Kinetic Studies on beta -Site Amyloid Precursor Protein-cleaving Enzyme (BACE)

CONFIRMATION OF AN ISO-MECHANISM*

Larisa ToulokhonovaDagger , William J. Metzler§, Mark R. Witmer, Robert A. CopelandDagger , and Jovita MarcinkevicieneDagger ||

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The steady-state kinetic mechanism of beta -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 delta  13.0 ppm and phi  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 beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, beta -secretase and gamma -secretase. The second cleavage activity (gamma -secretase) is strongly associated with the presenilin multisubunit complexes (1), whereas beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning, Expression, and Purification of Human beta -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 alpha -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-EIDLdown-arrow MVLDWHDK-DNP-OH (synthesized in-house) and MCA-EVNLdown-arrow 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 (epsilon 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,


y=a+bx+cx<SUP>2</SUP> (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,


Y=<FR><NU>100%</NU><DE>1+<FENCE><FR><NU>x</NU><DE><UP>IC</UP><SUB>50</SUB></DE></FR></FENCE><SUP>s</SUP></DE></FR> (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,
&ngr;=<FR><NU>V<SUB><UP>max</UP></SUB> · [<UP>S</UP>]</NU><DE>K<SUB>m</SUB> · (1+[I]/K<SUB>is</SUB>)+[<UP>S</UP>]</DE></FR> (Eq. 3)

&ngr;=<FR><NU>V<SUB><UP>max</UP></SUB> · [<UP>S</UP>]</NU><DE>(K<SUB>m</SUB>+[<UP>S</UP>]) · (1+[I]/K<SUB>i</SUB>)</DE></FR> (Eq. 4)

&ngr;=<FR><NU>V<SUB><UP>max</UP></SUB> · [<UP>S</UP>]</NU><DE>K<SUB>m</SUB> · (1+[I]/K<SUB>is</SUB>)+[<UP>S</UP>] · (1+[I]/K<SUB>ii</SUB>)</DE></FR> (Eq. 5)
where [S] and Km are the concentration and Michaelis constant of the low Km substrate; Vmax and nu  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,
F=<FR><NU>(&khgr;<SUP>2</SUP><SUB>1</SUB>−&khgr;<SUP>2</SUP><SUB>2</SUB>)/(&ngr;<SUB>1</SUB>−&ngr;<SUB>2</SUB>)</NU><DE>&khgr;<SUP>2</SUP><SUB>2</SUB>/&ngr;<SUB>2</SUB></DE></FR> (Eq. 6)
where chi <UP><SUB><IT>n</IT></SUB><SUP>2</SUP></UP> 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 nu  is the number of degrees of freedom (defined as nu  = 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,
Y=<FR><NU>Y<SUB><UP>max</UP></SUB></NU><DE>(1+H/K<SUB>a</SUB>+K<SUB>b</SUB>/H)</DE></FR> (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,
Y=<FR><NU>k<SUB><UP>cat</UP></SUB> · [<UP>S</UP>]</NU><DE>K<SUB>m</SUB> · (1+F<SUB>i</SUB> · E<SUB>k<SUB><UP>cat</UP></SUB>/K<SUB>m</SUB></SUB>)+[<UP>S</UP>] · (1+F<SUB>i</SUB> · E<SUB>k<UP>cat</UP></SUB>)</DE></FR> (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,


[<UP>P</UP>]=&ngr;<SUB>s</SUB>+<FR><NU>(&ngr;<SUB>i</SUB>−&ngr;<SUB>s</SUB>)·(1−&ggr;)</NU><DE>k<SUB><UP>obs</UP></SUB>·&ggr;</DE></FR>·<UP>ln</UP><FENCE><FR><NU>1−&ggr;·e<SUP>−k<SUB><UP>obs</UP></SUB><UP>·t</UP></SUP>)</NU><DE><UP>1−&ggr;</UP></DE></FR></FENCE> (Eq. 9)
where [P], [E], and [I] are the product, enzyme, and inhibitor concentrations, respectively; nu i is the initial velocity; nu s is the steady-state velocity, and kobs is the pseudo-first order rate constant for the approach to the steady state. gamma  = [E]/[I]·(1 - nu s/nu 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 (phi ) was determined by fitting the data to the Equation 10,
(yC)−1=&phgr;(1−X)/X+1 (Eq. 10)
where y is the signal intensity; C is a constant, and X is the mole fraction of H2O (23).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Chemical structures of the BACE inhibitors OM99-2 (A) and statine-valine (B).


