were prepared in Me2SO and stored at 4 °C for
about a month. The concentration of the low Km
substrate was calculated using the extinction coefficient for DNP
(
360 = 17,700 M
1·cm
1 at neutral pH).
Enzyme Assays--
Assays with the low Km
substrate were performed in 200-µl reaction mixtures containing the
substrate (1-40 µM) in 50 mM acetate buffer
with 0.25 mg/ml BSA, pH 4.5, at 25 °C. Me2SO content was
adjusted in all assays to be the same and not to exceed 6% (no loss of
enzyme activity was observed at this Me2SO concentration). Reactions were initiated by addition of 10 or 20 nM BACE
and quenched at various time points (depending on the experiment) by
either boiling for 2 min or by addition of 200 µl of 3% (v/v)
trifluoroacetic acid. The quenched reaction mixtures were subject to
HPLC separation (Waters Associates) monitoring absorbance at 360 nm.
Product and residual substrate peaks were well resolved with an
increasing gradient of 0-80% acetonitrile containing 0.1%
trifluoroacetic acid, allowing precise integration of the peak areas.
The area units were converted into molar concentration using a standard curve of known substrate concentrations. Because of nonspecific adsorption of product during the separation step, the calibration curves were non-linear. Therefore, sums of product and substrate peak
areas after the reaction were plotted against the known substrate concentrations at the beginning of the reaction, and non-linear calibration curves were constructed. These calibration curves showed
best fitting to the quadratic Equation 1,
|
(Eq. 1)
|
where y is sum of the product and substrate peak
areas after the reaction; x is substrate concentration in
µM before the reaction; and b and c
are coefficients derived from these data (a is assumed to be
zero, at zero substrate concentration). Then the coefficients
b and c were used to calculate product
concentration from the product peak area. All measurements with the
fluorescently labeled, high Km substrate were
performed as described in detail elsewhere (17).
Inhibition Studies--
The IC50 for steady-state
inhibition of BACE by Stat(V) was determined at several fixed substrate
concentrations in a reaction mixture (200 µl) containing buffer, low
Km substrate, and varying amounts of Stat(V) (0-5
µM). As in all assays, Me2SO content was
constant in all mixtures and did not exceed 6% of the final volume.
The reaction was initiated by addition of 10 nM BACE and
stopped after 15 min by boiling the reaction mixtures for 2 min. All
experiments were performed at 25 °C. The HPLC method was applied to
separate product and residual substrate peaks and to calculate their
molar concentrations as described above. IC50 values were
determined by fitting the data to the Cheng-Prusoff equation (18) as
shown in Equation 2,
|
(Eq. 2)
|
where Y is percent of inhibition at x
concentration of the inhibitor, and s is the Hill slope.
Product inhibition was studied in reaction mixtures (200 µl)
containing fixed concentrations of one of the products, varying amounts
of substrate (1-30 µM), Me2SO (to
adjust its content to 6% final), and standard buffer (50 mM acetate with 0.25 mg/ml BSA, pH 4.5). After mixing the assay components, BACE (20 nM final) was added to initiate
the reaction, and after 15-min 200 µl of 3% trifluoroacetic acid was used to stop it. By this method the reaction velocities were determined as a function of substrate and inhibitor concentration. These data were
analyzed by fitting them globally to the equations for competitive
(Equation 3), non-competitive (Equation 4), or mixed type (Equation 5)
inhibition, respectively,
|
(Eq. 3)
|
|
(Eq. 4)
|
|
(Eq. 5)
|
where [S] and Km are the concentration and
Michaelis constant of the low Km substrate;
Vmax and
are the values for maximal and
initial velocities; [I] corresponds to the inhibitor (one of the
products) concentration; Kis is the dissociation
constant for the enzyme-inhibitor complex; and Kii
is the dissociation constant for ternary enzyme-substrate-inhibitor complex. The goodness of fit was inspected visually and then evaluated statistically by the F-test (GraFit, Erithacus) as shown in Equation 6,
|
(Eq. 6)
|
where 
is the smallest possible
sum of squares deviations of the experimental values from the
calculated ones (n denotes the number of paired fits being compared), and
is the number of degrees of freedom (defined as
= N
n
1, where
N is the number of data points, and n is the
number of variables in the equation).
