From the
Departments of Pediatrics and
¶Pharmaceutical Sciences, The University of
Colorado Health Sciences Center, Denver, Colorado 80262 and the
Departments of Biochemistry and Molecular
Biology and Medical Genetics, Mayo Clinic and Mayo Foundation, Rochester,
Minnesota 55905
Received for publication, October 21, 2002 , and in revised form, April 16, 2003.
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ABSTRACT |
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INTRODUCTION |
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MCAD and SCAD are both inactivated by 2-alkynoyl-CoAs (68). The mechanism of inactivation of these two related dehydrogenases is proposed to involve the initial abstraction of a proton from C-4 of the inhibitor even though it is not mandatory that the catalytic base access C-4 during normal catalysis (5). The residues that are covalently modified by these mechanism-based inhibitors, in both MCAD and SCAD, are the glutamate bases that initiate the catalytic pathways by abstracting a C-2 proton prior to hydride transfer to the N-5 of FAD (6, 7).
In the experiments reported here, we investigated the rapid inactivation of GCD by 2-pentynoyl-CoA. The experiments show that the catalytic base, Glu-370, is modified in the inactivation process. The rapid inactivation of GCD is, therefore, not because of the covalent modification of another residue. The experiments provide rate constants for the formation and decay of a spectral intermediate in the inactivation pathway along with rate constant for inactivation and equilibrium constants for the binding of 2-pentynoyl-CoA and 3-thiaglutaryl-CoA. Because the rate of inactivation is extremely rapid, the latter constants were determined in the presence of a non-oxidizable analog, 3-thiaglutaryl-CoA, which competes for the active site. A rationale for the enhanced reactivity of GCD based on access of the catalytic base to C-4 of the inhibitor is proposed and is consistent with the rate of inactivation and formation of a charge transfer species in the reaction of 2-pentynoyl-CoA with GCD.
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EXPERIMENTAL PROCEDURES |
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Enzyme AssaysThe dehydrogenases were assayed
spectrophotometrically (Shimadzu UV-2401 PC spectrophotometer) at 25 °C by
monitoring the decrease in absorbance at 600 nm. The assays were performed in
50 mM potassium phosphate, pH 7.6, 45 µM
glutaryl-CoA, 3 µM electron transferring flavoprotein, and 50
µM DCPIP, the terminal electron acceptor, and started by adding
the enzyme (4). Rates were
calculated using 600 nm = 20.1
mM1 cm1 for the
reduction of the dye (4). In
these assays, electron transferring flavoprotein was used in place of 2
mM phenazine methosulfate.
Preparation of Acyl-CoAs2-Pentynoyl-CoA was synthesized
from 2-pentynoic acid by the mixed anhydride method
(10), purified as described
(11), and quantified using
260 nm = 20.9 mM1
cm1
(6). 3-Thiaglutaryl-CoA was
synthesized as described previously
(4). The purity of all
synthetic acyl-CoAs was
98% as determined by analytical high performance
liquid chromatography
(12).
Inactivation of GCDThe reaction of 2-pentynoyl-CoA with
GCDs was carried out in 10 mM potassium phosphate, pH 7.0,
containing 5% ethylene glycol at 4 °C with an enzyme concentration of 2
µM. All inhibition experiments with 2-pentynoyl-CoA were
conducted under pseudo first-order conditions. Samples were withdrawn from the
reaction mixtures and assayed at regular intervals to monitor the rate of
inactivation (13). In these
experiments, it is assumed that the inactivation reaction is terminated upon
diluting an aliquot of the reaction mixture into the assay that contains
saturating amounts of substrate
(13). In the initial
experiments, the rate of inactivation was extremely rapid with a
t of less than 10 s. Alteration of pH and
temperature of the inactivation mixture did not slow the inactivation
sufficiently. Therefore, further studies were carried out with varying
concentrations of 2-pentynoyl-CoA but in the presence of the non-oxidizable
substrate analog, 3-thiaglutaryl-CoA. Addition of this ligand to the reaction
mixtures decreased the rate of inactivation by competition of the analog with
2-pentynoyl-CoA at the active site.
