From the Department of Molecular and Medical
Genetics, Oregon Health Sciences University, Portland, Oregon 97201, the § Department of Chemistry, Reed College, Portland,
Oregon 97202, and the ¶ Department of Biochemistry, Indiana
University, Indianapolis, Indiana 46202
Received for publication, August 21, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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Fumarylacetoacetate hydrolase (FAH) catalyzes the
hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to
yield fumarate and acetoacetate as the final step of Phe and Tyr
degradation. This unusual reaction is an essential human metabolic
function, with loss of FAH activity causing the fatal metabolic disease hereditary tyrosinemia type I (HT1). An enzymatic mechanism involving a
catalytic metal ion, a Glu/His catalytic dyad, and a charged oxyanion
hole was previously proposed based on recently determined FAH crystal
structures. Here we report the development and characterization of an
FAH inhibitor, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid
(HMPOBA), that competes with the physiological substrate with a
Ki of 85 µM. The crystal structure of
FAH complexed with HMPOBA refined at 1.3-Å resolution
reveals the molecular basis for the competitive inhibition, supports
the proposed formation of a tetrahedral alkoxy transition state
intermediate during the FAH catalyzed reaction, and reveals a
Mg2+ bound in the enzyme's active site. The analysis of
FAH structures corresponding to different catalytic states reveals
significant active site side-chain motions that may also be related to
catalytic function. Thus, these results advance the understanding of an essential catabolic reaction associated with a fatal metabolic disease
and provide insight into the structure-based development of FAH inhibitors.
The aromatic amino acids Phe and Tyr are catabolized by a pathway
of six enzyme catalyzed reactions. The inherited metabolic diseases
phenylketonuria, alkaptonuria, and tyrosinemia types I, II, and III are
associated with mutations of these enzymes. Mutations in
fumarylacetoacetate hydrolase
(FAH1; EC 3.7.1.2) cause
hereditary tyrosinemia type I (HT1), the most severe disease associated
with Phe and Tyr catabolism (1). HT1 is an autosomal recessive disease
with an acute form that causes death in infancy due to liver failure
and a chronic form that causes an early age death due to hepatocellular
carcinoma, liver cirrhosis, and neurologic crises (2, 3). The lethal nature of HT1 is most likely due to the accumulation of the toxic intermediate fumarylacetoacetate in the absence of FAH activity (4-6).
This reactive metabolite may cause direct DNA (7) and tissue damage,
depletion of cellular glutathione levels (8, 9), and induction of
apoptosis (10). HT1 is currently treated by combined liver/kidney
transplantation (11) and pharmacologic inhibition of
hydroxyphenylpyruvate dioxygenase (12), which catalyzes an earlier step
in Phe/Tyr catabolism.
A mouse model of HT1 has been developed (5, 14) and can be treated
effectively using adenoviral and retroviral based gene therapy vectors
to restore FAH activity (14-16). These studies have demonstrated that
hepatocytes expressing FAH exhibit a strong selective advantage over
FAH-deficient cells in vivo. Therefore, it has been
suggested that metabolic overloading of the Phe/Tyr catabolic pathway
and/or inhibitors of FAH may be of use to improving the efficiency and
permanence of liver gene therapy strategies, in general (15). The
overexpression of FAH carried by a gene therapy vector harboring
another corrective gene or the expression of inhibitor-resistant
variants of FAH could be used as a selectable marker in the treatment
of liver diseases other than HT1.
FAH catalyzes the final reaction of Phe/Tyr catabolism, the formation
of fumarate and acetoacetate from fumarylacetoacetate hydrolysis (17, Scheme 1). FAH is a homodimer of 46 kDa
subunits that catalyzes the hydrolytic cleavage of carbon-carbon bonds in a variety of diketo acid substrates (18). In contrast to common
biochemical hydrolysis reactions involving amide and ester bonds,
hydrolytic cleavage of relatively stable carbon-carbon bonds is a
fairly unusual reaction. Only ten enzymes having this primary function
have been described by the Enzyme Commission (EC 3.7.1.1-3.7.1.10).
However, FAH is structurally distinct from other carbon-carbon bond
hydrolases known to be members of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-hydrolase fold family
(19, 20). In addition to the degradation of aromatic amino acids,
carbon-carbon bond hydrolysis reactions are also required for the
degradation of aromatic hydrocarbons by soil bacteria (21). Therefore,
further understanding of this type of reaction is of potential use in
bioengineering efforts aimed at the bioremediation of toxic hydrocarbon
wastes, such as polychlorinated biphenyls.
View larger version (4K):
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Scheme 1.