Solvent Kinetic Isotope Effects--
Solvent kinetic isotope
effects were investigated by parallel measurements of initial
velocities of substrate cleavage in 50 mM acetate with 0.25 mg/ml BSA prepared in H2O and D2O. To derive
pH/pD profiles for BACE activity, 10-fold concentrated buffers (without
BSA) were first prepared in H2O; the pH was adjusted to the
desired values, and these solutions were diluted in either H2O (pH buffer) or in D2O (pD buffer).
Lyophilized BSA was added after reading the pH of the diluted buffers
with a pH meter and calculating pD as pD = pHmeasured + 0.33 for 88% D2O (19). The enzyme stock solutions were
then prepared in the same buffers to match the desired pH/pD. Substrate
and inhibitor stock solutions were prepared in deuterated
Me2SO, which yielded 90% of the label in the final
reaction mixtures. Reactions were initiated by BACE addition (20 nM) into mixtures of specific pH (or pD) buffer and low
Km substrate (1-30 µM in 200 µl of
final volume). After 5 min the reactions were quenched by adding 200 µl of 3% trifluoroacetic acid, and the mixtures were analyzed by
HPLC as described above.
Solvent kinetic isotope effects on the inhibition by cleavage products
(P and Q) were performed using the same strategy. Buffer (prepared by
10-fold dilution into H2O or D2O) containing
variable concentrations of products P or Q and substrate were mixed
with the enzyme (prepared as well either in H2O or
D2O) and quenched (by adding trifluoroacetic acid) after 15 min of reaction. Product formation and substrate contents were analyzed
by HPLC as described earlier. Data for the
kcat/Km dependence on the
pH(D) were fitted to the Equation 7,
|
(Eq. 7)
|
where Y is the experimentally derived
kcat/Km value;
Ymax refers to the observed maximum of that
experimental value; and Ka and Kb
are the lower and higher Ka values for the two
titratable groups.
Kinetic solvent isotope effects on kcat
(D2Okcat) and
kcat/Km
(D2Okcat/Km)
were calculated using Cleland's program (20) as shown in Equation 8,
|
(Eq. 8)
|
where Y is the initial velocity; [S] is the
substrate concentration; kcat and
Km refer to the maximal turnover number and
Michaelis constant, respectively; and Fi is fraction of heavy atom label.
Ekcat/Km = ((D2Okcat/Km)
1) and Ekcatx = ((D2Okcat)
1).
Solvent kinetic isotope effects on the inhibition by cleavage products
(P and Q) were studied by using the same strategy. Buffer (prepared by
10-fold dilution into H2O or D2O) containing variable concentrations of products P or Q and substrate were mixed
with the enzyme (prepared as well either in H2O or
D2O) and quenched (by adding trifluoroacetic acid) after 15 min of reaction. Product formation and substrate contents were measured by the HPLC method described above, and the data were analyzed by
fitting to Equations 3-5.
Solvent Kinetic Isotope Effects on the Onset of Inhibition by
Stat(V)--
Stat(V) exhibited a time-dependent onset of
the inhibition during the catalysis, and we attempted to measure
solvent kinetic isotope effects on the kon of
this inhibition. The fluorescence intensity change upon cleavage of the
high Km substrate was monitored in parallel
experiments in 50 mM acetate with 0.25 mg/ml BSA at pH 4.5 and pD 5.05 (buffers were prepared from the same 10-fold concentrated
buffer with pH 4.5 as described above) using the same concentrations of
substrate (25 µM), enzyme (20 nM), and
different fixed concentrations of the inhibitor (0-90 nM).
Progress curves analysis was performed as described (17) to derive
kobs values for the approach to the
steady-state. Data were fit to Equation 9,
|
(Eq. 9)
|
where [P], [E], and [I] are the product,
enzyme, and inhibitor concentrations, respectively;
i is the
initial velocity;
s is the steady-state velocity, and
kobs is the pseudo-first order rate constant for
the approach to the steady state.
= [E]/[I]·(1
s/
i)2.
Proton NMR Experiments--
NMR spectra were recorded on a
Varian Inova 600 spectrometer equipped with a triple resonance 5-mm
probe with triax gradients. Spectra were recorded using the 1-1
sequence to suppress the water signal with minimal saturation (21).
