From the Department of Pediatrics, § Human
Medical Genetics Program, and
The Department of Pharmaceutical
Sciences, The University of Colorado Health Sciences Center, Denver,
Colorado 80262 and ¶ Department of Biochemistry, The Medical
College of Wisconsin, Milwaukee, Wisconsin 53226
Received for publication, August 22, 2000, and in revised form, October 5, 2000
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ABSTRACT |
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Glutaryl-CoA dehydrogenase catalyzes the
oxidation and decarboxylation of glutaryl-CoA to crotonyl-CoA and
CO2. Inherited defects in the protein cause glutaric
acidemia type I, a fatal neurologic disease. Glutaryl-CoA dehydrogenase
is the only member of the acyl-CoA dehydrogenase family with a cationic
residue, Arg-94, situated in the binding site of the acyl moiety
of the substrate. Crystallographic investigations suggest that Arg-94 is within hydrogen bonding distance of the Human glutaryl-CoA dehydrogenase
(GCD)1 is a homotetrameric
mitochondrial flavoprotein that catalyzes the Glutaric acidemia type I is an inherited neurologic disease resulting
from defects in GCD (1). An Arg-94 In this report, we have investigated the functions of Arg-94 and Ser-98
(Arg-138 and Ser-142 in the complete sequence containing the 44-amino
acid mitochondrial targeting sequence (3)). We determined steady-state
kinetic constants of wild type GCD, Arg-94 Materials--
Ferrocenium hexafluorophosphate
(FcPF6) was obtained from Aldrich. CoASH, glutaryl-CoA,
acetoacetyl-CoA, and hexanoyl-CoA were from Sigma.
[1,5-14C]Glutaric acid was from ICN Biochemicals and
[1,5-14C]glutaryl-CoA was prepared from the anhydride as
described (2). Glutaramic acid (glutaric acid monoamide) was
synthesized as described by Marquez et al. (12) from
glutaric anhydride and NH3. Synthetic glutaramic acid had a
melting point of 93 °C in agreement with the literature value,
93-94 °C (13), and the 1H NMR spectrum was as expected
(12). Glutaramyl-CoA was synthesized by the mixed anhydride method (14)
and purified by high pressure liquid chromatography (7).
3-Thiaglutaryl-CoA was synthesized as described by Dwyer et
al. (7). Porcine medium chain acyl-CoA dehydrogenase was purified
as described previously (15).
Purification of Proteins--
Wild type GCD and the Arg-94 Enzyme Assays--
The GCD proteins were assayed at 25 °C in
10 mM potassium phosphate, pH 7.6, with 200 µM FcPF6 as the electron acceptor;
glutaryl-CoA, hexanoyl-CoA, and glutaramyl-CoA were the varied
substrates. Enzyme activity was routinely determined using
Site-directed Mutagenesis--
Site-directed mutagenesis was
carried out using the QuikChange mutagenesis system (Stratagene)
according to the manufacturer's instructions. The mutagenized plasmids
were sequenced in the region of the mutations to verify introduction of
the mutation. Cassettes containing the mutated site were removed by
digestion with EcoRI and SacI and ligated into
the wild type plasmid that had been digested with the same
endonucleases. The expression vectors were then sequenced across
junctions and through the mutated cassette to demonstrate the mutation
and the absence of unwanted mutation. No unwanted mutations were
detected. DNA was sequenced using the dideoxynucleotide termination
method (19) using the Alf express system (Amersham Pharmacia Biotech).
