(Received for publication, March 31, 1995; and in revised form, May 18, 1995)
From the
The role of cysteine 266 in human 3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA) lyase, a residue that is homologous to a cysteine mapped to
the active site of prokaryotic HMG-CoA lyase by protein chemistry
approaches, has been investigated by site-directed mutagenesis. Both
the wild-type human enzyme and a C323S variant, in which a regulatory
sulfhydryl has been eliminated without any negative effect on catalytic
activity (Roberts, J. R., Narasimhan, C., Hruz, P. W., Mitchell, G. A.,
and Miziorko, H. M.(1994) J. Biol. Chem. 269,
17841-17846), were used as models. Mutant enzymes C266A,
C266A/C323S, C266S, and C266S/C323S were overexpressed in Escherichia coli and purified to homogeneity. In all cases,
kinetic characterization indicated that the K
3-Hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) catalyzes the
cleavage of HMG-CoA
Two reactive cysteines have
been identified on the basis of mechanistic and protein chemistry
studies on HMG-CoA lyases. Work with the avian enzyme indirectly
implicated a cysteine in in vitro regulation of the activity
of eukaryotic HMG-CoA lyase by controlling the thiol/disulfide state of
the enzyme(8) . A hyperreactive cysteine was mapped to the
C-terminal region of the avian protein and was found to be capable of
forming an intersubunit cross-link with the corresponding cysteine on
the other subunit of this dimeric enzyme(8) . This cysteine is
conserved in both human and mouse HMG-CoA lyases, but not in the
bacterial enzyme. Interestingly, the activity of the bacterial enzyme
is not highly dependent on thiol-reducing reagents, as are the
eukaryotic counterparts(9) . Mutation of this reactive cysteine
(C323S)
A second reactive cysteine has been mapped to
the active site. Utilizing both bacterial and avian lyases,
Hruz et al.(11) established that the substrate
analog 2-butynoyl-CoA stoichiometrically modifies the enzyme. Protein
chemistry studies on the modified prokaryotic protein established
Cys-237 as the target. Alignment of the available amino acid sequences
for HMG-CoA lyases suggests that the labeled cysteine corresponds to
Cys-266 of the human enzyme.
Recently, a recombinant system for
expression and isolation of human HMG-CoA lyase was
developed(10) . Utilizing this system, site-directed
mutagenesis was employed to replace, in both wild-type and C323S
enzymes, the Cys-266 sulfhydryl group. This report describes our
strategy for evaluating the structural integrity of the isolated mutant
proteins, which is a prerequisite to any meaningful interpretation of
the results of the accompanying kinetic characterization. The results
represent a significant confirmation of the active-site assignment
prompted by the original affinity labeling studies and implicate human
lyase Cys-266 in the chemistry of HMG-CoA cleavage.
The pH/rate profiles for wild-type and C323S lyases
were measured as follows. Enzyme was incubated for 15 min at room
temperature in the presence of 20 mM dithiothreitol and then
placed on ice until needed. After addition of the reduced lyase to the
assay mixture, the reaction was immediately initiated with HMG-CoA,
thus minimizing any instability related to changes in pH. The buffer
employed in the assay mixture was either 100 mM Tris-HCl, 0.10
mM EDTA for pH values between 6.6 and 8.9 or 100 mM glycine, 0.10 mM EDTA for pH values between 8.7 and 10.0.
The V
The pH
dependence of C266A/C323S lyase was measured using a radioactive assay (21) in order to improve sensitivity. The reaction mixture
contained the appropriate buffer (specified above), 10 mM MgCl
Human lyase samples in 10 mM potassium
phosphate, pH 7.8, containing 100 mM NaCl, 20% glycerol, and
1.0 mM dithiothreitol were concentrated to 100-150
µM (calculated on the basis of a 34-kDa subunit) using
Amicon Centriflo membrane cones. Glycerol was removed from the samples
prior to ESR experiments using Sephadex G-50 centrifugal
columns(22) . The ESR samples contained variable concentrations
of Mn
Figure 1:
pH dependence of human HMG-CoA lyase
activity.
The
specific activity of the purified Cys-266 mutants is summarized in Table 2. The serine-containing variants (C266S and C266S/C323S)
exhibited 700-1300-fold diminution in activity. In the case of the
alanine mutants (C266A and C266A/C323S), the diminution in activity in
comparison with wild-type (or C323S) lyase was even more prominent,
with a net effect of >10
Activities of wild-type and
C323S human lyases(10) , avian lyase(24) , and
bacterial lyase (9) are all markedly stimulated by divalent
cations. The activity of each Cys-266 variant was also found to be
dependent on the presence of a divalent cation. Wild-type and C323S
human lyases exhibit K
The K
Figure 2:
Double-reciprocal plot of the initial
velocity of human HMG-CoA lyase as a function of Mn
Since the C323S
variants were not stable for extended periods of time after the removal
of glycerol, the Mn
Figure 3:
Mn
Engineering mutations into HMG-CoA lyase has proven
informative in the context of evaluating regulatory (10) and
catalytic (this report) regions within the enzyme. Such an approach may
also prove useful as we model the point mutations associated with
defects in this protein that produce hydroxymethylglutaric aciduria in
humans. For this reason, attempts were made to develop methods useful
for evaluation of the structural integrity of mutant human HMG-CoA
lyase proteins.