Crystal structures of FAH and FAH complexed with its physiological
products, fumarate and acetoacetate, were recently determined (22). The
FAH structure consists of a 120-residue N-terminal domain of unknown
function and a 300-residue C-terminal domain defined by a novel
-sandwich roll structure that forms an active site in close
proximity to the dimer interface (Fig. 1,
A and B). The FAH active site occurs in a
solvent-filled cavity that is complementary in shape and charge to the
fumarylacetoacetate substrate (22). The base of the active site is
formed by a metal ion coordinated by four side-chain carboxyl groups at
the edge of the
-roll, whereas the sides are formed by helices and
turns located above the
-roll. The product acetoacetate binds to FAH by providing two oxygen ligands to a Ca2+ ion present in
the metal ion binding site. Fumarate binding involves an Arg and two
Tyr side chains near the entrance to the active site. These structural
observations, along with the finding that a D233V substitution causing
HT1 (23) and a E201G mutation causing a phenotype like HT1 in
mice,2 led to a proposed
mechanism involving the active site His-133 imidazole ring as a
general base catalyst in the direct activation of a nucleophilic water
molecule (22). The side chains of Arg-237, Gln-240, and Lys-253 are
proposed to stabilize the formation of a tetrahedral alkoxy transition
state intermediate, and the metal ion is proposed to function in
binding substrate and stabilizing an acetoacetate carbanion leaving
group.
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Molecules containing phosphorus have proven to be effective inhibitors of enzyme-catalyzed hydrolytic and condensation reactions. Substrate analogs containing phosphonate, phosphonoamidate, and phosphinate groups have been used as effective noncovalent inhibitors of aspartylproteases (24) and metalloproteases (25), whereas phosphonate and phosphinate bisubstrate inhibitors have been used to inhibit farnesyltransferase (26). Of particular interest to the enzymatic mechanism of FAH are studies indicating that the tetrahedral geometry associated with the phosphorus groups in these types of inhibitors closely approximates both geometric and electronic aspects of the transition state (24, 27, 28). Thus, the effectiveness of these compounds as inhibitors is presumably associated with similarities to the high energy reaction intermediates stabilized during catalysis.
The synthesis of an inhibitory phosphinate substrate analog of FAH and
its characterization using kinetic and crystallographic methods are
reported here. As an effective inhibitor of a carbon-carbon hydrolase,
HMPOBA represents the first example of a phosphorus-based analog
targeting this class of enzymes. These results provide a context for
future structure-based design of FAH inhibitors and provide additional
insight into the mechanism by which FAH catalyzes the hydrolysis of
carbon-carbon bonds.
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EXPERIMENTAL PROCEDURES |
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Inhibitor Synthesis and Characterization
Products of the synthesis (Scheme
2) were confirmed using gas
chromatography/mass spectrometry data acquired on a Hewlett Packard
5995 and by recording 1H, 13C, and
31P NMR data using a Bruker AC-300 (300.13 MHz)
spectrophotometer. Solvents were dried by passing over activated
alumina, and all reactions were performed under a positive pressure of
dry N2.
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Chloromethyl Methylphosphonate (Product 1)-- Oxalyl chloride (6.54 ml, 75.0 mmol) was added dropwise to a stirred solution of dimethyl methylphosphonate (6.2 g, 50.0 mmol) in CH2Cl2 (3 ml) with cooling at 0 °C. This reaction was left to warm to room temperature overnight. The volatiles were removed in vacuo to yield a yellow oil, which was suspended in THF (80 ml) and used directly. MS m/z 128.
4-(Methoxyphosphinoyl)-3-oxo-butanoic Acid, 1,1-Dimethylethyl
Ester (Product 2)--
t-Butyl acetoacetate (17 ml, 102.5 mmol) was added dropwise to a stirred slurry of NaH (2.46 g, 102.5 mmol) in THF (120 ml) with cooling at 0 °C. After 10 min the
reaction was cooled in a dry ice/acetone bath, whereupon
n-BuLi (1.6 M in hexanes, 64.1 ml, 102.5 mmol)
was added dropwise. The red-orange reaction was stirred for 40 min. The
phosphonochloridate, 1, from above was cooled in a dry
ice/acetone bath, whereupon the red-orange dianion solution was
transferred via cannula (dropwise over 15 min) while stirring. After
2 h, the reaction was quenched with HOAc (17.5 ml), water (200 ml)
was added, and THF was removed in vacuo. This mixture was
extracted with CH2Cl2 (3 × 200 ml), and
the organic extract was washed with 250 ml of H2O/EDTA
(0.01%) and dried in vacuo. Silica gel chromatography
(EtOAc/hexane, 1:1) of the rust-colored oil afforded 2 (70%
yield). Only data for the keto form are reported. 1H NMR
(CDCl3): 1.47 (9H, s), 1.58 (3H, d,
JHCP = 14.6 Hz), 3.28 (2H, d,
JHCP = 17.9 Hz), 3.58 (2H, s), 3.78 (3H, d,
JHCOP = 11.3Hz). 13C NMR
(CDCl3):
14.06 (1C, d, JCP = 97.9 Hz), 27.90 (3C, s), 45.46 (1C, d, JCP = 77.96 Hz), 51.33 (1C, d, JCOP = 6.79 Hz), 51.60 (1C, s), 82.34 (1C, s), 165.85 (1C, s) 98.12 (1C, d,
JCCP = 5.43 Hz). 31P NMR
(CDCl3):
48.5.
4-(Hydroxymethylphosphinoyl)-3-oxo-butanoic Acid, Dipotassium
Salt (HMPOBA, Product 3)--
The dry phosphinic ester (0.61 g, 2.45 mmol) was dissolved in CH2Cl2 (7 ml) and dry
cyclopentene (24.5 mmol, 2.2 ml). TMSI (7.83 mmol, 1.11 ml) was added
dropwise to the stirred solution at 0 °C, and the reaction was left
for 2 h. The volatiles were removed in vacuo, and
toluene (5 ml) was added and evaporated. The residue was dissolved in
THF (2 ml) and transferred via cannula to a stirred solution of
KHCO3 (4.73 mmol, 0.47 g) in water (10 ml).