Ligand Binding Studies--
The binding of 3-thiaglutaryl-CoA
and acetoacetyl-CoA to the glutaryl-CoA dehydrogenases and calculation
of binding constants were carried out as described previously (7). All
spectra were corrected for dilution due to addition of ligands. Binding
of 3-thiaglutaryl-CoA to wild type GCD and mutants was followed
spectrophotometrically at 825 and 450 nm, respectively. Similarly,
binding of acetoacetyl-CoA to Arg-94 Steady-state Kinetic Constants of Wild Type and Mutant GCDs with
Glutaryl-CoA and Alternate Substrates--
In the initial experiments,
we determined the steady-state kinetic constants of wild type GCD and
the three mutant dehydrogenases, Arg-94
kcat of the Arg-94
In contrast with these results, the steady-state kinetic constants of
the enzymes are less affected with hexanoyl-CoA as the substrate. The
45% decrease in Km of the Arg-94
The Km of wild type GCD for glutaramyl-CoA is
similar to the Km for glutaryl-CoA, but
kcat decreases. The structural similarity of the
resulting 2,3-enoyl-CoA amide analog product with the tightly bound
natural intermediate, glutaconyl-CoA, may limit steady-state turnover
due to a slow off-rate. The increased volume of the active site and
decreased strength of hydrogen bonding of the
The predominant effect of both substitutions for Arg-94 in GCD with
glutaryl-CoA as substrate is to reduce kcat to
2-3% of the wild type turnover, although Km for
glutaryl-CoA increases about 10- to 16-fold. The effects of the
mutations of Arg-94 on the steady-state kinetic constants could be
ascribed to a role in binding the
These experiments also suggest a catalytic basis for the pathogenicity
of the Arg-94 Binding and Deprotonation of 3-Thiaglutaryl-CoA and Acetoacetyl-CoA
by Wild Type, Arg-94
Proton abstraction from 3-thiaglutaryl-CoA by a Glu-370
Wild type GCD binds acetoacetyl-CoA with a submicromolar
Kd and abstracts the The three-dimensional structure of human GCD suggested that Arg-94
could play a role in the binding of glutaryl-CoA and glutaconyl-CoA in
the oxidative decarboxylation of glutaryl-CoA catalyzed by the enzyme
(2). When glutaryl-CoA was modeled into the active site of the enzyme,
the A likely function of Arg-94 is to effectively decrease the
pKa of the A second possible function of Arg-94 in GCD may be similar to that of
the conserved arginine residue, Arg-120, in prostaglandin endoperoxidases 1 (PGHS-1) and the homologous Arg-106 in PGHS-2. The
steady-state kinetic constants and structures of the two proteins are
almost identical. The crystal structures of ovine PGHS-1 and human and
murine PGHS-2 have been determined with substrate analogs (non-steroidal anti-inflammatory drugs) bound in the active sites (26-29). The carboxylate of the ligands interacts with the conserved arginine. Site-directed mutagenesis of Arg-120 alters the steady-state kinetic constants of the mutants, and additional kinetic analyses of
human Arg-106 In the case of the Gln and Gly substitutions of Arg-94 in GCD, the
Km values increase only 10- to 16-fold,
respectively, and decreases in
kcat/Km values of the mutant
enzymes are dominated by the 50-fold decreases of
kcat. The decreased kcat
could be due to the positioning of the substrate with respect to
Glu-370, the catalytic base, or with respect to the flavin so that the
efficiency of hydride transfer also decreases. Substitution of
aspartate for Glu-370 in glutaryl-CoA dehydrogenase results in a
similar decrease of kcat as that determined for
the Arg-94 To summarize, Arg-94 does not appear to play a major role in the
binding of glutaryl-CoA and, probably, glutaconyl-CoA. Other residues
in the active site and the presumed hydrogen bonds of the thioester
oxygen with the 2'-hydroxyl of the FAD and peptide amide hydrogen of
Glu-370 (9) apparently play more significant roles. The data can be
interpreted to indicate that Arg-94 electrostatically facilitates
deprotonation of substrate and 3-thiaglutaryl-CoA. Arg-94 may also
function to orient the substrate to facilitate proton abstraction. It
is not clear whether Arg-94 is required for positioning glutaryl-CoA
for hydride transfer to the flavin or stabilization of the transient
crotonyl-CoA anion.