Previous work with both bacterial HMG-CoA lyase (27) and avian HMG-CoA synthase (13) has demonstrated
the spin-labeled substrate analog R
While the constraint that ESR measurements of
Mn
Finally, the marked diminution in catalytic activity that
coincides with replacement of the Cys-266 sulfhydryl in the otherwise
structurally intact HMG-CoA lyase mutants invites comment. The enzyme
catalyzes a typical Claisen cleavage. Hanson and Rose (29) have
proposed that enzymatic reactions of this type involve general acid and
general base catalysts oriented on opposite sides of the substrate. The
pH/rate profile for wild-type human lyase is compatible with the
hypothesis that deprotonation of an amino acid side chain exhibiting a
pK
Alison Carstens expertly expressed and purified the
HMG-CoA lyase enzymes used in these studies. Grant Mitchell (Hopital
Ste. Justine, University of Montreal) provided plasmid pETHL-1, which
contains the human HMG-CoA lyase-encoding cDNA that was used to
construct the expression plasmids used in this study. Liane
Mende-Mueller (Medical College of Wisconsin Protein/Nucleic Acid
Facility) supervised production of the oligonucleotides used for
cassette mutagenesis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
value for HMG-CoA was not substantially different from the
value measured using wild-type human lyase, suggesting that no serious
structural perturbation occurs upon replacing Cys-266. A dissociable
divalent cation (Mn
or Mg
), which
is required for activity in both native and C323S enzymes, is also an
essential component for activity in each of the Cys-266 mutants. The
structural integrity of the human mutants was further indicated by
Mn
binding studies, which demonstrate similarities
not only in the activator cation binding stoichiometries, but also in
the K
values for Mn
as
determined for wild-type and mutant C266A or C266S proteins. Purified
C266A and C266A/C323S mutants both displayed
1.3
10
-fold diminution in specific activity, while the k
value was diminished in both C266S and
C266S/C323S by
9.9
10
-fold. This large
diminution in catalytic efficiency in enzyme variants that display no
substantial structural perturbations is in accord with an active-site
assignment to Cys-266 and qualifies its sulfhydryl group for
consideration as a component of the catalytic apparatus.
(
)to form acetyl-CoA and
acetoacetate. This reaction accounts for the formation of ketone
bodies, an alternate source of fuel during times of fasting or
starvation(1) . In addition, HMG-CoA lyase functions in the
final steps of leucine catabolism(2) . A deficiency in HMG-CoA
lyase can result in the inherited metabolic disease
hydroxymethylglutaric aciduria(3) , which, in some populations,
can represent 16% of hereditary organic acidurias(4) .
Elucidation of the cDNA sequences that encode a number of eukaryotic
enzymes, including the human, chicken, and mouse proteins, has been
reported(5, 6) . The sequence of HMG-CoA lyase from Pseudomonas mevalonii has also been deduced(7) .
Comparison of the deduced peptide sequences reveals that the human
lyase polypeptide exhibits 87% identity to mouse, 82% to chicken, and
52% to P. mevalonii lyases.
(
)in the human enzyme did not impair
catalytic function, but led to a complete loss of the enzyme's
ability to form an intersubunit cross-link and to substantial
diminution in the requirement for thiol reagents to maintain
activity(10) .
Materials
[C]Acetic anhydride was purchased from
DuPont NEN. Q-Sepharose anion-exchange resin and Superose-12
(preparatory grade) resin were products of Pharmacia Biotech Inc.
Phenyl-agarose was purchased from Sigma. Restriction enzymes were a
product of New England Biolabs Inc. Qiaex and Qiagen were purchased
from QIAGEN Inc. (Chatsworth, CA). HMG-CoA was synthesized from the
anhydride, prepared from the free acid (Fluka) according to Goldfarb
and Pitot (12) . All other chemicals were reagent-grade.
Methods
Enzymatic Preparation of
3-[
Radiolabeled
HMG-CoA was enzymatically synthesized (13) from acetoacetyl-CoA
and radiolabeled [1-C]Hydroxy-3-methylglutaryl-CoA
C]acetyl-CoA(14) .
The product (6000 dpm/nmol) was purified by DEAE-Sephadex
chromatography and isolated in a salt-free form using a Sep-Pak
reverse-phase cartridge(8) .