Signals were acquired over 10,000 scans with a sweep width of 30 kHz,
8192 time domain points, and a relaxation delay of 4 s. Spectra
were collected at 5, 10, 15, and 20 °C. All BACE solutions were 50 µM protein in either Buffer A (10 mM
phosphate, pH 7.0, 3 mM KCl, 137 mM NaCl),
Buffer B (50 mM sodium acetate, pH 5.3), or Buffer C (50 mM sodium acetate, pH 4.5). 1.5 equivalents of OM99-2 (10 mM in d6-Me2SO
(Cambridge Isotope Laboratories, Inc.)) were added to the BACE solution
yielding a final ligand concentration of 75 µM.
Me2SO concentration did not exceed 0.75%.
The D/H fractionation factors were determined at 20 °C by
integrating the peak intensities of the downfield resonances in a
series of samples containing different D2O/H2O
solvent compositions. In these experiments BACE solutions were prepared
in Buffer C, pH 4.5, as both 100% H2O and 100%
D2O (99.9%, Isotec) stock solutions. For D2O
stock solutions, the observed pH (at 20 °C) was lowered by 0.4 units. Appropriate volumes of the H2O and D2O
stock solutions were mixed to yield the desired
D2O/H2O composition. Corrections to the signal
intensity for mole fraction H2O were made as described by
Markley and Westler (22). The fractionation factor (
) was determined
by fitting the data to the Equation 10,
|
(Eq. 10)
|
where y is the signal intensity; C is a
constant, and X is the mole fraction of H2O
(23).
 |
RESULTS |
Inhibition Studies--
Mechanistic studies of BACE using the
fluorogenic substrate described previously (24) were severely limited
because of the inner filter effects and the high Km
of this substrate. Another peptidic substrate with a much lower
Km has been reported recently (25), and we were able
to advance mechanistic studies using this substrate and HPLC-based
detection method. Under our assay conditions, the latter substrate was
cleaved by BACE with kcat of 0.32 ± 0.02 s
1 and Km of 5.2 ± 0.7 µM.
A number of peptides, based on the amino acid sequence around the
amyloid precursor protein
-cleavage site and containing a
hydroxyethylene (including OM99-2 (16)) or statine (including Stat(V)
(26)) isostere (Fig. 1), were tested as
inhibitors in the assay using this low Km substrate.
Both of the above-mentioned inhibitors showed tight binding behavior
with Ki, calculated using Morrison's quadratic
equation, of 2 and 20 nM for OM99-2 and Stat(V),
respectively. The observed Ki (IC50) for competitive inhibitors, which bind exclusively to the free enzyme, should be linearly dependent on substrate concentration (27). However,
upon varying Stat(V) (0-5 µM) and substrate
concentrations (5-25 µM), we found that the
IC50 was independent of substrate concentration (Fig.
2). Similar inhibition pattern was
observed for a number of hydroxyethylene-containing peptides (data not shown). This type of pattern has traditionally been characteristic of
non-competitive inhibitors that bind to both the free enzyme and the
enzyme-substrate complex.

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Fig. 2.
The effect of substrate concentration on the
IC50 value of the statine-valine inhibitor.
IC50 values were individually calculated for each set of
experiments at a fixed substrate concentration and varied Stat(V)
concentrations (0-5 µM) by using Equation 2.
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To follow up on this unexpected finding we conducted product inhibition
studies. Initial velocity was measured varying substrate and one of the
two cleavage products (Ac-EIDL or Ac-MVLDWHDR). Data collected using
Ac-EIDL as an inhibitor were best fit globally (reconfirmed by F-test,
Grafit, Erithacus) to a noncompetitive inhibition pattern (Fig.
3A) with
Kis = Kii = 3.2 ± 0.4 mM. The second product, Ac-MVLDWHDR, likewise displayed a
noncompetitive inhibition pattern (Fig. 3B) with the values of Kis = Kii = 1.48 ± 0.08 mM.

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Fig. 3.
A, inhibition of BACE (20 nM) by 0 ( ), 0.5 ( ), 1 ( ), and 2 ( )
mM product P (N-terminal product). The lines are
global fits of the data to the non-competitive inhibition pattern
revealed to be the best fit after comparing with other inhibition modes
by using Equations 3-5. B, inhibition of BACE (20 nM) by 0 ( ), 0.5 ( ), 1 ( ), and 2 ( )
mM of product P2 (C-terminal portion). The graph
represents the best global fit of the data to the non-competitive
inhibition mode after analyzing the data by using Equations 3-5.
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|
Solvent Kinetic Isotope Studies--
Kinetic parameters for the
BACE-catalyzed reaction were measured over the pH range of 3.5-6.0.