-carboxylate of
glutaryl-CoA. Substitution of Arg-94 by glycine, a disease-causing
mutation, and by glutamine, which is sterically more closely related to arginine, reduced kcat of the mutant
dehydrogenases to 2-3% of kcat of the wild
type enzyme. Km of these mutant dehydrogenases for
glutaryl-CoA increases 10- to 16-fold. The steady-state kinetic constants of alternative substrates, hexanoyl-CoA and
glutaramyl-CoA, which are not decarboxylated, are modestly
affected by the mutations. The latter changes are probably due to
steric and polar effects. The dissociation constants of the
non-oxidizable substrate analogs, 3-thiaglutaryl-CoA and
acetoacetyl-CoA, are not altered by the mutations. However, abstraction
of a
-proton from 3-thiaglutaryl-CoA, to yield a charge transfer
complex with the oxidized flavin, is severely limited. In contrast,
abstraction of the
-proton of acetoacetyl-CoA by Arg-94
Gln mutant dehydrogenase is unaffected, and the resulting
enolate forms a charge transfer complex with the oxidized flavin. These
experiments indicate that Arg-94 does not make a major contribution to
glutaryl-CoA binding. However, the electric field of Arg-94 may
stabilize the dianions resulting from abstraction of the
-proton of
glutaryl-CoA and 3-thiaglutaryl-CoA, both of which contain
-carboxylates. It is also possible that Arg-94 may orient
glutaryl-CoA and 3-thiaglutaryl-CoA for abstraction of an
-proton.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
dehydrogenation and decarboxylation of glutaryl-CoA, an intermediate in the oxidation of lysine and tryptophan (1-3). The products of the reaction are
crotonyl-CoA and CO2 (2). The introduction of the double bond requires abstraction of an
-proton, presumably the
pro-R
-hydrogen, by analogy with other acyl-CoA
dehydrogenases (4, 5), followed by hydride transfer from the
-carbon
to the flavin and decarboxylation of the enzyme-bound intermediate
glutaconyl-CoA (5). Investigations of related bacterial glutaryl-CoA
dehydrogenases indicate that decarboxylation requires electron transfer
from the dehydrogenase to its electron acceptor, electron transfer flavoprotein, or an artificial electron acceptor capable of accepting one electron (5, 6). Recent studies of the human enzyme indicate that
Glu-370 is the catalytic base that abstracts the
-proton (7).
Previous work by Gomes et al. (5) with GCD from
Pseudomonas fluorescens suggests that the
-proton is
subsequently transferred from the conjugate acid of the catalytic base
to the crotonyl-CoA anion following decarboxylation of the
-carboxylate. The decarboxylation reaction catalyzed by GCD is
unique among the acyl-CoA dehydrogenases.
Gly mutation has been identified
as a disease-causing mutation in one patient with glutaric acidemia
type I, suggesting that Arg-94 plays an important role in the reaction
catalyzed by the dehydrogenase (8). Arg-94 is located at the
"bottom" of the substrate-binding site, and molecular modeling
indicates that it is within hydrogen bonding distance of the
-carboxylate of glutaryl-CoA (9). This position is occupied by a
glycine residue in many acyl-CoA dehydrogenases (10) or, in the case of
medium chain acyl-CoA dehydrogenase, by a glutamine residue (9-11).
With the exception of Arg-94 in human glutaryl-CoA dehydrogenase, no
acyl-CoA dehydrogenases contain a cationic residue in the active site
(10). Based on the x-ray structure of the dehydrogenase, it was
proposed that Arg-94 participates in the binding of glutaryl-CoA. It
was also suggested that the delocalized charge of the guanidinium group might also stabilize a transient crotonyl-CoA anion prior to
protonation or might stabilize, developing a negative charge at the
-carbon in the transition state for decarboxylation (9).
Gly, Arg-94
Gln, and
Ser-98
Ala mutants of GCD using glutaryl-CoA and two alternate
substrates, hexanoyl-CoA and glutaramyl-CoA, that are not
decarboxylated. We also investigated the binding of non-oxidizable
glutaryl-CoA analogs, 3-thiaglutaryl-CoA and acetoacetyl-CoA, by wild
type GCD and the mutants. These studies suggest that Arg-94 functions
to effectively decrease the pKa of the
-proton in
the low dielectric in the interior of the protein. Electrostatic
stabilization by Arg-94 would favor formation of
-anions of the
substrate and substrate analog, 3-thiaglutaryl-CoA, both of which
contain
-carboxylates. Arg-94 may also orient the substrate for
efficient proton abstraction at the
-carbon from glutaryl-CoA.