CO2 was removed under vacuum, and the pH was adjusted to 7 with KOH. Following evaporation in vacuo, residual volatiles were removed by successive evaporation with CH3CN (2 × 5 ml) and CCl4 (5 ml). The residue was dried in
vacuo overnight to yield 0.86 g of a pale yellow solid
(137%). The solid (0.422 g) was dissolved in H2O (5 ml),
and the phosphorus concentration (0.2 M) was assayed (29).
The excess mass was shown by neutron activation analysis to result from
I (presumably as KI). 1H NMR
(H2O/D2O, 9:1):
3.59 (2H, s), 3.14 (2H, d,
JHCP = 17.4 Hz), 1.35 (3H, d,
JHCP = 14.2Hz). 13C NMR
(H2O/D2O, 9:1):
18.91 (1C, d,
JCP = 97.9 Hz), 51.59 (1C, d,
JCP = 71.6 Hz), 56.60 (1C, s), 177.00 (1C, s),
207.74 (1C, d, JCCP = 32.27 Hz). 31P
NMR (H2O/D2O, 9:1):
33.34.
Kinetic Assays
Kinetic assays utilized human FAH expressed with an N-terminal
poly-His tag using the vector pQE-30 (Qiagen, Chatsworth, CA). Bacteria containing the pQE-FAH plasmid were grown in LB media and
induced overnight with 0.5 mM isopropyl
-D-thiogalactopyranoside. The bacteria were harvested by
centrifugation and lysed by sonication. Lysates were centrifuged, and
recombinant FAH was purified from the supernatant using metal chelate
chromatography by eluting with a solution containing 50 mM
sodium phosphate, pH 6.0, 300 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 100 mM
EDTA. FAH activity was assayed spectrophotometrically using acetopyruvate (AP) (Sigma Chemical Co.) and fumarylacetoacetate (FAA)
prepared enzymatically from homogentisic acid (Sigma) as described previously (17, 18, 30). Assays were initiated by adding
recombinant His-tagged FAH to reaction mixtures that covaried AP
(0.625, 0.833, 1.25, 2.5, and 5.0 mM) with HMPOBA (0.0, 250, 500, and 750 µM) or covaried FAA (3, 8, and 15 µM) with HMPOBA (0, 125, 250, 500, or 750 µM). The disappearance of AP and FAA was followed at 292 and 330 nm for 2-3 min at 37 °C, respectively. Extinction
coefficients of 9,510 and 13,500 cm
1
M
1 were used for AP and FAA, respectively.
Initial rates were fit to the competitive inhibition model by nonlinear
regression analysis using SigmaPlot (Jandell Scientific).
Expression, Crystallization, and Structure Determination
Mouse FAH was expressed as a glutathione
S-transferase fusion protein (22) using pGEX4T-1 (Amersham
Pharmacia Biotech) and was purified using glutathione-Sepharose
(Amersham Pharmacia Biotech). Mouse and human FAH share 88% sequence
identity, with strict conservation of the active site residues. The
fusion protein was digested using bovine thrombin (Sigma) in 20 mM Tris, 10 mM CaCl2, pH 7.4. The free FAH was separated from GST using a Mono-Q column (Amersham Pharmacia Biotech). FAH was at least 90% pure as assessed by Coomassie Blue-stained SDS-polyacrylamide electrophoresis gels and was
concentrated using a Centricon ultrafiltration device (Amicon). FAH
concentrations were determined using an extinction coefficient of 1.31 ml·cm1·mg
1 at 280 nm. FAH was
crystallized by the hanging drop vapor diffusion method at room
temperature by combining 10 µl of a 3 mg/ml FAH solution with 5 µl
of a precipitant solution containing 17-18% polyethylene glycol 8000, 0.03-0.15 M nickel acetate, 0.00-0.24 M
sodium acetate, 0.1 M sodium cacodylate, pH 6.5 (22).
Acetate was maintained at a constant 0.3 M concentration.
FAH crystallizes under these conditions in the space group
P21 with unit cell dimensions of 64.1 × 109.5 × 67.5 Å3 and
= 102.4°.
Crystals of FAH were soaked with 0.1 M HMPOBA in
precipitant solution for 16 h at room temperature prior to data
collection. Data were collected on beamline X12C at the Brookhaven
National Laboratory National Synchrotron Light Source at 100 K using a cryoprotectant solution containing 30% polyethylene glycol 400, 0.3 M sodium acetate, 0.1 M sodium cacodylate, pH
6.5. Data were integrated, scaled, and merged using DENZO and SCALEPACK
(31). Details of the data collection and processing are given in Table I below. The structure of FAH complexed with HMPOBA was determined by
refining coordinates of the mouse FAH structure (22) against data
collected from a single soaked crystal. The positions of the ligand in
the FAH active site were apparent in electron density maps calculated
using 2Fo Fc and
Fo
Fc coefficients. Iterative
cycles of model building using the program O (32) were followed by
restrained refinement using the programs REFMAC (33) and
XPLORv3.8 (34) and automated water building using the program ARPP
(33). A model of FAH complexed with HMPOBA consisting of 835 amino acid
residues, 1 Ni2+, 2 Mg2+, 2 Ca2+, 2 HMPOBA molecules, 2 acetate molecules, and 833 water molecules has been
refined to a crystallographic R-factor of 0.181 (Rfree = 0.199) at 1.30-Å resolution. Details
of the model refinement are given below in Table I. Unrestrained
refinement and refinement, including model H atoms did not improve the
Rfree residual. The figures were created using O
(32) and MOLSCRIPT (35).