The pseudo first-order rate constant of inactivation, kapp, was calculated using KaleidaGraph 3.5. The data was further analyzed from a plot of kapp versus the concentration of 2-pentynoyl-CoA in the presence of 3-thiaglutaryl-CoA by the "competitive inhibition" model shown below in Scheme I (14), where E, I, L, E·I, E I, and E·L refer to enzyme, inhibitor (2-pentynoyl-CoA), ligand (3-thiaglutaryl-CoA), a reversible enzyme-inhibitor complex, an irreversible enzyme-inhibitor covalent complex, and a reversible enzyme-ligand complex, respectively.
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Ki and KL are
dissociation constants for inhibitor and ligand, respectively, and
ki is the first-order rate constant for the
inactivation process. The pseudo first-order rate constant of inactivation,
kapp, is related to
ki, Ki, and
KL by Equation
1.
![]() | (Eq. 1) |
These parameters were evaluated by fitting all the data to Equation 1, which is of the same form as that describing competitive inhibition (14), by multiple non-linear regression using GraFit 4.0 (15). Each of the data sets was fitted simultaneously to Equation 1 with ki, KL, and Ki as the shared parameters.
Stopped-flow Kinetics and Data AnalysesThe rapid reaction kinetics of 2-pentynoyl-CoA with the dehydrogenases were monitored with an Applied Photophysics SX.18MV-R stopped-flow reaction analyzer equipped with a 256-element photodiode array detector and xenon lamp. The dead time of the instrument is about 1.0 ms in this configuration using a cell (20 µl) with a 2.0-mm path length. All concentrations cited refer to final concentrations after mixing. Wild type or E370D GCD was reacted with 2-pentynoyl-CoA at 4 or 25 °C in 10 mM phosphate buffer, pH 7.0, 5% ethylene glycol. The reaction was monitored over 500 s at 4 °C and 100 s at 25 °C on a log time scale with spectra accumulated in the 330 to 1050-nm region at a spectral resolution of about 3.3 nm yielding a data matrix of 500 x 198.
The data were analyzed using Pro-K global analysis/simulation software supplied with the instrument. Global optimization of reaction parameters was achieved by using the Marquardt-Levenberg algorithm and fitting to models by numerical integration. The data were analyzed simultaneously at all wavelengths by curve-fitting to an irreversible sequential first-order model, represented in Scheme II, below, where kf and ks are the fast and slow first-order rate constants related to the spectral species, A, B, and C.
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The absorbance at any given wavelength, A, is
then given by a sum of two exponentials, shown below in
Equation 2, where the
kf and ks, being
independent of wavelength, are averaged.
![]() | (Eq. 2) |
Mass SpectrometryThe covalent modification of GCD by 2-pentynoyl-CoA was detected by mass spectrometry. Wild type GCD (16.6 µM) was inactivated with a 5-fold excess of 2-pentynoyl-CoA in 10 mM ammonium acetate, pH 7.5, at room temperature for 5 min. As a control wild type enzyme without inhibitor was processed similarly. The molecular mass of a subunit of native and inactivated wild type GCD was determined by ESI mass spectrometry as described below. The ESI mass spectral experiments were carried out by coupling a protein-trapping column to a Q-Tof2TM mass spectrometer (Micromass Ltd., Manchester, United Kingdom). Mass spectra were acquired with the time-of-flight analyzer at pusher frequency 16129 Hz covering the mass range from 1000 to 2500 atomic mass units and accumulating data for 5 s per cycle. Time to mass calibration was made with CsI cluster ions acquired under the same conditions. The cone voltage was 60 eV. The collision cell was maintained at 12 eV without gas. Protein samples were desalted by loading protein onto a protein-trapping column (15 x 1 mm) hand-packed with a polymeric reverse phase resin (PRP-1; Hamilton Co., Reno, NV) with 1% acetic acid at 150 µl/min flow rate. Elution of proteins was carried out with 90% methanol/0.5% formic acid at a flow rate of 20 µl/min, using a syringe pump (Harvard Apparatus 22). Mass spectrometric data were processed using MassLynx software provided by Micromass Ltd.