Saturating kinetics in the substrate concentration range of 1-33
µM were observed over the pH range of 3.5-5.0.
Increasing pH resulted in an increased Km to the
extent that no saturation could be observed above pH 5.0; therefore,
kcat/Km was estimated from
the slope of the velocity
substrate concentration plot. For
this reason we were not able to evaluate the
kcat dependence on pH. The pK values for kcat/Km calculated from
fitting the data to Equation 7 were 3.54 ± 0.13 and 5.21 ± 0.11, confirming the expectation of general acid and general base
catalysis. Repeating the entire pH profile in D2O-based
buffer (78% 2H) gave an increase in the pK
value of the general base (4.43 ± 0.05), whereas no shift of the
general acid value (5.21 ± 0.06) was observed (Fig.
4A). An equilibrium solvent
deuterium isotope effect usually is accountable for an increase in
pK values by ~0.4
0.6 units (28). The larger changes in
pK seen here suggest that additional factors contribute to
the almost unit increase of the lower pK, whereas the higher
pK is invariant. From the data in Fig. 4A we
chose values of pH = 4.5 and pD = 5.03 (as isotopically
equivalent in a pH-independent region) to analyze further the solvent
kinetic isotope effect on kcat. An increased reaction velocity was observed in the D2O solution (Fig.
4B), and these data revealed an inverse solvent kinetic
isotope effect on kcat (0.67 ± 0.11), with
very modest, if any, effect on
kcat/Km (0.84 ± 0.31).

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Fig. 4.
A,
kcat/Km dependence on pH(D)
of cleavage of the low Km substrate by BACE in
H2O ( ) and D2O ( ). The curves
were drawn through the experimental points by fitting to Equation 7.
The calculated pK values in H2O were 3.54 ± 0.13 and 5.21 ± 0.11, whereas those in D2O were
4.43 ± 0.05 and 5.21 ± 0.06. B, solvent kinetic
isotope effect on the BACE-catalyzed reaction. Initial velocity values
at pH 4.5 ( ) and pD 5.03 ( ) were transformed into Lineweaver-Burk
coordinates, and solvent kinetic isotope effects on
kcat
(D2Okcat) and
kcat/Km
(D2Okcat/Km)
were calculated by fitting the data to Equation 8.
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Solvent Isotope Effect on Product Inhibition--
Even though
solvent kinetic isotope effects on substrate or inhibitor binding are
very rare and usually small (28), we decided to explore the effect of
D2O on product inhibition parameters. The reactions were
run in parallel in H2O and 82% D2O, pH 4.5 and
pD 5.17, varying substrate and product Ac-MVLDWHDR concentrations. Data
for the reaction in H2O solution fit well to noncompetitive inhibition, yielding Kis = Kii = 1.87 ± 0.11 mM. Inhibition by Ac-MVLDWHDR in
deuterated solution appeared to be stronger, and these data could be
fit successfully to either noncompetitive inhibition
(Kis = Kii = 0.60 ± 0.03 mM) or mixed type inhibition equations
(Kis = 0.35 ± 0.09 mM,
Kii = 0.80 ± 0.02 mM). The
calculated D2OKis (as
ratio of Kis in water and D2O) was
5.1 ± 1.1 and
D2OKii = 2.3 ± 0.1.
Solvent Isotope Effect on Inhibition by Stat(V)--
Slow onset
inhibition of BACE by Stat(V) has been observed previously (17) and
could be monitored by the following reaction progress curves of
fluorogenic substrate cleavage at S < Km. Inhibition onset kinetics were compared in H2O and
D2O solutions at different concentrations of inhibitor, and
the data were fit to Equation 9 to calculate
kobs. The dependence of the first order rate
constant (kobs) on inhibitor concentration was
linear and very similar in H2O and D2O
solutions (Fig. 5); the calculated values
of kon ((1.9 ± 0.5) × 105 M
1 s
1 in
H2O and (1.7 ± 0.5) × 105
M
1 s
1 in D2O),
koff ((5 ± 1) × 10
4
s
1 in both solvents) and subsequently
Ki* (26.4 versus 28.1 nM)
were solvent-independent. Therefore, no obvious solvent isotope effects
were observed on BACE inhibition by Stat(V) during the catalytic
turnover.

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Fig. 5.