Supplementary NMR Data
NMR data were obtained using a Bruker AC-300 (300.13 MHz) spectrophotometer. 1H (300.13 MHz) and 13C (75.45 MHz) resonances were recorded in parts per million downfield from tetramethylsilane. 31P (121.49 MHz) spectra were recorded in parts per million downfield from 85% phosphoric acid for CDCl3 samples, and downfield from 1% phosphoric acid for D2O samples.
4-(Methoxyphosphinoyl)-3-oxo-butanoic Acid, 1,1-Dimethylethyl
Ester (Product 2)--
1H NMR (CDCl3) keto:
1.47 (9H, s, -OC(CH3)3), 1.58 (3H, d,
JHCP = 14.6 Hz, CH3P), 3.28 (2H, d,
JHCP = 17.9 Hz, -P(O)CH2C(O)-), 3.58 (2H, s, -CH2C(O)O-), 3.78 (3H, d,
JHCOP = 11.3Hz, -OCH3); enol:
1.49 (9H, s, -OC(CH3)3), 1.60 (3H, d,
JHCP = 14.6Hz, CH3P-), 2.74 (2H, d,
JHCP = 17Hz, -P(O)CH2C(O)-), 3.58 (2H, s, -CH2C(O)O-), 3.78 (2H, d,
JHCOP = 11.3 Hz, CH3P), 12.41 (1H,
s, -OH). 13C NMR (CDCl3) keto:
14.06 (1C,
d, JCP = 97.9 Hz, CH3P), 27.90 (3C,
s, -OC(CH3)3), 45.46 (1C, d,
JCP = 77.96 Hz, -P(O)CH2C(O)-), 51.33 (1C, d, JCOP = 6.79 Hz,
CH3OP), 51.60 (1C, s, -C(O)CH2C(O)-), 82.34 (1C, s, OC(CH3)3), 165.85 (1C, s,
-CH2C(O)O-), 98.12 (1C, d, JCCP = 5.43 Hz). 31P NMR (CDCl3):
48.5.
4-(Hydroxymethylphosphinoyl)-3-oxo-butanoic Acid, Dipotassium
Salt (HMPOBA, Product 3)--
1H NMR
(H2O/D2O, 9:1): 3.59 (2H, s,
C4H2), 3.14 (2H, d, JHCP = 17.4 Hz, C9H2), 1.35 (3H, d,
JHCP = 14.2Hz, C15H2).
13C NMR (H2O/D2O, 9:1):
18.91 (1C, d, JCP = 97.9 Hz, C15), 51.59 (1C, d, JCP = 71.6 Hz, C9), 56.60 (1C, s, C4), 177.00 (1C, s, C2), 207.74 (1C, d,
JCCP = 32.27 Hz, C5).
31P NMR (H2O/D2O, 9:1):
33.34.
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RESULTS |
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Inhibitor Synthesis and Characterization--
HMPOBA was
synthesized according to Scheme 2 with a 70% yield via a three-step
synthesis from dimethyl methylphosphonate. The phosphonochloridate
(product 1) was made by reaction of dimethyl methylphosphonate with
oxalyl chloride. Dianions of -keto esters have previously been shown
to react with acyl chlorides to form terminally acylated products (36).
Similarly, reaction of this phosphonochloridate with the dianion of
t-butyl acetoacetate resulted in phosphinylation of the
terminal carbon to yield the phosphinic ester (product 2). Two
equivalents of dianion were used in the reaction, because a proton is
made acidic upon phosphinylation. Deprotection of the phosphinic ester
with TMSI followed by basic work-up circumvented decarboxylation, which
occurs in acid, and the di-potassium salt of HMPOBA was produced
(product 3).
Kinetic Assays--
Hydrolysis of acetopyruvate and the
physiological substrate was followed spectrophotometrically by loss of
absorbance at 292 and 330 nm, respectively. Initial velocities varied
as a function of substrate concentration in the absence of inhibitor
according to simple Michaelis-Menten kinetics. Substrate inhibition of
FAH by AP (17) was not observed at concentrations 15-fold higher than
the Km. Steady-state kinetic assays performed as a
function of HMPOBA concentration were fit according to the competitive inhibition pattern. Global fitting of the data measured at three FAA
and five inhibitor concentrations yielded a Ki of 30.3 ± 7.7 µM and a Km of
0.4 ± 0.1 µM (data not shown). Global fitting of
data measured at five AP and four inhibitor concentrations yielded a
Ki of 84.8 ± 4.2 µM and a
Km of 1.3 ± 0.1 mM (Fig.
2). The Ki obtained
from AP assays is likely to be more accurate, given difficulties
associated with making measurements below the Km of
FAA.