Identification of the modified peptide was accomplished by MALDI-TOF mass spectrometry of the trypsin-digested protein. Native and modified wild type GCD with 2-pentynoyl-CoA (as above) were reduced with dithiothreitol and alkylated with iodoacetate in 6 M guanidinium chloride (16). After extensive dialysis against distilled water, the proteins were lyophilized and resuspended in 10 mM NH4HCO3. The proteins were then digested overnight with 2% (w/w) TPCK-trypsin at room temperature (17). The digested samples were analyzed by MALDI-TOF mass spectrometry (Voyager DE-PRO; PerSeptive Biosystems, Framingham, MA) in linear and reflector modes using sinapic acid (10 mg/ml in an 80:20 (v/v) mixture of acetonitrile and 0.1% trifluoroacetic acid, 0.5 ml) as the MALDI matrix. Spectra were externally calibrated to angiotensin I and the ACTH fragments 117, 1839, and 738. The amino acid sequence of human GCD precursor protein (18), which includes the 44-amino acid mitochondrial targeting sequence, can be accessed through the NCBI protein data base under NCBI accession number Q92947 [GenBank] .
Quantitation of Free CoAS
AnionThe release of CoAS anion from
covalently inactivated GCD was quantitated by reaction with DTNB by two
methods. The first method was that described by Freund et al.
(6). In the second method, GCD
was inactivated by addition of 10-fold molar excess of 2-pentynoyl-CoA at 4
°C for 1 h in 300 µl of 10 mM potassium phosphate buffer, pH
7.0, containing 5% ethylene glycol. The enzyme was removed from the reaction
mixture by filtering through Centricon centrifugal membrane filter (YM-30),
and the free CoAS anion in the filtrate was quantitated
using 0.1 mM DTNB as above, again using 412 nm =
14.1 mM1 cm1
(19). The results obtained by
the two methods agreed within ± 10%.
Data Base Searches and Molecular ModelingThe amino acid sequence of the mature GCD (18) was used to search against the P. fluorescens genome in the data base of the United States Department of Energy Joint Genome Institute using TBLASTN (20). The Pseudomonas GCD was located in Contig302 using the following parameters: percent identity, 40% and minimum matching length, 20.
The distances between the nearest carboxylate oxygen of the glutamate catalytic base to the C-4 of the substrate in the crystal structures of MCAD (Protein Data Bank number 3MDE [PDB] ) (21), SCAD (Protein Data Bank number 1JQI [PDB] ) (22), and IVD (Protein Data Bank number 1IVH [PDB] ) (1, 23) were determined using Insight II 97.0 package of modeling software on a Silicon Graphics Indigo 2 work station as described (1, 24). MCAD and SCAD were co-crystallized with ligands octanoyl-CoA and acetoacetyl-CoA, respectively (21, 22), whereas isovaleryl-CoA was modeled into the active site of wild type IVD as described (1, 24).
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RESULTS |
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We also compared the pseudo first-order rate constants for the inactivation
of wild type GCD and E370D GCD (Fig.
2). The reactions were conducted at 4 °C in incubations
containing 135 µM 3-thiaglutaryl-CoA, 20 µM
2-pentynoyl-CoA, and 2 µM dehydrogenase in 10 mM
potassium phosphate, pH 7.0, 5% ethylene glycol. The values of
kapp for inactivation of wild type and E370D GCD
are 1.16 ± 0.07 and 0.26 ± 0.01
min1, respectively. The 4.5-fold lower rate
constant of inactivation of the mutant may be explained by the difference in
distance (1 Å) from the
-carboxylate of Glu-370 to the C-4
proton (or C-2) compared with the
-carboxylate of Asp-370.