Arg-94 apparently does not play a major role in the binding of the
substrate. Ser-98 also lies at the bottom of the active site and is
conserved in several members of the acyl-CoA dehydrogenase family (long
chain,
-methyl-butyryl-, and short chain acyl-CoA dehydrogenases)
(10). Glu-99 occupies a similar position in the three-dimensional
structure of medium-chain acyl-CoA dehydrogenase and participates in a
hydrogen bonding network with tightly bound water molecules at the base
of the active site in the absence of acyl-CoA ligands (11).
Substitution of Ser-98 by alanine has little effect on the steady-state
kinetic constants of the dehydrogenase. The latter data suggest that
the polarity of the
-hydroxyl group of Ser-98 is not essential for
substrate binding or a rate-limiting step in turnover.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Gly and Ser-98
Ala mutants were expressed in Escherichia
coli and purified as described (7). The absorption spectrum of
Arg-94
Gly GCD
(A269 nm:A370 nm:A447 nm::5.3:0.65:1.00) was very similar to that of the wild type dehydrogenase
(A269 nm:A369 nm:A447 nm::5.0:0.62:1.00). The Arg-94
Gln mutant of GCD was expressed in E. coli
grown at 23 °C. Optimal expression was dependent on co-expression of the chaperones, GroEL/ES, from the plasmid reported by Gatenby (16). Arg-94
Gln was purified as described previously (7), except
that the gradient on DEAE-Sepharose was extended from 150 to 170 mM potassium phosphate, pH 7.0. The ratios of absorption maxima of the purified Arg-94
Gln GCD were
A268 nm:A370 nm:A446 nm::5.3:0.67:1.0. The extinction coefficients of FAD at the visible absorption maxima, 446 or 447 nm, in wild type, Arg-94
Gly, and Arg-94
Gln
dehydrogenases are 14.5 × 103 (7), 14.2 × 103, and 14.8 × 103
M
1 cm
1, respectively. The
latter values were determined after release of the FAD from the
proteins with 0.1% sodium lauryl sulfate (17). The errors in these
determination were ±0.6%. The Ser-98
Ala mutant dehydrogenase was
expressed and purified as the wild type dehydrogenase. The ratios of
absorbance maxima were 268 nm:368 nm:449 nm::5.3:0.59:1.00.
The extinction coefficient at 449 nm of the bound flavin is 16.7 × 103 M
1 cm
1. The
ratios of the maxima in absorption spectra of the mutants compared with
the wild type dehydrogenase indicate that the mutants do not lose
FAD, and the FAD:subunit ratio is unity.
300 nm = 4.3 × 103
M
1 cm
1 for FcPF6
(18). GCD specific activity was also assayed by following the release
of 14CO2 from
[1,5-14C]glutaryl-CoA (2) in the standard reaction
containing 10 mM potassium phosphate, pH 7.6, 25 µM [1,5-14C]glutaryl-CoA and 200 µM FcPF6.
Gln GCD was followed at 322 nm
(7).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Gly, Arg-94
Gln, and
Ser-98
Ala with glutaryl-CoA, hexanoyl-CoA, and glutaramyl-CoA
(Table I). Decarboxylation is obviously
not a step in the steady-state turnover of hexanoyl-CoA and
glutaramyl-CoA. All reactions were assayed in 10 mM
potassium phosphate, pH 7.6, with FcPF6 as the electron
acceptor.