View larger version (7K):
[in this window]
[in a new window]
 
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.

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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   A, inhibition of BACE (20 nM) by 0 (open circle ), 0.5 (), 1 (), and 2 (black-square) 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 (open circle ), 0.5 (), 1 (), and 2 (black-square) 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.

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).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4.   A, kcat/Km dependence on pH(D) of cleavage of the low Km substrate by BACE in H2O (open circle ) 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 (open circle ) 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.

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.


View larger version (15K):
[in this window]
[in a new window]
 
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 (open circle ) and pD 5.05 (). Inset, progress curves of substrate (25 µM) cleavage by 20 nM BACE in H2O buffer in the presence (diamond ) or absence (open circle ) of 90 nM inhibitor and in D2O buffer in the presence (black-diamond ) or absence () of 90 nM inhibitor. Data were fitted to Equation 9.

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 delta  >15.0 ppm) and low deuterium fractionation factors (phi  < 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 delta  = 11.8 ppm. No other downfield resonance is observed. When complexed with OM99-2, a potent inhibitor of BACE, an additional resonance appears at delta  = 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).


View larger version (12K):
[in this window]
[in a new window]
 
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.

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 phi  value for the proton resonating at delta  = 13.0 ppm is 0.6, indicating it is participating in a relatively strong hydrogen bond. For comparison, the phi  value for the proton resonating at delta  = 11.8 ppm is 2.2, which is comparable with the phi  typically observed for a backbone amide proton in free exchange with water. The delta  and phi  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.


View larger version (10K):
[in this window]
[in a new window]
 
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 (phi ).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (11K):
[in this window]
[in a new window]
 