Alternatively, the difference in rate constants may also be explained by the
distance from the Michael donor, carboxylate, to the Michael acceptor,
2,3-pentadienoyl-CoA, which is an intermediate formed in the inactivation
process (6). It is also
possible that there is a difference in the orientation of the carboxylate as
it functions as a catalytic base, conjugate acid, or nucleophile in the
Michael addition (25).
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Release of CoAS Anion and Stoichiometry of ModificationAs is the case with MCAD (6), the CoAS anion is liberated upon covalent modification of wild type GCD and E370D GCD by 2-pentynoyl-CoA. The amount of CoAS anion released from the inactivated wild type GCD was 0.98 ± 0.10 per site as determined by reaction with DTNB as described under "Experimental Procedures." The stoichiometry of CoAS anion released from inactivated E370D GCD was 1.12 ± 0.11 per site.
Evidence for covalent modification of the protein was obtained by mass spectrometry of the unmodified wild type and modified enzymes. The average mass of the unmodified subunit determined by ESI mass spectrometry was 43601.2 Da; the average mass of the subunit based on the primary sequence deduced from the DNA coding sequence in the expression vector is 43598.0 Da (18). Dissociation of the tetrameric enzyme to subunits and loss of FAD occurs during the processing of the sample (see "Experimental Procedures"). The average mass of the inactivated subunit determined by ESI mass spectrometry is 43699.1 Da (data not shown), an increase of 97.9 Da, corresponding to C5H6O2, which is consistent with the mass of the expected adduct following release of CoAS anion.
Identification of Glu-370 as the Site of Chemical ModificationTable I shows the observed masses of the tryptic peptides from native GCD along with their amino acid sequence. The numbering of the peptides reported here is for mature human GCD without the 44-amino acid mitochondrial targeting sequence but includes the amino-terminal methionine required for expression from the vector in the case of peptide 125 (see footnotes in Table I). Comparison of tryptic maps of the native and 2-pentynoyl-CoA modified enzyme by MALDI-TOF mass spectrometry showed an additional peptide (m/z = 2775.2) that is 98.6 mass units greater than the peptide (m/z = 2676.6) containing residues 359382 (see Fig. 3 and Table I). The average mass predicted for the latter peptide, [M + H]+, is 2677.0. The peptide 359382 has seven residues that could act as a catalytic base in the enzymatic reaction: His-359, Glu-364, Glu-370, His-373, Asp-374, His-376, and Arg-382. Of these, only Glu-370 is positioned appropriately to function as the catalytic base (26). Further, Glu-370 in human GCD occupies the same position as Glu-376 in MCAD and Glu-368 in SCAD (18, 27). Finally, substitution of Glu-370 by glutamine drastically reduces the catalytic activity of GCD to 0.04% of wild type and substitution by aspartate reduces activity 14-fold (4) and rate constant of inactivation 4.5-fold. Some native peptide was also detected in the analysis of peptides from the inactivated protein although the reaction was run to completion as judged by the complete loss of catalytic activity and absence of significant native monomer as determined by ESI mass spectrometry (Fig. 3B). The covalent derivative of the glutamate in porcine short chain acyl-CoA dehydrogenase is known to be alkali-labile, as are the acyl-CoA dehydrogenases modified by 3-alkynoyl-CoAs (3, 28). Some adduct is likely lost during reductive alkylation and trypsin digestion, which are conducted at pH 8. Incubation of the peptides generated from unmodified and modified GCD at room temperature for 16 h in 0.1 M NH4OH resulted in the complete conversion of the modified peptide to the unmodified peptide but no change in the profile of the other peptides (Fig. 3C). This result is consistent with the contention that Glu-370 is the modified residue and that Glu-370 reacts with the allene intermediate in a Michael addition forming an ester linkage.
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Spectral Changes and Rapid Kinetics of Interaction of GCD with
2-Pentynoyl-CoAFig.