Steady-state kinetic constants of wild type, Arg-94 Gln, Arg-94
Gly, and Ser-98
Ala glutaryl-CoA dehydrogenases with
glutaryl-CoA, hexanoyl-CoA, and glutaramyl-CoA
Gly mutant with
glutaryl-CoA as substrate decreases to about 3% that of the wild type
dehydrogenase, and Km for glutaryl-CoA increases
16-fold. Glutamine was also substituted for Arg-94, because glutamine
is sterically similar to arginine, and the amide group maintains the
polar character at the mutated site, although the cationic
charge is absent. Modeling of glutaryl-CoA into the active site
suggests that Arg-94 is within hydrogen bonding distance of the
-carboxylate of glutaryl-CoA (9). However, the glutamine side chain
is at least 2.3 Å shorter than that of arginine; therefore, the
-carboxylate of glutaryl-CoA and the amide of Gln-94 cannot interact
as strongly as in the wild type dehydrogenase. Substitution of Arg-94
by glutamine increases Km for glutaryl-CoA 10-fold,
and kcat decreases to 2% of
kcat of wild type GCD. However, the kinetic
constants of Ser-98
Ala GCD with glutaryl-CoA as substrate are very
similar to those of wild type GCD (Table I). Ser-98 apparently plays no
major role in the steady-state kinetic pathway of the dehydrogenase.
Gly mutant may
be attributed to the increased volume at the bottom of the active site,
which is likely occupied by water molecules in the mutant protein. The
mutation would eliminate an unfavorable interaction between the charged
guanidinium group of Arg-94 and the alkyl chain of hexanoyl-CoA.
kcat of Arg-94
Gly GCD decreases somewhat
(25%), but the specificity of the mutant enzyme reflected by
kcat/Km is essentially
unchanged when compared with the wild type GCD. By the same criterion,
the specificity of the Arg-94
Gln mutant actually increases.
Moreover, the kinetic constants of this mutant enzyme are very similar
to those of medium-chain acyl-CoA dehydrogenase in which a glutamine
residue occupies the same position as Arg-94 in GCD (11, 20). The
residues comprising the active sites of GCD and medium-chain acyl-CoA
dehydrogenases are reasonably conserved with the exception of Arg-94
and a negatively charged residue, Glu-99, in medium-chain acyl-CoA
dehydrogenase (10, 11, 21). Glutaryl-CoA is not a substrate for
medium-chain acyl-CoA dehydrogenase, perhaps due to the unfavorable
interaction of Glu-99 with the
-carboxylate of glutaryl-CoA. The
Km of Arg-94
Gln GCD for hexanoyl-CoA decreases
further with the elimination of Arg-94, and the cavity in Arg-94
Gly GCD, presumably containing bound water, is occupied by the side
chain of glutamine. kcat of this mutant with
hexanoyl-CoA does decrease relative to that of wild type GCD, perhaps
due to the decreased dissociation of product as in medium chain
acyl-CoA dehydrogenase (22) or positioning of substrate.
-amide may cause the
increase of Km for glutaramyl-CoA, but the increased
volume of the active site of Arg-94
Gly GCD may promote the rate of
dissociation of the 2,3-enoyl-CoA product. The Km
for glutaramyl-CoA of Arg-94
Gln is increased relative to wild type
GCD but is significantly less than the Arg-94
Gly mutant, which has
the large cavity at the bottom of the active site. The substitution of
glutamine at position 94 yields a mutant with a turnover that is
similar to the wild type dehydrogenase, perhaps limiting turnover by
decreasing the rate of dissociation of the glutaconyl-CoA analog. The
specificity of the Arg-94
Gln mutant is essentially identical to
that of the Arg-94
Gly mutant with the
-amide analog substrate
likely reflecting the absence of interaction between the amide and
Arg-94 seen in the wild type dehydrogenase.
-carboxylate, or, perhaps, to
stabilization of the transient crotonyl-CoA carbanion prior to
protonation to yield crotonyl-CoA (9). Stabilization of such a
negatively charged intermediate is not required in the oxidation of
either hexanoyl-CoA or glutaramyl-CoA. The effects of the mutations
can also be evaluated from the resulting changes in
kcat/Km (Table I).