Solvent isotope effect on the onset of
Stat(V) inhibition during catalytic turnover. First order rate
constants, kobs, were obtained from the progress
curves of high Km substrate (25 µM)
cleavage by 20 nM BACE at different inhibitor
concentrations performed at pH 4.5 ( ) and pD 5.05 ( ).
Inset, progress curves of substrate (25 µM)
cleavage by 20 nM BACE in H2O buffer in the
presence ( ) or absence ( ) of 90 nM inhibitor and in
D2O buffer in the presence ( ) or absence ( ) of 90 nM inhibitor. Data were fitted to Equation 9.
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Protein Proton NMR Studies--
Low barrier hydrogen bonds display
distinctive physicochemical properties that are readily amenable to
detection by NMR. Two such properties are unusually downfield proton
chemical shifts (typically
>15.0 ppm) and low deuterium
fractionation factors (
< 0.8). To determine whether the
formation of an LBHB may be participating in the mechanism of action of
BACE, proton NMR studies were initiated. Fig.
6 shows the downfield region of the
proton NMR spectrum of BACE. In the free protein, the furthest
downfield signal resonates at
= 11.8 ppm. No other downfield
resonance is observed. When complexed with OM99-2, a potent inhibitor
of BACE, an additional resonance appears at
= 13.0 ppm. The
presence and resonance frequency of this peak was unchanged across a
range of pH values (7.0, 5.3, and 4.5) and temperatures (5, 10, 15, and
20 °C).

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Fig. 6.
Downfield region of the 1H NMR
spectrum of 50 µM BACE in 90%
H2O at 20 °C. The full spectrum was acquired a
sweep width of 30,000 kHz (~30 to 20 ppm). Comparison of the
spectrum in the absence (A) and presence (B) of
75 µM OM99-2, a potent inhibitor of BACE.
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To measure the strength of the newly formed hydrogen bond, the D/H
fractionation factors at pH 4.5 were determined from the slope of the
lines obtained in plots of the integrated signal intensity of the
downfield resonances against the D2O/H2O
composition (Fig. 7). In the BACE-OM99-2
complex, the
value for the proton resonating at
= 13.0 ppm
is 0.6, indicating it is participating in a relatively strong hydrogen
bond. For comparison, the
value for the proton resonating at
= 11.8 ppm is 2.2, which is comparable with the
typically
observed for a backbone amide proton in free exchange with water. The
and
values observed for the downfield proton in the BACE-OM99-2
complex suggest the formation of a strong hydrogen bond coinciding with
the formation of the state close to transition and that the hydrogen
bond may not be as strong as the LBHBs observed previously in other
enzyme-inhibitor complexes. Possible reasons for this may include
sub-optimal hydrogen bonding geometry and imperfectly matched
pK values of the hydrogen bond donor and acceptor in the
BACE-OM99-2 complex.

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Fig. 7.
Determination of the D/H fractionation factor
for the downfield signals in BACE. The relative intensities of the
signals at 13.0 and 11.8 ppm (Fig. 6) are plotted as a function of the
mole fraction H2O (X) according to Loh and
Markley (23). The slope of the line is the D/H fractionation factor
( ).
|
|
 |
DISCUSSION |
For more than a decade, significant attention has been paid to the
concept of structure-based ligand design, in which the crystal- or
NMR-based structure of a target protein is utilized for the de
novo design and/or optimization of the ligands intended to fit
well into a specific pocket (typically the catalytic active site of an
enzyme). Nevertheless, the structural information obtained by x-ray
crystallography and NMR spectroscopy represent structural "snapshots" of the enzyme active site, and do not always account for the conformational dynamics of enzymes and other target proteins. Both the solvated ligand (e.g. substrate molecules) and the
solvated enzyme can exist in multiple conformational states prior to
complex formation. Interconversions among such conformational states
are often associated with specific steps in catalysis, and in some cases these requisite conformational changes can impose rate
limitations on turnover. Ligands that specifically stabilize the
conformational state preceding the rate-determining step in catalysis
can offer great advantages with respect to increased binding affinity
and target selectivity. Thus, the combination of kinetic, chemical, and
structural information provides the most complete opportunity for
design and optimization of ligands for specific targets.