4A shows stopped-flow spectral data in which 460
µM 2-pentynoyl-CoA was mixed with 54 µM wild type
GCD at 4 °C in 10 mM potassium phosphate, pH 7.0, 5% ethylene
glycol. The flavin absorbance at 447 nm decreases, and the long wavelength
absorption (max
791 nm) is immediately observed on
mixing; the latter absorbance decays over a period of about 500 s.
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Singular value decomposition analysis of the spectral kinetic data provided at least three significant singular values. A minimal description of the data indicates a two-step model with three spectral species (A, B, and C). Global analyses of these data according to Scheme II yielded the two pseudo first-order rate constants, kf = 61.98 ± 0.11 min1 and ks = 0.94 ± 0.01 min1. The convergence occurred in five iterations with a variance of 5.126 x 104. Similarly, data obtained at 25 °C, when analyzed as above, yielded the two first-order rate constants, kf = 231.3 ± 1.07 min1 and ks = 4.15 ± 0.01 min1 showing increased values of kf and ks by factors of 3.73 and 4.42, respectively, compared with the data at 4 °C (data at 25 °C not shown).
Rate constants determined by single wavelength analysis (791 nm) are
identical to those obtained by global analysis
(Fig. 4B). The
spectral species, B, with absorbance at 791 nm reaches a maximum of
96% and an apparent steady state in about 2 s
(Fig. 4B). This
apparent steady state persists between 2 and 6 s
(Fig. 4B). The
calculated spectrum of species B
(Fig. 5A) indicates
that it is the spectral intermediate that exhibits the charge transfer band at
long wavelength. Formation of species C occurs only after about 6 s
(Fig. 5B). The value
of 791 nm is 2.1
mM1cm1, which is
comparable with the charge transfer species exhibited by MCAD and SCAD during
inactivation by 2-pentynoyl-CoA
(6,
8).
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The results are consistent with the formation of a delocalized acyl-CoA
anion (max
791 nm) that forms a charge transfer
complex with the electron-deficient flavin
(29). This charge transfer
species may be either the delocalized C-4 anion generated by deprotonation or
the proposed enolate (29) that
forms after attack of the catalytic glutamate on the intermediate allene
(30). Both anions can be
stabilized by hydrogen bonding of the enolate oxygen to the 2'-hydroxyl
of FAD and the backbone NH of Glu-370
(26). The enolate subsequently
decays with the release of CoAS anion from the covalently
modified protein (6,
8). The charge transfer species
that accompanies inactivation in GCD forms much faster than the corresponding
species of MCAD or SCAD (6,
8).
When the mutant dehydrogenase, E370D (37 µM), was mixed with
560 µM 2-pentynoyl-CoA at 4 °C there was no increase in long
wavelength absorbance (Fig. 6).
Nonetheless, the mutant is irreversibly inactivated, and the final spectrum is
similar to that of the wild type dehydrogenase following inactivation.
Singular value decomposition analysis indicated three spectral species. Thus
global analysis, according to Scheme
II, yields two pseudo first-order rate constants,
kf = 14.72 ± 0.05
min1 and ks = 0.19
± 0.01 min1
(Fig. 6A). The
convergence occurred in five iterations with a variance of 4.209 x
104. These rate constants are 4.2- and 3.3-fold
slower than those determined with the wild type GCD and comparable with the
difference in rate constants of inactivation of wild type and E370D GCDs. The
calculated spectra of the individual species yielded max =
446 nm and
446 nm = 14.7 mM1
cm1 for A, and
max =
456 nm for B (
= 14.3 mM1
cm1) and C (
= 12.4
mM1 cm1)
(Fig. 6B). The
concentration of B reaches a maximum of 95% and remains in this
apparent steady state level between 15 and 21 s. Formation of the spectral
species, C, occurs after 21 s and does not reach completion during
the 500-s duration of this experiment. The absence of a long wavelength
intermediate is similar to the observation when MCAD is inactivated with
propiolyl-CoA, phenylpropiolyl-CoA, or 2-octynoyl-pantetheine and points out
the steric requirement for charge transfer complex formation
(6).