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Structure of the FAH·HMPOBA Complex-- The crystal structure of mouse FAH complexed with HMPOBA was determined by soaking an enzyme crystal with the inhibitor. The inhibitor was readily identified in electron density maps calculated at near atomic resolution (1.3 Å) using crystallographic phases from a previously determined structure of mouse FAH (22) and the structure factors measured from the soaked crystal. Crystallographic data and refinement statistics are given in Table I. Refinement of this model against the 1.3-Å data have enabled atomic positions in the model to be defined by electron density that discriminates between carbon, nitrogen, and oxygen atoms (Fig. 3) with an overall coordinate error estimated at less than 0.05 Å.
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The HMPOBA binds the FAH active site through hydrogen bond and/or
charge interactions between the pro-S phosphinoyl oxygen atom and the Gln-240 N2, Arg-237 NH1 and Lys-253 N
side
chain atoms, which are separated by respective distances of 2.9, 2.9, and 2.8 Å from the inhibitor oxygen atom (Fig.
4). The pro-R phosphinoyl oxygen atom interacts with His-133 at a distance of 2.7 Å and a water
molecule at a distance of 2.8 Å bound between the Arg-237 NH2 and Thr-350 O
1 atoms (Fig. 4). HMPOBA provides two
ligands to the FAH active site Ca2+ present in the
catalytic metal ion binding site through the carbonyl oxygen atom (O8)
and one carboxyl oxygen atom (O1). The HMPOBA carboxyl oxygen atoms are
also located at 2.9 and 3.0 Å from the main chain nitrogen atom of
Thr-350. Although the enzyme ligand interactions appear to be dominated
by the electrostatic interactions described above, several
complementary van der Waals contacts are also made between the
inhibitor and FAH. The methylene carbon bonded to the phosphorus atom
is located at respective distances of 3.9 and 4.0 Å from the C
1 and
C
2 atoms of Leu-247, which originates from the opposite FAH subunit
as part of the dimer interface (Fig. 1B). The inhibitor
carbonyl carbon atom is located 3.8 Å from the Tyr-159 C
2 atom. The
inhibitor methylene carbon atom positioned between the carbonyl and
carboxyl groups is located 3.7 Å from the Tyr-128 C
1 atom and 3.2 Å from the Phe-127 O atom. The HMPOBA methyl carbon atom is located
3.8 Å from the Tyr-128 C
1 atom.
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Active Site Metal Ions--
In addition to the Ca2+
occupying the catalytic metal ion binding site, a second divalent metal
ion was identified in the active site during the refinement of the
FAH·HMPOBA complex when a water molecule bound to the Asp-233 side
chain was found to have an abnormally low temperature factor and an
associated positive peak in Fo Fc difference maps. The isotropic temperature factors of equivalent water molecules in each active site refined to
3.0 and 3.3 Å2, whereas the surrounding oxygen atoms had
temperature factors ranging from 8.3 to 9.6 Å2.
Furthermore, the three carbonyl, one carboxyl, and one hydroxyl oxygen
atoms surrounding each of the water molecules provided an excess of
hydrogen bond acceptors. Reassignment of this peak to Mg2+
in both active sites eliminated the positive feature from the Fo
Fc difference map and
resulted in refined temperature factors for the Mg2+ ions
of 8.7 Å2. Examination of several other FAH structures
refined between 1.55 and 1.90 Å (22) indicate similar anomalies with
the water bound to the Asp-233 side chain, and in every case
reassignment to Mg2+ is consistent with the refined
temperature factors and difference maps. Thus, FAH contains a binuclear
metal binding site centered about the Asp-233 side chain (Fig. 4), with
one carboxyl oxygen binding the catalytic metal ion and the other
binding Mg2+.
The hexagonal coordination geometry for the catalytic metal ion site is
best described as a distorted square bipyramid, whereas the pentavalent
Mg2+ coordination is best described as a distorted square
pyramid. Details of the coordination geometries are given in Table
II. The largest distortions of the
octahedral Ca2+ coordination involve ligands from the bound
inhibitor. These distortions are likely to be due to covalent bonding
restraints on the positions of the O1 and O8 atoms within the -keto
acid portion of HMPOBA. The O1 and O8 atoms form a six-membered ring with the carbonyl, methylene and carboxyl carbon atoms, and the Ca2+ that is strained by both a 73°
O1-Ca2+-O8 angle and a nearly planar configuration of
all but the methylene carbon atom.
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Alternate Active Site Side-chain Conformations--
FAH crystal
structures representing free enzyme, the enzyme·HMPOBA complex, and
the enzyme·product complex reveal distinct side-chain conformations
for several catalytic residues within the active site. The CA positions
of free FAH, represented by the structure of the
selenomethionine-substituted enzyme refined at 1.9 Å, were
superimposed with those of the FAH·HMPOBA complex with an overall
r.m.s. deviation of 0.21 Å, whereas those of the FAH·product
complex refined at 1.9 Å were superimposed with the FAH·HMPOBA
structure with an r.m.s. deviation of 0.33 Å. The active site of the
free FAH structure and the FAH·HMPOBA complex contain an acetate
bound to Tyr-128 and Arg-142 (Figs. 3 and 4), and a second acetate
binds the Ca2+ ion in the free FAH structure (22). The
largest side-chain motions in the active site are associated with
Tyr-128, Glu-199, Ser-235, Gln-240, and Lys-253. The position of the
Glu-199 O2 atom in the HMPOBA complex differs by 2.2 Å relative to
both the free and product complex structures (Fig.