Aspartyl proteases have been shown to contain unusually extended and
mobile active sites that bind inhibitors in unpredictable ways due to
multiple conformations of the active site (29, 30). The best studied
conformers of the aspartyl proteases are the free enzyme and the
enzyme-statine or enzyme-hydroxyethylene complexes. These two forms can
differ in the co-planarity (from crystal structure (31, 32)) and/or
ionization state of the catalytic aspartates. Although the crystal
structure resolution of the free enzyme and aspartyl protease-ligand
complexes are insufficient to localize protons, the generally accepted
chemical mechanism predicts that if the enzyme starts its catalytic
cycle with Asp-32 as a general acid and Asp-228 as a general base (BACE
amino acid numbering, species EH in
Scheme 1), the amide hydrate tetrahedral
intermediate (species FHA in Scheme 1) would be in the
opposite protonation state. Asp-228 is protonated after abstracting a
proton from the catalytic water molecule, and Asp-32 is ionized as a
result of substrate carbonyl protonation. It should be noted that
direct evidence for such a monoprotonated form of a tetrahedral
intermediate-mimicking enzyme-hydroxyethylene complex was obtained for
endothiapepsin using neutron diffraction methods (33).
After protonation of the leaving amine and collapse of the tetrahedral
intermediate (step 4 in Scheme 1), the enzyme returns to its original
protonation state (species GH in the Scheme 1) but still
lacks the catalytic water molecule. It has been proposed that
reprotonation or rehydration of the enzyme active site (step 7 in
Scheme 1) may be a rate-limiting step during pepsin (12) and HIV
protease catalysis (10).

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|
Scheme 1.
Proposed catalytic mechanism of BACE.
EH is a free monoprotonated form of the enzyme, which after
substrate binding (step 1) undergoes loop closure (step 2) to form a
tightened enzyme-substrate complex FHS. Activation of the
catalytic water by the active site base and substrate carbonyl
protonation by the general acid leads to formation of a tetrahedral
intermediate FHA (step 3). Collapse of FHA is followed by (or
concomitant with) peptide bond breaking, protonation of the leaving
amine, and restoration of the enzyme to the initial state, albeit
without the active site water (step 4, GHPQ). Free enzyme
(GH) is restored to a catalytically relevant form by water
incorporation (step 7). We chose to picture step 4 as a single bond
breaking event but note that we cannot exclude the possibility that
some sequential steps are occurring and other intermediate transitions
(F or FH2) may exist.
|
|
Our product inhibition data (both cleavage products showed
noncompetitive inhibition patterns) are consistent with a
uni-bi-iso-mechanism (34, 35). This mechanism predicts that one
substrate yields two products and that there is a kinetically
significant conformational change required during catalysis. Isoenzyme
mechanisms related to the protonation/deprotonation have been well
described for proline racemase (36), fumarase, and carbonic anhydrase
(37) and a number of aspartyl proteases (13). It is very possible that
similar mechanism functions during
-secretase catalyzed cleavage,
because a non-competitive inhibition by intermediate analogs was
observed recently (38).
If the change of conformation involves rate-limiting proton
transfer(s), this should be manifested in a solvent kinetic isotope effect. A very small and statistically insignificant inverse SKIE was
observed on the kcat/Km of
BACE (Fig. 4A and Table I),
suggesting either that there were no rate-limiting proton transfer
steps up to the first irreversible step (step 4 in Scheme 1) or that
there were multiple proton transfers that offset each other. Loop
closing subsequent to substrate or inhibitor binding could be
accompanied by multiple water displacements, yielding an inverse
solvent isotope effect, although both normal and inverse effects have
been reported to accompany conformational changes in other systems (39,
40). Alternatively, this effect could be offset by a normal isotope
effect originating from amide hydrate intermediate formation (two
protons). These possibilities could be further distinguished by
studying proton inventory effects on
kcat/Km. Unfortunately, we
were not able to perform such experiments, because by changing the
H2O/D2O ratio, the pH/pD is altered in such a
way that parameters change not because of the label ratio but rather
because of the effective pH change. The absence of a solvent kinetic
isotope effect on kon for BACE inhibition by
Stat(V) (Fig. 5 and Table I) suggests that this inhibitor binds equally
well to all isoforms of the enzyme. Thus rate-limiting and proton
transfer-associated interconversions of these enzyme forms are not
reflected in onset of the inhibition.