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DISCUSSION |
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The mechanism of inactivation of MCAD and SCAD by 2-alkynoyl-CoAs involves initial abstraction of a proton at C-4 followed by isomerization of the delocalized C-4 anion to 2,3-pentadienoyl-CoA and covalent modification of the glutamate catalytic base by a Michael addition (7, 8). It is likely that the pKa of the C-4 protons of 2-pentynoyl-CoA is comparable with that of a C-2 proton of the natural acyl-CoA substrates. Proton transfer from C-2 to the base catalyst of GCD initiates the catalytic pathway of GCD, as is the case with MCAD and SCAD (31). However, this protonated glutamate of GCD is also thought to function as a conjugate acid catalyst to protonate the crotonyl-CoA anion following decarboxylation of the enzyme-bound intermediate, glutaconyl-CoA (8, 9). The proposed mechanism for inactivation of acyl-CoA dehydrogenases by 2-alkynoyl-CoAs is shown below in Scheme III.
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Inactivation involves a Michaelis complex, E·I1, between the enzyme and inhibitor, a delocalized C-4 anion charge transfer complex with the enzyme, E·I2*, and a non-covalent complex between 2,3-pentadienoyl-CoA and the enzyme, E·I3. Attack of Glu-370 on the 2,3-diene yields a covalent complex between a delocalized enolate of the acyl-CoA. This enolate is expected to participate in a second charge transfer complex, E I4*, with the oxidized flavin. E I4* subsequently hydrolyzes to free CoA and the five carbon carboxylic acid covalently bound to the catalytic glutamate through an ester bond, E I5. With the exception of the charge transfer complex that absorbs at 790 nm, which could be either E·I2* or E I4*, multiple intermediates could not be identified in this investigation or in any previous work. However, it is not unreasonable that the rapid formation of the charge transfer complex reflects the formation of the C-4 anion. The decay of this long wavelength-absorbing species reflects the decay of the covalent enolate that involves hydrolysis of the thioester. The bracketed regions in Scheme III indicating kf and ks are the observed first-order rate constants referring to the formation and decay of spectral species B in Scheme II that is detected by stopped-flow experiments. The individual reactions in Scheme III cannot be resolved, because there are few spectral changes associated with the different chemical steps. Also, binding of 2-pentynoyl-CoA is rapid and could not be observed. The first spectral species in the global analysis, A, is the Michaelis complex. The spectral species with the charge transfer band, B, reflects both C-4 anion and the enolate. The bound 2,3-pentadienenoyl-CoA is not expected to form a charge transfer complex, because it is electron-deficient. The scheme proposes two anionic forms forming charge transfer band that are separated by a non-absorbing species at long wavelength. Yet a decrease in absorbance at 790 nm is not detected suggesting that the intermediate allene reacts rapidly with Glu-370, and the two charge transfer complexes cannot be resolved. The decay of the charge transfer spectral intermediate presumably occurs upon substitution of a water molecule for the liberated CoA. The final spectral species, C, is the covalently inactivated enzyme. Therefore, the only rate constants that can be resolved are for the formation of the delocalized C-4 anion (kf = 61.98 min1) and the decay of the enolate because of hydrolysis of the thioester bond and release of CoAS anion (ks = 0.94 min1).
Formation of the apparent C-4 anion is rate-limiting in the inactivation of
MCAD by 2-octynoyl-CoA, because formation of this charge transfer complex and
the rate constant of inactivation both show a primary deuterium isotope effect
of 67 when MCAD is reacted with (4,4-d2)
2-octynoyl-CoA (6). The ratio,
kf/ki, is 2.3 for
this reaction. The corresponding ratio for GCD is 15. Assuming that formation
of the C-4 anion by GCD is also reflected by formation of the charge transfer
species (max
790 nm), formation of the C-4 anion is
not rate-limiting in the inactivation of GCD.