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 gamma -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.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Solvent kinetic isotope effects (SKIE) on the reaction catalyzed by BACE and 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 delta  13.0 ppm was observed (Fig. 6) when BACE was mixed with the inhibitor, and this proton showed a fractionation factor (phi ) of 0.6 (Fig. 7). It should be noted that interpreting the delta  and phi  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 phi  and hydrogen bond strength may be more appropriate than delta . Factor phi  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 phi  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 delta  and phi : for Asp-Hisdelta 1, delta  = 18.1 and phi  = 0.4, whereas for Hisepsilon 2-Ser, delta  = 13.2 and phi  = 0.69 (22). In the present study, the values of delta  and phi  measured for the downfield proton in the BACE-OM99-2 complex (delta  = 13.0, phi  = 0.6) are comparable with those measured for the LBHB between Hisepsilon 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, beta -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517[CrossRef][Medline] [Order article via Infotrieve]
2. Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A., and Tang, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1456-1460[Abstract/Free Full Text]
3. Yian, R., Blenkowski, M. J., Shuck, M. E., Miao, H., Tory, M. C., Pauley, A. M., Brashler, J. R., Stratman, N. C., Mathews, W. R., Buhl, A. E., Catrer, D. B., Tomasselli, A. G., Parodi, L. A., Heinricson, R. L., and Gurney, M. E. (1999) Nature 402, 533-537[CrossRef][Medline] [Order article via Infotrieve]
4. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Maendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J.-C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286, 735-741[Abstract/Free Full Text]
5. McGregor, G. A., Markandu, N. D., Roulstone, J. E., and Jones, J. C. (1981) Nature 291, 329-331[Medline] [Order article via Infotrieve]
6. Huff, J. R. (1991) J. Med. Chem. 34, 2305-2314[Medline] [Order article via Infotrieve]
7. Citron, M. (2000) Mol. Med. Today 6, 392-397[CrossRef][Medline] [Order article via Infotrieve]
8. Francis, S. E., Sullivan, D. J., and Goldberg, D. E. (1997) Annu. Rev. Microbiol. 51, 97-123[CrossRef][Medline] [Order article via Infotrieve]
9. Suguna, K., Padlan, E. A., Smith, C. W., Carlson, W. D., and Davies, D. R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7009-7013[Abstract]
10. Hyland, L. J., Tomaszek, T. A., and Meek, T. D. (1991) Biochemistry 30, 8454-8463[Medline] [Order article via Infotrieve]
11. Cho, Y. K., Rebholz, K. L., and Northrop, D. B. (1994) Biochemistry 33, 9637-9642[Medline] [Order article via Infotrieve]
12. Rebholz, K. L., and Northrop, D. B. (1991) Biochem. Biophys. Res. Commun. 176, 65-69[Medline] [Order article via Infotrieve]
13. Northrop, D. B. (2001) Acc. Chem. Res. 34, 790-797[CrossRef][Medline] [Order article via Infotrieve]
14. Piana, S., and Carloni, P. (2000) Proteins Struct. Funct. Genet. 39, 26-36[CrossRef][Medline] [Order article via Infotrieve]
15. Cleland, W. W. (2000) Arch. Biochem. Biophys. 382, 1-5[CrossRef][Medline] [Order article via Infotrieve]
16. Ghosh, A., Shin, D., Downs, D., Koelsh, G., Lin, X., Ermolieff, J., and Tang, J. (2000) J. Am. Chem. Soc. 122, 3522-3523[CrossRef]
17. Marcinkeviciene, J., Luo, Y., Graciani, N., Combs, A. P., and Copeland, R. A. (2001) J. Biol. Chem. 276, 23790-23794[Abstract/Free Full Text]
18. Cheng, Y., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-4108[CrossRef][Medline] [Order article via Infotrieve]
19. Schowen, K. B., and Schowen, R. L. (1982) Methods Enzymol. 87, 551-606[Medline] [Order article via Infotrieve]
20. Cleland, W. W. (1979) Methods Enzymol. 63, 103-138[Medline] [Order article via Infotrieve]
21. Hore, P. J. (1983) J. Magn. Reson. 55, 293-300
22. Markley, J. L., and Westler, W. M. (1996) Biochemistry 35, 11092-11097[CrossRef][Medline] [Order article via Infotrieve]