Glutaryl-CoA is, predictably, the best substrate of wild type GCD. When
glycine or glutamine is substituted for Arg-94, the values of
kcat/Km indicate that
hexanoyl-CoA is the best of the three substrates and that the mutant
enzymes are only slightly less efficient with glutaramyl-CoA as
substrate. kcat/Km of the
mutants with glutaryl-CoA as substrate is strongly influenced by the
large decrease of kcat, again suggesting the
possible involvement of the delocalized positive charge of the
guanidinium group in stabilizing the transient crotonyl-CoA anion. The
decrease in kcat does not seem to be of
sufficient magnitude to indicate a role for Arg-94 in the stabilization
of a developing negative charge at the
-carbon in a decarboxylation
transition state. It is important that the Arg-94
Gly and Arg-94
Gln mutants are capable of catalyzing decarboxylation in the
standard radiochemical assay (Table II).
Although not extrapolated to infinite substrate concentration, the
decreased specific activities are of the same magnitude as the
decreases in kcat shown in Table I. Thus, Arg-94 is not absolutely required for decarboxylation of glutaconyl-CoA.
Specific activity of enzymatic decarboxylation of glutaryl-CoA by wild
type, Arg-94 Gly and Arg-94
Gln glutaryl-CoA dehydrogenases
Gly mutation in humans (8). Substitution of lysine
for Arg-94 is also a disease-causing mutation (23); however, this
mutant subunit was unstable when expressed in E. coli, and
the mutation may affect the folding pathway of the mutant subunit. The
stability of this subunit in human cells is not known.
Gly, and Arg-94
Gln GCDs--
The
function of Arg-94 was further investigated by determining the effects
of the Arg-94
Gly and Arg-94
Gln mutations on the binding of
3-thiaglutaryl-CoA, a nonoxidizable analog of glutaryl-CoA (7). The
obvious difference between the wild type and mutants is that binding of
the analog to the mutant proteins yields no detectable charge transfer
complex with a maximum at about 825 nm that is observed in the
titration of the wild type protein (Fig.
1, A-C). The charge transfer
species presumably results from the interaction of the
-carbanion of
3-thiaglutaryl-CoA following abstraction of a
-proton of the analog
with the electron deficient oxidized flavin (24, 25). In the case of
the Arg-94
Gln dehydrogenase a weak charge transfer band
(
max
650 nm) may be present (Fig. 1C).
The analog binds to both mutant proteins as demonstrated by the
perturbation of the flavin spectra of the two mutants proteins,
accompanied by a 7-nm blue shift in the spectra. The
concentration-dependent binding of 3-thiaglutaryl-CoA to
the wild type, Arg-94
Gly, and Arg-94
Gln dehydrogenases is
shown in Fig. 1 (A-C). The Kd values of
complexes of 3-thiaglutaryl-CoA with wild type GCD and the two mutants
are given in Table III. These data
indicate that Arg-94 is not a major determinant for glutaryl-CoA
binding, because its absence has no affect on the dissociation
constants of the complexes with mutant proteins when compared with the
wild type. Rather, both mutations alter the equilibrium for the
abstraction of the
-proton of 3-thiaglutaryl-CoA.
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Fig. 1.
Spectrophotometric titrations of wild type,
Arg-94 Gly, and Arg-94
Gln GCDs with 3-thiaglutaryl-CoA.
The spectra are numbered and indicated in
parentheses with the concentrations of ligand. A,
the wild type GCD, 9.1 µM, was titrated with
3-thiaglutaryl-CoA (in µM) as follows: (1) 0;
(2) 2.0; (3) 5.9; (4) 9.9;
(5) 16.8; (6) 32.7; (7) 44.5;
(8) 56.4. B, Arg-94
Gly GCD, 12.2 µM, was titrated with 3-thiaglutaryl-CoA (in
µM) as follows: (1) 0; (2) 2;
(3) 6; (4) 14; (5) 30; (6)
96. C, Arg-94
Gln GCD, 14.4 µM, was
titrated with 3-thiaglutaryl-CoA (in µM) as follows:
(1) 0; (2) 2; (3) 6; (4)
18; (5) 72; (6) 202. All titrations were
conducted at 25 °C in 125 mM potassium phosphate, pH
7.6. Some spectra are omitted for clarity but all spectral data were
used to calculate the data shown in Table III.
