Mechanism-based Inactivation of Human Glutaryl-CoA Dehydrogenase by 2-Pentynoyl-CoA

RATIONALE FOR ENHANCED REACTIVITY*

K. Sudhindra Rao {ddagger}, Mark Albro {ddagger}, Jerry Vockley § and Frank E. Frerman {ddagger} ¶ ||

From the Departments of {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
2-Pentynoyl-CoA inactivates glutaryl-CoA dehydrogenase at a rate that considerably exceeds the rates of inactivation of short chain and medium chain acyl-CoA dehydrogenases by this inhibitor and related 2-alkynoyl-CoAs. To determine the rate of inactivation by 2-pentynoyl-CoA, we investigated the inactivation in the presence of a non-oxidizable analog, 3-thiaglutaryl-CoA, which competes for the binding site. The enhanced rate of inactivation does not reflect an alteration in specificity for the acyl group, nor does it reflect the covalent modification of a residue other than the active site glutamate. In addition to determining the inactivation of catalytic activity a spectral intermediate was detected by stopped-flow spectrophotometry, and the rate constants of formation and decay of this charge transfer complex ({lambda}max {approx} 790 nm) were determined by global analysis. Although the rate-limiting step in the inactivation of the other acyl-CoA dehydrogenases can involve the abstraction of a proton at C-4, this is not the case with glutaryl-CoA dehydrogenase. Glutaryl-CoA dehydrogenase is also differentiated from other acyl-CoA dehydrogenases in that the catalytic base must access both C-2 and C-4 in the normal catalytic pathway. Access to C-4 is not obligatory for the other dehydrogenases. Analysis of the distance from the closest carboxylate oxygen of the glutamate base catalyst to C-4 of a bound acyl-CoA ligand for medium chain, short chain, and isovaleryl-CoA dehydrogenases suggests that the increased rate of inactivation reflects the carboxylate oxygen to ligand C-4 distance in the binary complexes. This distance for wild type glutaryl-CoA dehydrogenase is not known. Comparison of the rate constants of inactivation and formation of a spectral species between wild type glutaryl-CoA dehydrogenase and a E370D mutant are consistent with the idea that this distance in glutaryl-CoA dehydrogenase contributes to the enhanced rate of inactivation and the 1,3-prototropic shift catalyzed by the enzyme.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Several years ago, Schaller et al. (1) reported that 2-pentynoyl-CoA rapidly inactivates Paracoccus denitrificans glutaryl-CoA dehydrogenase (GCD)1 with t1/2 less than 1 min. We also observed an extremely rapid rate of inactivation of human GCD by 2-pentynoyl-CoA. The oxidative reaction catalyzed by GCD is similar to other acyl-CoA dehydrogenases in that it oxidizes the acyl-CoA to the 2-enoyl-CoA, glutaconyl-CoA (2). However, the catalytic pathway of GCD differs from other members of the acyl-CoA dehydrogenase family, because it also catalyzes the decarboxylation of glutaconyl-CoA to crotonyl-CoA. The latter reaction is formally the substitution of a proton at C-4 for CO2. Glu-370 in human GCD abstracts a proton from C-2, prior to hydride transfer from C-3 to the N-5 of FAD, and then functions as a conjugate acid catalyst, Glu-370H+, to transfer the same proton to C-4 of the transient, delocalized crotonyl-CoA anion following decarboxylation of glutaconyl-CoA (2, 3). Several aspects of the mechanism have been investigated with GCD isolated from Pseudomonas fluorescens (3). Given the sequence identity between this bacterial GCD and human GCD (64%) and the level of identity and conservative replacements (77%), a common reaction mechanism is likely. Unlike the glutamate base in MCAD and SCAD, Glu-370 in human GCD is proposed to access both C-2 and C-4 of the bound acyl-CoA in the normal catalytic pathway (3). Also, mutants of human GCD are available that may also be useful for understanding a possible basis for the extremely rapid rate of inactivation of this dehydrogenase (4, 5).

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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—2-Pentynoic acid was purchased from GFS Chemicals. Glutaryl-CoA, acetoacetyl-CoA, CoASH, DTNB, DCPIP, sinapic acid, angiotensin I, and ACTH fragments 1–17, 18–39, and 7–38 were purchased from Sigma. TPCK-trypsin and iodoacetic acid were purchased from Pierce. All other chemicals were of reagent grade. Human wild type and E370D GCDs were expressed in Escherichia coli, purified, and quantitated spectrophotometrically as described before (4). Human electron transferring flavoprotein was expressed in E. coli and purified as described by Griffin et al. (9).

Enzyme Assays—The 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 {Delta}{epsilon}600 nm = 20.1 mM–1 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-CoAs—2-Pentynoyl-CoA was synthesized from 2-pentynoic acid by the mixed anhydride method (10), purified as described (11), and quantified using {epsilon}260 nm = 20.9 mM–1 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 GCD—The 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 t1/2 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, EI, 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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SCHEME I
 

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 Analyses—The 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.