The results presented here are consistent with the modification of Glu-370 in the active site of human GCD. Therefore, the high rate of inactivation of GCD relative to that observed with other acyl-CoA dehydrogenases is not the result of covalent modification of a residue other than the glutamate base catalyst. Also, it is unlikely that the large difference in the rate constant of inactivation results from a difference among acyl group specificities of the three dehydrogenases. The enzymes have different chain length specificities, and GCD exhibits specificity for the carboxylate substituent at C-4 (4, 11). Differences among the active site cavities of MCAD, SCAD, IVD, and GCD have not been extensively compared (32); however, the active sites of both GCD and SCAD are shallower than that of MCAD, and the cavity of GCD is different in that a cationic residue, Arg-94, is present at the bottom of the binding pocket (21, 22, 26, 32). The shallow cavity may influence the capacity of the enzyme to polarize the inhibitor (29). That polarization could influence the pKa of hydrogens at C-4 or render C-3 of the resulting 2,3-dieneoyl-CoA a better acceptor in the Michael reaction with Glu-370 (29). This is also unlikely to be the dominant factor, because the rate constants of inactivation and formation of the long wavelength intermediate of GCD considerably exceed the rate constants found with SCAD (8). Also, ki for inactivation of MCAD by 2-pentynoyl-CoA and 2-octynoyl-CoA are identical (6).
Abeles and co-workers (3)
proposed the catalytic mechanism of GCD in their investigations of P.
fluorescens GCD. Their work indicated that a general base catalyzes a
1,3-prototropic shift, because no significant amount of tritium from solvent
is incorporated into the crotonyl-pantetheine product
(3). The sequence conservation
between human and Pseudomonas GCDs includes residues corresponding to
Glu-370 and Arg-94 of human GCD. In human GCD, Arg-94 is within hydrogen
bonding distance of the -carboxylate of glutaryl-CoA and functions as
an electrostatic catalyst (5,
26). Other conserved residues
in the active site of the bacterial enzyme correspond to Ser-98, Val-99,
Leu-103, Phe-133, Leu-246, and Tyr-369 of the human enzyme
(26). Given the sequence
identity between the bacterial GCD and human GCD (64%) and the level of
identity and conservative replacements (77%), a common reaction mechanism is
almost certain. Therefore, Glu-370 of human GCD is expected to access both C-2
and C-4 of the bound acyl-CoA in the catalytic cycle
(3). This access is not
essential for catalysis by the other dehydrogenases. Thus, there may be
structural differences among MCAD, SCAD, IVD, and GCD. These differences
permit Glu-370 in GCD to reach both C-4 and C-2 of glutaryl-CoA and
crotonyl-CoA more easily to ensure an efficient 1,3-prototropic shift. This is
reflected in the rapid inactivation of GCD by 2-pentynoyl-CoA. The relative
rate constants of inactivation of wild type MCAD and GCD by 2-octynoyl-CoA and
2-pentynoyl-CoA, respectively, can be compared with the rate constants of
inactivation of the corresponding mutants in which the glutamate base
catalysts have been replaced by aspartate residues. The E370D mutant of human
GCD is inactivated about 4-fold more slowly than the wild type, whereas the
E376D mutant of MCAD is inactivated 16-fold more slowly
(33). These data are
consistent with the idea that the carboxylate base of GCD has easier access to
C-4 of the 2-alkynoyl-CoA than the carboxylate base of MCAD.