5). This Glu-199 motion is largely the
result of respective 20° and 50° rotations about the
side-chain
2 and
3 angles in the
HMPOBA complex relative to the free and product complex structures. The
position of the Ca2+ ligand, Glu-199 O
1, does not change
significantly between the structures. A 100°
3
rotation changes the position of the Lys-253 N
atom by 1.6 Å in the
HMPOBA complex relative to the free structure. The Gln-240 side-chain
N
2 atom also moves about 1.6 and 1.3 Å toward the active site
entrance in the product complex relative to the respective free and
HMPOBA complex structures. The Tyr-128 OH atom moves by 1.2 Å in the
FAH·HMPOBA and FAH·product complexes relative to that of the free
enzyme, but the position of this atom differs by less than 0.3 Å between the structures of the two complexes. The Arg-142
NH2 and Tyr-244 OH atoms near the entrance to active site
also move between 0.7 and 0.9 Å in the complexes relative to the free
enzyme. Moderate shifts (0.6-0.8 Å) in the positions of the Val-137,
Arg-237, Pro-246, and Leu-247 side-chain atoms are also observed. In
contrast, the main-chain CA atoms of the residues mentioned above
deviate by less than 0.4 Å, with the exception of Arg-142, which has
CA positional differences of 0.6 and 0.5 Å in the product and HMPOBA
complexes relative to the free structure, and Ser-235, having a CA
positional difference of 0.5 Å between the HMPOBA and product
complex.
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DISCUSSION |
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Mechanistic Implications of Phosphorus-based Hydrolase Inhibitors-- Our studies of the phosphinoyl compound HMPOBA demonstrate the potential for this class of molecules to effectively inhibit a hydrolase that acts on carbon-carbon bonds. Phosphorus-containing compounds have previously been characterized as noncovalent inhibitors and transition state analogs of other hydrolases, including aspartyl- and metalloproteases (24, 25). A variety of crystallographic, kinetic, and structure/function data suggests that inhibitors containing tetrahedral phosphorus groups can share characteristics of tetrahedral transition state intermediates (24, 27, 28). Although the Ki for HMPOBA is higher than expected for a true transition state analog, it is reasonable to conclude that our results approximate many of the geometric and electronic features expected for an intermediate of FAH-catalyzed reactions.
The FAH·HMPOBA complex shares many features in common with the
previously proposed mechanism of FAH (22). According to this mechanism,
substrate binding to the catalytic metal ion positions a carbonyl
carbon atom for nucleophilic attack by hydroxide to give rise to a
tetrahedral alkoxy intermediate (Fig. 6).
Consistent with this mechanism, the HMPOBA acetoacetyl group
interacts directly with Ca2+ present in the catalytic metal
binding site, and the tetrahedral phosphinoyl group occupies the
position predicted for the alkoxy intermediate (22). The HMPOBA
pro-S oxygen atom is likely to be ionized and accepting
three hydrogen bonds from the Arg-237, Gln-240, and Lys-253 side chains
(Fig. 4). These side chains are proposed to function as an
oxyanion hole, stabilizing the negative charge acquired at this
position during catalysis (Fig. 6). The pro-R oxygen atom
occupies the position of a water molecule hydrogen bonded to the
His-133 and Glu-199 side chains (Fig. 5) in the free FAH structure
(22). This water molecule is proposed to function as the active site
nucleophile (22) as part of a catalytic triad involving the His-133 and
Glu-365 side chains (Fig. 4). Glu-199 may function along with His-133
to direct the nucleophilic attack by orienting the lone pairs of
electrons on the water molecule in the direction of the carbonyl carbon
atom. Therefore, the FAH·HMPOBA structure shares several features of
the proposed catalytic intermediate, and provides further support for
the involvement of a metal ion and the His-133, Arg-237, Gln-240, and
Lys-253 side chains in catalysis.
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Several features of the FAH·HMPOBA structure are likely to account
for a relatively high Ki. The most significant factor is likely to be the lack of a carboxyethylene group in HMPOBA
(Schemes 1 and 2), which could interact with the charged or polar
groups of Tyr-128, Arg-142, and Tyr-244 (Figs. 3-6). The importance of
these interactions is suggested by the binding of acetate (Figs. 3 and
4) or fumarate at this location in all FAH structures reported to date
(22). Potential van der Waals interactions between the fumaryl group
and nonpolar atoms present in the side chains of Tyr-128, Val-137,
Phe-141, Tyr-244, and Pro-246 are also absent from the HMPOBA complex.
Slight differences in the tetrahedral phosphorus center relative to the
predicted alkoxy intermediate (Fig. 6) may also exist. The
pro-R oxygen atom is likely to be charge neutral and
accepting two hydrogen bonds from the protonated imidazole of His-133
and a water molecule (Fig. 4). This arrangement differs slightly
from the proposed tetrahedral intermediate, where the protonated
His-133 N2 atom is adjacent to a hydroxyl proton, rather than a
ketone oxygen (Fig. 6). Differences in the atomic radii of tetrahedral
C and P could also have slight steric effects. Additional studies are
underway to assess these possibilities and to improve inhibitor binding
by adding groups to the HMPOBA framework to exploit
electrostatic and hydrophobic interactions near the entrance to the
active site.