The absorbance at any given wavelength, A{lambda}, 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)
The amplitudes, AA{lambda}, AB{lambda}, and AC{lambda}, allow the calculation of the spectrum and kinetic profiles of the three spectral species, A, B, and C. Because interchanging the values of the rate constants kf and ks leads to non-meaningful calculated spectra, the assignment of fast and slow phases to either kf or ks becomes unequivocal.

Mass Spectrometry—The 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 1–17, 18–39, and 7–38. 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 Anion—The 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 {epsilon}412 nm = 14.1 mM–1 cm1 (19). The results obtained by the two methods agreed within ± 10%.

Data Base Searches and Molecular Modeling—The 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Kinetics of Inactivation of GCD by 2-Pentynoyl-CoA—The inactivation of human wild type GCD by 2-pentynoyl-CoA was extremely rapid with a t1/2 that was less than 10 s at 4 °C. To characterize the inactivation quantitatively, the apparent rate of inactivation was determined at several different concentrations of a second ligand that binds to the active site. The ligand chosen was 3-thiaglutaryl-CoA, a redox-inactive analog of glutaryl-CoA (3). Fig. 1 shows a plot of kapp versus the concentration of inhibitor in the presence of protection afforded by the substrate analog, 3-thiaglutaryl-CoA. The analysis of the 20 experimental data points when fitted to the competitive inhibition model converged in five iterations with a reduced {chi}2 = 2.6 x 106. The maximum value for the first-order rate constant of inactivation, ki (extrapolated to zero concentration of 3-thiaglutaryl-CoA and infinite inhibitor concentration), is 4.3 ± 0.4 min1. This rate constant is too rapid to be measured in the absence of protection by a competing ligand. The apparent dissociation constants for the binding of 2-pentynoyl-CoA, Ki, and 3-thiaglutaryl-CoA, KL, were 6.9 and 22.8 µM, respectively. This indicates that the inhibitor, 2-pentynoyl-CoA, binds tightly, and the value for 3-thiaglutaryl-CoA agrees with our earlier determinations (4, 5).



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FIG. 1.
Rates of inactivation of human GCD as a function of 2-pentynoyl-CoA in the presence of varying concentrations of 3-thiaglutaryl-CoA. The concentrations of 3-thiaglutaryl-CoA in the inactivation reactions were 90, 135, 200, and 266 µM. The data were analyzed according to the competitive inhibition model using GraFit 4.0. The smooth curves are the best fit to the experimental data yielding the parameters ki = 4.3 min1, Ki = 6.9 µM, and KL = 22.8 µM.

 

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 {gamma}-carboxylate of Glu-370 to the C-4 proton (or C-2) compared with the {beta}-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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FIG. 2.
Comparison of the rates of inhibition of wild type GCD and E370D GCD by 2-pentynoyl-CoA in the presence of 3-thiaglutaryl-CoA. The rates of inactivation of wild type GCD (•) and E370Q GCD ({blacksquare}) were determined in reaction mixtures containing 2 µM enzyme, 20 µM 2-pentynoyl-CoA, and 135 µM 3-thiaglutaryl-CoA at 4 °C in 10 mM potassium phosphate buffer, pH 7.0, containing 5% ethylene glycol. The slopes were used to determine kapp for each enzyme species.

 

Release of CoAS Anion and Stoichiometry of Modification—As 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 Modification—Table 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 1–25 (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 359–382 (see Fig. 3 and Table I). The average mass predicted for the latter peptide, [M + H]+, is 2677.0. The peptide 359–382 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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TABLE I
Tryptic map of native, mature human glutaryl-CoA dehydrogenase in the mass range 2000–4000 Da

 


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FIG. 3.
MALDI-TOF mass spectra of tryptic peptides of native and 2-pentynoyl-CoA inactivated glutaryl-CoA dehydrogenase. The native enzyme (A) and enzyme inactivated with 2-pentynoyl-CoA (B) were reduced and alkylated, digested with trypsin, and analyzed by mass spectrometry. The peptides were then treated with 0.1 M ammonium hydroxide for 16 h at 25 °C, and the peptides were analyzed. Panel C shows the relevant region of the mass spectrum of the modified enzyme treated with base. The native peptide, residues 359–382 with m/z of 2676.63, shows a 98.6-Da increase after modification and loses this mass, 98.6 Da, upon treatment with ammonium hydroxide. Other peptides observed are reported in Table I.

 

Spectral Changes and Rapid Kinetics of Interaction of GCD with 2-Pentynoyl-CoA—Fig. 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 ({lambda}max {approx} 791 nm) is immediately observed on mixing; the latter absorbance decays over a period of about 500 s.