Turnover number (kcat), on the other hand, was
significantly affected by the solvent nature, showing an inverse
kinetic isotope effect of 0.67 (Fig. 4B and Table I). This
is an unprecedented finding for aspartyl proteases, because modest and
normal kinetic isotope effects on kcat have been
reported for HIV protease (10), renin (41), and pepsin (12). The slow
step in these cases was associated with the "recharging" of the
enzyme after bond breaking, most likely reflecting enzyme reprotonation
to the initial state and/or regaining of the catalytic water. The
inverse solvent isotope effect on kcat seen for
BACE could arise either because of a low fractionation factor of
proton(s) in the reactant state or a high fractionation factor in the
transition or product state (28). The reactant state reflected in
kcat/Km is the free enzyme
(EH in Scheme 1), whereas the reactant state reflected in
kcat is the tetrahedral intermediate
(FHA in Scheme 1) (19). The transition state for
kcat/Km would be FHA and that for kcat could be either GH or EH in Scheme 1.
Because the SKIE on kcat/Km
is ambiguous, we will not discuss it further. We suggest that an
inverse SKIE on kcat is a result of a short, strong hydrogen bond in the FHA complex (reactant state for
kcat) that fractionates with a low factor. LBHB
have been documented for the transition states of serine proteases
(42), triose-P-isomerase, citrate synthase, and other enzymes (43), and
for these enzymes the existence of a LBHB was reconfirmed by NMR
measurements. Aspartyl proteases have also been suggested to feature
LBHB in the free enzyme state (13), but no direct evidence of this
feature has yet been demonstrated. Nevertheless a short and strong
hydrogen bond was recently reported for endothiapepsin-statine,
gem-diol, and other inhibitor complexes (44). We were not
able to obtain any data confirming or dismissing this hypothesis for
free BACE (i.e. no downfield NMR signal in free enzyme), but
we propose the possibility of formation of a short, strong hydrogen
bond in one of the intermediates. This hypothesis is supported by
proton NMR studies, assuming that the enzyme-OM99-2 complex mimics the tetrahedral (FHA) or other intermediate complex. A downfield NMR signal
at
13.0 ppm was observed (Fig. 6) when BACE was mixed with the
inhibitor, and this proton showed a fractionation factor (
) of 0.6 (Fig. 7). It should be noted that interpreting the
and
values in terms of absolute hydrogen bond strength is an inexact task.
A theoretical study of the relationship between the proton chemical
shift and hydrogen bond strength found an excellent linear correlation
between the hydrogen bond strength and predicted chemical shift in
model LBHB complexes (45). This correlation held only for compounds
within the same class, however, and the authors cautioned about
comparing the LBHB proton chemical shifts of structurally unrelated
compounds. The correlation between
and hydrogen bond strength may
be more appropriate than
. Factor
is reduced by as much as a
4-fold for LBHBs relative to exchangeable protons in proteins (46).
Whether sufficient data exist to allow direct comparisons of
and
hydrogen bond strengths for LBHBs in chemically different environments
remains unclear. For example, in chymotrypsinogen A, two protons shown
to be involved in LBHBs were found to have significantly different
values for
and
: for Asp-His
1,
= 18.1 and
= 0.4, whereas for His
2-Ser,
= 13.2 and
= 0.69 (22). In the present study, the values of
and
measured for the downfield proton in the BACE-OM99-2 complex
(
= 13.0,
= 0.6) are comparable with those measured for the LBHB between His
2-Ser in chymotrypsinogen A.
Formation of this tetrahedral intermediate (featuring a strong short
hydrogen bond) during catalysis is unlikely to be rate-limiting (no
SKIE on kcat/Km). On the
other hand, either the collapse (protonation of the leaving group) or
enzyme recharging (correct enzyme reprotonation or rehydration)
most likely does impose a rate limitation, and this is what is
manifested in the SKIE on kcat. We suggest that
there are at least three enzyme forms that can bind peptidomimetic
inhibitors as follows: EH (correct protonation, hydrated), FH
(alternative protonation, dehydrated), and GH (correct protonation,
dehydrated). Our product inhibition data in D2O (Table I)
suggest that there is a kinetic component to the noncompetitive inhibition constant expression (47), which is potentially indicative of
a rate-limiting interconversion between enzyme forms during catalysis.
The species featuring a strong hydrogen bond (most likely complex with
FH) is favored in D2O where its accumulation results in
more potent inhibition and a large SKIE on Kii (13).