Schaller et al. (1) showed that IVD is not irreversibly inactivated by 2-pentynoyl-CoA unless the catalytic base is moved from its normal position on the G helix to the loop between the J and K helices. Those investigators proposed that wild type IVD probably failed to react, because the active site base could not reach the proton at C-4 of 2-pentynoyl-CoA (1). GCD represents the other extreme of the reactivity spectrum from IVD. Table II summarizes the rate constants of inactivation, ki, and the spectral rate constants, kf and ks, for the formation and decay of the long wavelength-absorbing intermediates of MCAD, SCAD, IVD, and GCD in the reactions with 2-pentynoyl-CoA, or 2-octynoyl-CoA in the case of MCAD. The distances from the nearest carboxylate oxygen of the catalytic base glutamate to C-4 of the bound ligand were determined from the crystal structures or molecular modeling. In these structures, the ligands are octanoyl-CoA in the case of porcine MCAD and acetoacetyl-CoA in the case of rat SCAD (21, 22). Isovaleryl-CoA was modeled into the structure of IVD. The data indicate an inverse relationship between the distance from the carboxylate oxygen to ligand C-4 and the rate constant of inactivation by a 2-alkynoyl-CoA. We do not have this distance for GCD; however, the 4-fold difference in kapp for inactivation of wild type GCD versus E370D GCD indicates that the same general relationship between distances, ki and kf, holds for GCD. These data suggest that the distance from the carboxylate oxygen of Glu-370 GCD to C-4 is less than that in SCAD, i.e. less than 3.5 Å. This rationale does not consider the most favorable angle of approach to the hydrogen by the oxygen.
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Examples of other enzymes that catalyze proton shifts are triose-phosphate
isomerase and 3-oxo-5-steroid isomerase
(34,
35). Glu-165 in
triose-phosphate isomerase functions as a catalytic base to abstract a C-1
proton from the substrate in the Michaelis complex
(34). The distance between the
carboxylate oxygen of Glu-165 and C-1 and C-2 of the substrate, 3.0 Å,
is shorter than the distance normally associated with C-H ... O
hydrogen bonds and optimal for proton transfer between C-1 and the carboxylate
oxygen (34). The typical
distance for C ... O hydrogen bonds is 3.5 Å, though shorter
distances, between 2.0 and 3.0 Å, are observed in other cases
(36,
37), as in the case of the
Glu-165 carboxylate oxygen and C-1 of dihydroxyacetone phosphate
(34). An oxygen atom
approaching a carbon atom along the C-H bond is not expected to be closer than
3.7 Å unless there is a cohesive interaction
(36,
37). These cohesive
interactions are also strongly dependent on the stereochemistry as expected
for hydrogen bonds (36,
38). Asp-38 of
3-oxo-
5-steroid isomerase catalyzes a 1,3-prototrophic shift
(35). Asp-38 is located 2.8
Å above the 4C
proton
(35). The carboxylate residues
of both isomerases also exhibit considerable conformational flexibility
(34,
35).
The reactivity of GCD with 2-pentynoyl-CoA may reflect a similarly short distance between the carboxylate oxygen of Glu-370 and C-4 of the inhibitor, as well as some conformational mobility of Glu-370. The data presented here provide support for the proposed 1,3-prototropic shift catalyzed by the general base catalyst in GCD and a structural basis for that efficient proton shift. We also suggest that the greatly enhanced rate of inactivation of GCD by 2-pentynoyl-CoA compared with other members of the acyl-CoA dehydrogenase family results from structural differences that reflect the efficiency of the proton shift, a unique aspect of the GCD mechanism among the acyl-CoA dehydrogenases.
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FOOTNOTES |
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* This work was supported in part by the National Institutes of Health Grant
NS39339 (to F. E. F.). The costs of publication of this article were defrayed
in part by the payment of page charges. This 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 Pediatrics, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Box C-233, Denver, CO 80262. Tel.: 303-315-7269; Fax: 303-315-8080; E-mail: Frank.Frerman{at}UCHSC.edu.
1 The abbreviations used are: GCD, glutaryl-coenzyme A dehydrogenase; ACTH,
adrenocorticotropic hormone; DCPIP, 2,6-dichlorophenol indophenol; DTNB,
5,5-dithiobis (2-nitrobenzoic acid); ESI, electrospray ionization; MALDI-TOF,
matrix-assisted laser desorption ionization time-of-flight; MCAD, medium chain
acyl-CoA dehydrogenase; SCAD, short chain acyl-CoA dehydrogenase; IVD,
isovaleryl-CoA dehydrogenase; TPCK, L-1-tosylamido-2-phenylethyl
chloromethyl ketone.
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ACKNOWLEDGMENTS |
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