Active Site Metal Ions-- The structure of the FAH·HMPOBA complex is consistent with both catalytic and structural requirements for divalent metal cations. In addition to substrate binding, the catalytic metal ion is expected to stabilize the enolate form (Fig. 6) of the acetoacetate carbanion leaving group (22). Charge neutralization and prevention of carboxylate side-chain repulsion in this region of the FAH structure are other likely roles for the catalytic metal ion. Ca2+ occupancy of the catalytic metal binding site is consistent with several different FAH structures refined at 1.3- to 1.9-Å resolution. The catalytic metal binding site must have significantly higher affinity for Ca2+ than the crystallization additive Ni2+; however, exogenous Ca2+ is added during the FAH purification ("Experimental Procedures"). Studies to identify the physiologically relevant catalytic metal ion are underway.
The Ca2+ coordination geometry (Table II) is
consistent with values obtained from small molecule analogs of
Ca2+·carboxylate complexes; however, the observed
Mg2+ coordination is unusual. The HMPOBA O1 and O8 atoms,
the Asp-233 O2 atoms, and the Glu-201 O
2 atoms are planar ligands
of Ca2+. The Asp-126 O
2 and Glu-199 O
1 atoms are
axial ligands. The average carboxylate oxygen distance to the
Ca2+ of 2.31 ± 0.04 Å (n = 10)
compares favorably with a mean value of 2.37 Å and range of 2.27-2.49
Å recently reported in a survey of the Cambridge Structural Data base
(CSD (13)). This finding further supports the accuracy of the
1.3-Å refinement, which placed no restraints on the metal ion
coordination geometries. The significantly longer distance between the
HMPOBA carbonyl oxygen (Table II) and Ca2+ may be due to
the lack of a formal charge on O8. Although metal·carbonyl oxygen
complexes were not part of the CSD survey (13), the distances between
Mg2+ and the main-chain carbonyl oxygen atoms (Table II)
are about 0.3 Å longer than those found in representative CSD
structures.3 The Asp-233
O
1, Trp-234 O, Gly-256 O, and Thr-257 O
atoms are planar ligands
of Mg2+. The Lys-253 O is the axial ligand. The distances
between Mg2+ and the Asp-233 O
1 and the Thr-257 O
atoms are, respectively, 0.3 and 0.6 Å longer than the mean distance
reported for relevant Mg2+ complexes (13).
The newly identified Mg2+ appears to have a role in
stabilizing the structure of the -roll that forms the central
scaffold of the FAH active site and a possible role in intersubunit
communication. In contrast to Ca2+, Mg2+ is not
added exogenously and is not accessible to solvent within the active
site. The Mg2+ is located within the
-roll structure
near the dimer interface, linking main-chain atoms at the end of a
helix between Phe-250 and Phe-255 with two
-strands between Thr-257
and Ile-259 and between Phe-226 and Ala-236 (Fig. 1). Mg2+
may also stabilize a loop between Trp-234 and Lys-253 that positions Pro-246 and Leu-247 within the active site of the opposite subunit. Although half-site reactivity or catalytic cooperativity has not been
reported, the FAH structure seems suited to such functions. Interactions between the Leu-247 side chain of one subunit and HMPOBA
bound in the opposite subunit were described above. Pro-246 also makes
an intersubunit interaction with Tyr-244 of the fumaryl binding site
and interacts with fumarate in the FAH·product complex (22).
Therefore, changes within the fumaryl and/or acetoacetyl binding sites
could be transmitted through the 234/253 loop to the active site in the
opposite subunit. The catalytic residues Arg-237, Gln-240, and Lys-253
and the D233V, W234G, and P249T mutations causing HT1 (19) also occur
in this region of the structure. Therefore, Mg2+ appears to
be important for joining secondary structures within the
-roll, as
well as having potential roles in communication between active sites
and positioning the side chains of the oxyanion hole.
Mg2+ could have additional catalytic effects. Mg2+ could maintain a favorable overall charge within the active site by balancing the formal negative charges associated with the dicarboxylic acid substrate and the four carboxylate ligands to the catalytic metal ion. Assuming Arg-142, Arg-237, and Lys-273 are protonated, the net formal charge of the substrate-bound active site in the presence of two divalent cations is +1. Finally, Mg2+ could affect properties of the catalytic metal ion, such as Lewis acidity, through the shared Asp-233 carboxylate ligand. The importance of this residue in normal FAH function is indicated by the HT1-associated D233V mutation (23), which is anticipated to affect both the catalytic metal ion and Mg2+ binding sites.
Side-chain Mobility-- A comparison of FAH structures bound to different ligands indicates conformational mobility within the FAH active site. Four of the five active site residues having the largest positional differences also have central roles in the proposed mechanism. This observation suggests that these conformational differences may be significant to substrate binding or product release, formation of the leaving group, and proton transfer to the leaving group. The observed positional deviations are significantly larger than the estimated coordinate errors of the refined structures and involve side chains that are well defined by electron density. Indeed, these side chains have temperature factors that are less than 24.0 Å2, indicating they occupy distinct, stable conformations in different structures, rather than being mobile within a given structure.