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FIG. 4.
Rapid kinetic analysis of the reaction of 2-pentynoyl-CoA with wild type GCD. In panel A, wild type enzyme (54 µM) and 2-pentynoyl-CoA (460 µM) were mixed at 4 °C in the stopped-flow spectrophotometer with a photodiode array detector. The times of accumulation of the selected spectra are as follows: 1, 0.0032 s; 2, 0.5613s; 3, 4.9950 s; 4, 20.780 s; 5, 140.70 s; and 6, 475.90 s. The reaction was carried out in 10 mM phosphate buffer, pH 7.0, 5% ethylene glycol. Global analyses of the data according to Scheme II, a minimal step mechanism, yielded kf = 61.98 ± 0.11 min1 and ks = 0.94 ± 0.01 min1. In panel B, the data were analyzed at 791 nm as a function of time. The data at this single wavelength were fit to the equation describing an irreversible sequential first-order model, A -> B -> C. The analysis yielded kf = 60.86 ± 0.96 min1 and ks = 0.64 ± 0.01 min1. The inset in panel B shows the same data over the first 6 s to show the formation of the charge transfer species. Note that the absorbance remains at its maximum value during the 2- to 6-s period.

 

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 {epsilon}791 nm is 2.1 mM–1cm1, 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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FIG. 5.
Changes in the concentration of the individual species as a function of time during the reaction of wild type GCD with 2-pentynoyl-CoA. These analyses are based on the experiment shown in Fig. 4A. Panel A shows calculated spectra of the individual species obtained by global analyses. Panel B shows the time dependence of the concentrations of the three spectral species, a, b, and c, defined in Scheme II and corresponding to the spectra in panel A. The inset of panel B shows data over the first 6 s of the reaction.

 

The results are consistent with the formation of a delocalized acyl-CoA anion ({lambda}max {approx} 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 {lambda}max = 446 nm and {epsilon}446 nm = 14.7 mM–1 cm1 for A, and {lambda}max = 456 nm for B ({epsilon} = 14.3 mM–1 cm1) and C ({epsilon} = 12.4 mM–1 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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FIG. 6.
Rapid kinetic analysis of the reaction of 2-pentynoyl-CoA with E370D GCD. In panel A, 37 µM enzyme and 560 µM 2-pentynoyl-CoA were mixed at 4 °C in the stopped-flow spectrophotometer with a photodiode array detector. The times of accumulation of the selected spectra are as follows: 1, 0.0032 s; 2, 0.5613 s; 3, 4.9950 s; 4, 20.780 s; 5, 140.70 s; and 6, 475.90 s. The reaction was carried out in 10 mM phosphate buffer, pH 7.0, 5% ethylene glycol. Global analyses of the data according to Scheme II yielded kf = 14.72 ± 0.05 min1 and ks = 0.19 ± 0.01 min1. Panel B shows calculated spectra of the three species, a, b, and c, from global analyses of data in panel A. The inset of panel B shows changes in the concentration of the individual species as a function of time.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanism-based inhibitors have been useful tools for the investigation of acyl-CoA dehydrogenase catalysis and, in one case, permitted discrimination among otherwise very closely related members of the acyl-CoA dehydrogenase family based on a single structural difference related to function (1). Like P. denitrificans GCD (1), human GCD is rapidly inactivated by 2-pentynoyl-CoA with t1/2 less than 10 s at 4 °C. The rate constant of inactivation greatly exceeds those of MCAD and SCAD by 2-pentynoyl-CoA and 2-octynoyl-CoA at 25 °C (68). Significantly, 2-pentynoyl-CoA does not inactivate IVD (1). The location and function of Glu-370 in GCD are identical to the homologous catalytic glutamate residue located on the loop between the J and K helices in MCAD and SCAD (22). The catalytic glutamate is positioned on the G helix in IVD but has the same function as those in GCD, MCAD, and SCAD (22).

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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SCHEME 3
 

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, EI4*, with the oxidized flavin. EI4* subsequently hydrolyzes to free CoA and the five carbon carboxylic acid covalently bound to the catalytic glutamate through an ester bond, EI5. With the exception of the charge transfer complex that absorbs at 790 nm, which could be either E·I2* or EI4*, 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 6–7 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 ({lambda}max {approx} 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 {gamma}-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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TABLE II
The relation between the carboxylate oxygen to ligand C-4 distance and the rate constants of inactivation (ki) by 2-alkynoyl-CoAs, the formation (kf), and the decay (ks) of the long wavelength absorbing intermediate(s) during the inactivation reaction.

 

Examples of other enzymes that catalyze proton shifts are triose-phosphate isomerase and 3-oxo-{Delta}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-{Delta}5-steroid isomerase catalyzes a 1,3-prototrophic shift (35). Asp-38 is located 2.8 Å above the 4C{beta} 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.


    FOOTNOTES
 
The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number Q92947 [GenBank] .

* 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. Back

|| 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. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Todd D. Williams and Homigol Biesiada of the Mass Spectrometry Laboratory, University of Kansas, Lawrence, Kansas for acquiring ESI mass spectra. We also thank Dr. Kim Fung and Dr. Mark Duncan, Biochemical Mass Spectrometry Facility, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado for acquiring MALDI-TOF mass spectra.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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