Constants for the binding of 3-thiaglutaryl-CoA with wild type,
Arg-94 Gly and Arg-94
Gln glutaryl-CoA dehydrogenases
Asp mutant
of GCD is also significantly decreased (
97%) (7). Glu-370 is the
catalytic base in GCD that abstracts the
-proton of the substrate.
The distance of the aspartate
-carboxylate in the Glu-370
Asp
GCD from the
-carbon of 3-thiaglutaryl-CoA apparently alters the
equilibrium for proton abstraction. The Glu-370
Asp mutation
results in a 90-95% decrease in kcat,
depending on pH (7), which is similar to the reduction of
kcat of the Arg-94
Gly and Arg-94
Gln
dehydrogenases. Arg-94
Gly and Arg-94
Gln mutations affect
proton abstraction from 3-thiaglutaryl-CoA but do not significantly
alter the Kd values. Arg-94 may facilitate
abstraction of the
-proton by neutralization of the negative charge
of the
-carboxylate, effectively decreasing the
pKa of the
-proton in the low dielectric of the active site. Arg-94 may also function to orient the substrate for
efficient abstraction of the
-proton.
-proton of the
3-ketoacyl-CoA with formation of the enzyme-bound enolate (7). The wild
type dehydrogenase-enolate complex exhibited decreased absorbance at
447 nm due to perturbation of the flavin upon acetoacetyl-CoA binding,
increased absorbance at 322 nm due to the enolate, and increased
absorbance in the 550-nm region due to formation of the charge transfer
complex between the enolate and electron-deficient oxidized flavin (7). Titration of the Arg-94
Gln GCD with acetoacetyl-CoA (Fig.
2) yields results that are very similar
to those obtained with wild type GCD (7). Acetoacetyl-CoA, lacking the
-carboxylate, would not be expected to require Arg-94 to facilitate
deprotonation of an
-proton, for binding, or orientation of the
ligand with respect to Glu-370. The Kd of the
complex is 0.1 µM, and the stoichiometry of binding is
0.87 per mol of enzyme flavin. The values for wild type GCD are 0.4 and
0.90 µM in 50 mM potassium phosphate at the
same pH (7).
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Fig. 2.
Spectrophotometric titration of Arg-94 Gln GCD with acetoacetyl-CoA. Arg-94
Gln GCD, 10.8 µM, was titrated with acetoacetyl-CoA at 25 °C in 125 mM potassium phosphate, pH 6.4. The concentrations
(µM) of acetoacetyl-CoA were as follows: (1)
0; (2) 1.1; (3) 2.2; (4) 3.3;
(5) 4.4; (6) 5.5; (7) 6.6;
(8) 8.1; (9) 11.0; (10) 19.8;
(11) 33.0. Some spectra are omitted for clarity but all
spectral data were used to calculate the binding constants.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxylate is within hydrogen bonding distance of the
guanidinium group of Arg-94 (9). The possibilities were also considered
that Arg-94 could stabilize a transition state involving developing a
negative charge at the
-carbon prior to decarboxylation or stabilize
the intermediate crotonyl-CoA anion (9). The experiments presented here
show that the major effect of substitution of Arg-94 by glycine or
glutamine is on kcat of the mutant enzymes. Both
mutant enzymes have about 2-3% residual activity with glutaryl-CoA as
substrate, but have smaller effects on Km for
glutaryl-CoA. It is reasonable to conclude that Arg-94 does not
participate in stabilization of a decarboxylation transition state,
because the decrease in kcat is at least an order of magnitude less than might be expected for such an effect. When
decarboxylation does not enter steady-state turnover, as with
hexanoyl-CoA and glutaramyl-CoA, substitution at position 94 has
comparatively little effect on the steady-state kinetic constants of
the enzyme. It is of interest that the dehydrogenating activity of GCD
determined with hexanoyl-CoA is 2-fold greater than the turnover of
glutaryl-CoA, although the chemical steps for
,
-dehydrogenation
are essentially identical. We speculate that the greater intrinsic
dehydrogenation activity with the alternate substrate indicates that
decarboxylation is rate-limiting in the turnover of glutaryl-CoA. The
decarboxylation of glutaconyl-CoA and complete turnover involves
oxidation of the dehydrogenase flavin, decarboxylation, protonation of
the crotonyl-CoA anion intermediate, and product dissociation.