A rate-limiting proton transfer accompanying FH collapse could be
slowed in D2O resulting in accumulation of FH and increased binding as well. The inhibition constant Kis
measures inhibitor dissociation from all free enzyme forms (mostly EH
and GH) that are in equilibrium; any solvent isotope effect on
Kis must have a thermodynamic origin. GH will
accumulate in D2O, resulting in tighter inhibitor binding
and a large SKIE on inhibition (Table I).
Taken together, our data suggest that BACE catalyzes proteolysis
involving a rate-limiting enzyme isomerization step(s), which occurs
after tetrahedral intermediate formation. Solvent kinetic isotope
effects and proton NMR results point toward collapse of the amide
hydrate and restoration of the initial enzyme protonation state as
being the slow step. We do not have any strong evidence that
reprotonation of the enzyme occurs after product release. A prevailing
hypothesis relates restoration of the general base by protonation of
the leaving product amine and restoration of the general acid by proton
abstraction to yield the carboxyl product carbonyl. It is quite
possible that these two steps occur sequentially along the reaction
pathway resulting in multiple transitions. Our results reinforce
(i.e. the presence of a short, strong hydrogen bond) the
inference that hydroxyethylene inhibitors bind more potently to the FH
form of the enzyme (oppositely monoprotonated and dehydrated).
Nevertheless, it is difficult to define the extent to which
hydroxyethylene complexes resemble the true tetrahedral intermediate of
catalysis, because only one hydroxyl is present in the enzyme-inhibitor
complexes. This could be a reason for the observed distortion of the
aspartate coplanarity (32) in these complexes, and thus the potential
of the inhibitor is not maximized. Hence, compounds that bind the
monoprotonated form of the enzyme (the same FH), but engage the
aspartates in a more coplanar interaction, could be even more
attractive as inhibitors and possibly feature an even stronger and
shorter hydrogen bond (48). On the other hand, depending upon the
sequence of reprotonation of the catalytic aspartates, the transitions
can emerge as dianionic (F
if the amine protonation is
first and not rate-limiting) or diprotonated (FH2, if
Asp-32-H regeneration is first and not rate-limiting). Either of these
forms would be stabilized by distinct classes of inhibitors. Given the
fact that the BACE active site is a large and flexible cavity,
designing an optimal inhibitor is a subtle interplay between
identifying the right isostere and optimizing adjacent parts of the
molecule. The ability to take advantage of the short and strong
hydrogen bonds that engage the catalytic aspartates depends on finding
optimal interactions between the enzyme and the rest of the inhibitor
molecule, allowing proper positioning of the isostere.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Nilsa Graciani and
Dale Collins (Combinatorial Chemistry group) for the peptide synthesis
and Dr. Janet Chang and Dharti Kothari for large scale production of
BACE-Ig. We thank Drs. Andrew M. Stern, Mark Harpel, Zhihong Lai, Lorin Thompson (Bristol Myers-Squibb), Dexter Northrop (University of Wisconsin, Madison), and Daniel Quinn (University of Iowa) for valuable discussions.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Chemical
Enzymology, Bristol-Myers Squibb Pharmaceutical Company, P. O. Box
80400, Wilmington, DE 19880-0400. Tel.: 302-467-5031; Fax: 302-467-6820; E-mail: jovita.marcinkeviciene@bms.com.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M210471200
2
A non-competitive inhibition constant for an
iso-mechanism reflects inhibitor binding to all enzyme isomers
appearing during the catalysis. Expression of the Ki
in this case is not a simple dissociation constant between the enzyme
isomers and inhibitor but rather a complex parameter, which is affected
by the isomerization rate constant (kinetic contribution) and
steady-state concentration of the isomeric form of enzyme. Thus, the
conformer preceding a slow isomerization step will accumulate, and
inhibitors, which bind to this particular entity, will yield more
inhibition compared with compounds binding to the short lived enzyme
forms. For detailed mechanistic analysis and equations see Refs. 35 and
47. Identifying unique conformers for the specific aspartyl proteases
might render more specific inhibitors in spite of high amino acid
sequence homology at the active site and commonly shared ground state
enzyme structures.
 |
ABBREVIATIONS |
The abbreviations used are:
BACE,
-APP-cleaving enzyme;
APP, amyloid precursor protein;
SKIE, solvent
kinetic isotope effect;
LBHB, low barrier hydrogen bond;
DNP, 2,6-dinitrophenol;
PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;
HPLC, high pressure liquid chromatography;
HIV, human
immunodeficiency virus.
 |
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