Entry to the FAH active site appears restricted in the FAH structures,
indicating that a conformational change is required for substrate
binding. A small opening to the active site is defined by a triangular
arrangement of the Arg-142 NH1, Pro-246 C, and Tyr-244 OH atoms
separated by distances of 5.3-5.5 Å. Considering the van der Waals
radii of the atoms, this opening is not large enough to accept
substrates, products, or inhibitors. However, ligand binding to FAH
within the crystal lattice suggests the entrance to the active site
does open. The entrance to the active site is likely to occur near the
end of a helix formed between Ser-130 and Gly-143. This region has some
of the highest temperature factors found in the FAH structures and
shows some of the largest r.m.s. deviations between main-chain
positions. Furthermore, a G369V mutation causing HT1 is located
adjacent to this region (23). A transient opening of Phe-141 and
Arg-142 away from the active site would expose an elongated entrance.
Conversely, occupancy of the fumarate binding site would cause the
active site to close by creating the hydrogen bond network between
Tyr-128, Arg-142, and Tyr-144. It is also interesting to note that,
although fumarate binding might be expected to affect the conformation
of residues near the entrance to the active site, HMPOBA binding also
resulted in significant conformational changes in Tyr-128, Arg-142, and Tyr-244 relative to the free structure. Therefore, ligand occupancy of
the acetoacetate binding site can apparently induce conformational changes near the entrance to the active site.
Conformational changes affecting the oxyanion hole may accompany the transformation of the tetrahedral alkoxy intermediate to the planar fumarate carboxyl group (Fig. 6). Substantial differences in the Arg-237, Gln-240, and Lys-253 side-chain positions are observed between the different FAH structures. The separation of products following the breakdown of the tetrahedral intermediate may be indicated in comparing the HMPOBA and product structures, which reveals a 2.3-Å difference between the positions of the HMPOBA phosphorus and the fumarate carboxyl carbon atoms. Thus, fumarate formation may pull Gln-240 in the direction of the entrance to the active site.
Side-chain mobility may also be related to proton transfer to the
carbanion leaving group. This step may involve the protonated forms of
either the fumarate carboxyl group or the Lys-253 amine group. His-133
is unlikely to serve as the general acid, because the N2 atom is
located over 5.1 Å away from the HMPOBA equivalent of the acetoacetate
methyl group, the C9 atom. The superimposed structures reveal that a
fumarate carboxyl oxygen is located 2.9 Å from the HMPOBA C9 atom.
This distance would allow for direct or water-mediated proton transfer
between the protonated carboxyl and the leaving carbanion. Another
possibility involves the Lys-253 rotation away from an interaction with
the Ser-235 O
in the free structure to interact with HMPOBA as part
of the oxyanion hole. This motion may be coupled with a change in the
conformation of Glu-199, which pivots about the catalytic metal ion to
accept a hydrogen bond from Lys-253 in the HMPOBA structure (Fig. 5) and suggests Lys-253 could function as the proton donor. Indeed, a
preferred rotamer conformation resulting from a 90° rotation about
the side chain
2 angle brings the Lys-253 N
atom
within 3.0 Å of the HMPOBA C9 atom.
Summary--
Studies of more exact transition state analogs,
currently under development in our laboratories, are expected to
address many of these issues. In particular, a crystal structure of FAH
complexed with an analog, which incorporates a competent mimic of the
fumaryl portion of FAA should allow us to probe the role of the 140/147 loop in the closing/opening portion of the active site and to ascertain
the related functions of Phe-141 and Arg-142 in covering the respective
binding sites of acetoacetate and fumarate. Additionally, it is
expected that such an analog will serve as a more potent inhibitor of
FAH, with profound therapeutic properties for hepatic repopulation,
far superior to that observed with HMPOBA.
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ACKNOWLEDGEMENTS |
---|
We thank Stephen Frantz of the Reed College Reactor Facility and Jill Dorocke, Kara Manning, and Soya Gamsey for their assistance in this work.
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FOOTNOTES |
---|
* This work was supported by NIDDK, National Institutes of Health (NIH) grants (to D. E. T. and M. G.), by an NIGMS, NIH grant (to R. W. M.), and by grants from the Indiana Affiliate of the American Heart Association and the Grace M. Showalter Research Trust Fund (to D. E. T.).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.
The atomic coordinates and the structure factors (code 1HYO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed: Dept. of
Biochemistry, Indiana University, Indianapolis, IN 46202. Tel.:
317-274-1551; Fax: 317-274-4686; E-mail: dtimm@iupui.edu.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M007621200
2 D. Johnson personal communication.
3 M. Harding, personnal communication.
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ABBREVIATIONS |
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The abbreviations used are:
FAH, fumarylacetoacetate hydrolase;
AP, acetopyruvate;
n-BuLi, n-butyllithium;
CSD, Cambridge Structural Database;
FAA, fumarylacetoacetate;
HMPOBA, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid;
LB, Luria broth;
THF, tetrahydrofuran;
TMSI, iodotrimethylsilane;
GST, glutathione
S-transferase;
MS, mass spectrometry;
r.m.s., root mean
square;
CA, carbon atom.
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