23. Loh, S. N., and Markley, J. L. (1994) Biochemistry 33, 1029-1036[Medline] [Order article via Infotrieve]
24. Mallender, W. D., Yager, D., Onstead, L., Nichols, M. R., Eckman, C., Sambamurti, K., Kopcho, L., Marcinkeviciene, J., Copeland, R. A., and Rosenberry, T. L. (2000) Mol. Pharm. 59, 619-626[Abstract/Free Full Text]
25. Turner, R. T., III, Koelch, G., Hong, L., Castenheira, P., Ghosh, A., and Tang, J. (2001) Biochemistry 40, 10001-10006[CrossRef][Medline] [Order article via Infotrieve]
26. Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M., Dovey, H. F., Frigon, N., Hong, J., Jacobson-Croak, K., Jewett, N., Keim, P., Knops, J., Lieberburg, I., Power, M., Tan, H., Tatsuno, G., Tung, J., Schenk, D., Seubert, P., Suomensaart, S. M., Wang, S., Walker, D., Zhao, J., McConlogue, L., and John, V. (1999) Nature 402, 537-540[CrossRef][Medline] [Order article via Infotrieve]
27. Chou, T. C. (1974) Mol. Pharmacol. 10, 235-247[Abstract]
28. Quinn, D. M., and Sutton, L. D. (1991) in Enzyme Mechanisms from Isotope Effects (Cook, P. F., ed) , pp. 73-126, CRC Press, Inc., Boca Raton, FL
29. Oefner, C., Binggeli, A., Breu, V., Bur, D., Clozel, J.-P., D'Arcy, A., Dorn, A., Fischli, W., Gruninger, F., Guller, R., Hirth, G., Marki, H. P., Mathews, S., Muller, M., Ridley, R. G., Stadler, H., Vieira, E., Wilhelm, M., Winkler, F. K., and Wostl, W. (1999) Chem. Biol. 6, 127-131[CrossRef][Medline] [Order article via Infotrieve]
30. Marcinkeviciene, J., Kopcho, L. M., Yang, T., Copeland, R. A., Glass, B. M., Combs, A. P., Falahatpisheh, N., and Thompson, L. (2002) J. Biol. Chem. 277, 28677-28682[Abstract/Free Full Text]
31. Cooper, J. B., Khan, G., Taylor, G., Tickle, I. J., and Blundell, T. L. (1990) J. Mol. Biol. 214, 199-222[Medline] [Order article via Infotrieve]
32. Hong, L., Koelsch, G., Lin, X., Wu, S., Terzyan, S., Ghosh, A., Zhang, X. C., and Tang, J. (2000) Science 290, 150-153[Abstract/Free Full Text]
33. Coates, L., Erskin, P. T., Wood, S. P., Myles, D. A. A., and Cooper, J. B. (2001) Biochemistry 40, 13149-13157[CrossRef][Medline] [Order article via Infotrieve]
34. Segel, I. H. (1993) Enzyme Kinetics , pp. 100-113, John Wiley & Sons, Inc., New York
35. Rebholz, K. L., and Northrop, D. B. (1995) Methods Enzymol. 249, 211-240[Medline] [Order article via Infotrieve]
36. Albery, W. J., and Knowles, J. R. (1986) Biochemistry 25, 2572-2577[Medline] [Order article via Infotrieve]
37. Rebholz, K. L., and Northrop, D. B. (1994) Arch. Biochem. Biophys. 312, 227-233[CrossRef][Medline] [Order article via Infotrieve]
38. Tian, G., Sobotka-Briner, C. D., Zysk, J., Liu, X., Birr, C., Sylverster, M. A., Edwards, P. D., Scott, C. D., and Greenberg, B. D. (2002) J. Biol. Chem. 277, 31499-31505[Abstract/Free Full Text]
39. Stein, R. L. (1985) J. Am. Chem. Soc. 107, 6039-6043
40. Wang, M. S., Gandour, R. D., Rodgers, J., Haslam, J. L., and Showen, R. L. (1975) Bioorg. Chem. 4, 392-395
41. Green, D. W., Aykent, S., Gierse, J. K., and Zupec, M. E. (1990) Biochemistry 29, 3126-3133[Medline] [Order article via Infotrieve]
42. Cassidy, S. C., Lin, J., and Frey, P. A. (1997) Biochemistry 36, 4576-4584[CrossRef][Medline] [Order article via Infotrieve]
43. Cleland, W. W., and Kreewoy, M. M. (1994) Science 264, 1887-1890[Medline] [Order article via Infotrieve]
44. Coates, L., Erskin, P. T., Crump, S. P., Wood, S. P., and Cooper, J. B. (2002) J. Mol. Biol. 318, 1405-1415[CrossRef][Medline] [Order article via Infotrieve]
45. Kumar, G. A., and McAllister, M. A. (1998) J. Org. Chem. 63, 6968-6972[CrossRef][Medline] [Order article via Infotrieve]
46. Kreevoy, M. M., and Liang, T. M. (1980) J. Am. Chem. Soc. 102, 3315-3322
47. Northrop, D. B., and Rebholz, K. L. (1997) Arch. Biochem. Biophys. 342, 317-321[CrossRef][Medline] [Order article via Infotrieve]
48. Fraser, M. E., Strynadka, N. C. J., Bartlett, P. A., Hanson, J. E., and James, M. N. G. (1992) Biochemistry 31, 5201-5214[Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.