-proton of glutaryl-CoA and
3-thiaglutaryl-CoA. The
-anions of glutaryl-CoA and
3-thiaglutaryl-CoA are in close proximity to the
-carboxylates.
Electrostatic stabilization would facilitate the formation of the
-anion in the low dielectric of the enzyme interior. This
hypothesis is supported by kinetic data that show that substitution of
Arg-94 has comparatively little effect on the steady-state kinetic
constants of the mutant enzymes with alternative substrates that do not
contain a
-carboxylate group. In the same vein, the charge transfer
complex between the 3-thiaglutaryl-CoA anion with the dehydrogenase
flavin is not stabilized by the Arg-94
Gly or Arg-94
Gln
mutants. In contrast, the Arg-94
Gln mutant deprotonates
acetoacetyl-CoA and stabilizes the enolate. Absence of the
-carboxylate permits deprotonation of acetoacetyl-CoA in the absence
of Arg-94.
Gln PGHS-2 with alternate fatty acid substrates suggested that Arg-106 might position the substrate for abstraction of
the 13-pro-S-hydrogen (30, 31). Furthermore, the ratio of
products of the Arg-120vGln and Arg-120
Leu mutants of human PGHS-2
is altered so that Arg-106 appears to position arachidonate such that
bis-oxygenation is favored over monooxygenation (31). Similarly, Arg-402 has been proposed to function in the binding and
positioning of arachidonate in human 15-lipoxygenase (32).
Gly and Arg-94
Gln mutants (7). Dwyer et
al. (7) proposed that the decreased kcat of
the Glu-370
Asp is due to the unfavorable equilibrium of the
abstraction of the
-proton based on similar studies with
3-thiaglutaryl-CoA. The binding of the non-oxidizable substrate
analogs, 3-thiaglutaryl-CoA and acetoacetyl-CoA, support the idea that
Arg-94 positions the substrate for efficient deprotonation. The Arg-94
Gly and Arg-94
Gln mutant proteins bind the non-oxidizable substrate analog, 3-thiaglutaryl-CoA, with affinity equal to the wild
type, but abstraction of the
-proton is severely limited. A role in
positioning the substrate for hydride transfer is more difficult to
determine, because the initial step in the reaction pathway of the
Glu-370
Asp mutant is compromised. Also consistent with conclusions
from kinetic data with alternative substrates, the binding and
deprotonation of acetoacetyl-CoA by Arg-94
Gln GCD is essentially
unaffected by the mutation. The evidence for this is based on the
appearance of the charge transfer complex when the enolate is formed on
the enzyme. Finally, preliminary data indicates that oxidized human
glutaryl-CoA dehydrogenase has enoyl-CoA hydratase activity and
hydrates glutaconyl-CoA, the tightly bound intermediate in the
reductive half-reaction of the dehydrogenase
flavin.2 Paracoccus
denitrificans GCD and pig kidney medium-chain acyl-CoA dehydrogenase also exhibit enoyl-CoA hydratase activity with
crotonyl-CoA (6, 33). Hydratase activity of Arg-94
Gln GCD with
glutaconyl-CoA as substrate is reduced 30-fold. These data suggest that
Arg-94 also positions glutaconyl-CoA for hydration in the active site of the dehydrogenase.
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FOOTNOTES |
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* This work was supported by grants from the United States Public Health Service (NS39339 to F. E. F. and GM29076 to J. J. P. K.) and by a grant from the Denver Children's Hospital Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Pediatrics, Box C233, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. Tel.: 303-315-7269; Fax: 303-315-8080; E-mail: frank.frerman@uchsc.edu.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M007672200
2 J. B. Westover and F. E. Frerman, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: GCD, glutaryl-CoA dehydrogenase; PGHS, prostaglandin H synthase; FcPF6, ferrocenium hexafluorophosphate.
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