Construction of Mutants Containing Substitutions for
Cysteine 266
Construction of the C266S, C266S/C323S, C266A, and
C266A/C323S mutations was performed as follows. Either the pTrcHL-1 or
pTrcHL-C323S plasmid (10) was digested with NcoI and BamHI, and the resulting 925-base pair fragment containing
the coding region corresponding to the mature matrix form of the
protein was isolated and purified using the Qiaex kit and protocol
(QIAGEN Inc.). pTrc99A was digested with NcoI and BamHI; the resulting 4.1-kilobase vector fragment was purified
as described above. The 925-base pair fragments, containing either the
wild-type coding sequence or C323S, were digested with PstI
and AccI, which have unique restriction sites within the
coding sequence. The double digestion generated three fragments with
sizes of
680, 95, and 150 base pairs. The C266S or C266S/C323S and
the C266A or C266A/C323S mutations were generated with a synthetic
oligonucleotide duplex, spanning the 95-base pair region between the
unique PstI and AccI restriction sites. The synthetic
cassette was generated by annealing six oligonucleotides (three for
each strand). The cysteine codon was altered to either serine
(TCT) or alanine (GCT). The generation of a viable
plasmid encoding the desired mutation at position 266 of the human
HMG-CoA lyase gene was then accomplished by a four-way ligation of the
following: the appropriate synthetic cassette, the isolated 680-base NcoI/PstI fragment, the isolated 150-base AccI/BamHI fragment, and the 4.1-kilobase fragment
isolated from the NcoI/BamHI-digested pTrc99A
plasmid. The resulting expression vectors, pTrcHL-C266S,
pTrcHL-C266S/C323S, pTrcHL-C266A, and pTrcHL-C266A/C323S, were
sequenced in both directions (15) to confirm that only the
appropriate mutations were encoded in the region where the synthetic
cassette replaces the wild-type sequence.
Purification of Human Lyase Mutants
Recombinant
wild-type human lyase, C323S lyase, and the C266A, C266S, C266A/C323S,
and C266S/C323S variants were expressed and purified as described by
Roberts et al.(10) . All mutants purified identically
to the wild-type enzyme. The isolated enzymes were >95% pure.
Protein concentrations were determined by the Bradford method (16) using bovine serum albumin as a standard.
SDS-polyacrylamide gel electrophoresis was run under denaturing
conditions as described by Laemmli (17) utilizing an 11%
acrylamide running gel and a 4.5% acrylamide stacking gel.
Kinetic Characterization of Human HMG-CoA Lyase
Mutants
Enzymatic activity of HMG-CoA lyase and the engineered
variants was determined using the citrate synthase-coupled assay of
Stegink and Coon (18) as modified by Kramer and Miziorko (19) except that 100 µM HMG-CoA was used to
initiate the reaction. When the K value
of Mn
was determined, all components of the assay
mixture, including buffers, were treated with Chelex 100 to remove
trace metals.
value for HMG-CoA lyase was determined at
the specified pH values by linear regression analysis of the
Lineweaver-Burk plots. The pH/rate profile data were fit using a
nonlinear regression analysis algorithm(20) .
, HMG-CoA lyase (8.2 µg of C266A/C323S)
preincubated with 20 mM dithiothreitol, and 100
µM [
C]HMG-CoA in a final volume of
200 µl. The reaction was initiated by addition of the enzymatically
synthesized [
C]HMG-CoA (6000 dpm/nmol) at 30
°C. At various time points, aliquots were removed and acidified
with 6 N HCl. After heating to dryness, the acid-stable
radioactivity attributable to [
C]HMG from
unreacted substrate was determined by liquid scintillation counting.
Depletion of acid-stable radioactivity is a measure of enzymatic
cleavage of substrate to form acid-volatile product (acetoacetate). The
radioactive assay, under standard pH conditions (Tris-HCl, pH 8.2), was
also utilized to confirm the activity of the wild-type, C323S, C266A,
C266S, and C266S/C323S enzymes.
ESR Methodology for Structural Characterization of
HMG-CoA Lyase Mutants
Measurement of Mn binding to human HMG-CoA lyase by ESR was performed as follows.
X-band ESR spectra were recorded using a Varian Century-Line 9-GHz
spectrometer with a TE
cavity at 22 °C and a
modulation amplitude of 10 G, modulation frequency of 100 KHz, and
microwave power of 60 milliwatts. The field sweep was 1000 G, and the
time constant was 0.25 s. A quartz microflat cell was used for all
measurements.
(25-182 µM) with either 100
µM (wild-type) or 68 µM (mutant) HMG-CoA
lyase sites. Mn
bound to HMG-CoA lyase was calculated
by comparing the amplitudes of the sample spectra with the
corresponding amplitudes observed with a solution containing an equal
concentration of Mn
in buffer (10 mM potassium phosphate, 100 mM NaCl, 1.0 mM dithiothreitol, pH 7.8). The data were subjected to Scatchard
analysis; data plots were fit using linear regression analysis.
Rationale for Mutagenesis of Cysteine 266
Hruz et al.(11) were able to identify Cys-237 of the
bacterial enzyme as a residue potentially important for catalysis. The
stoichiometry of the affinity label 2-butynoyl-CoA bound to the
recombinant human enzyme (1.0 per subunit(10) ) paralleled the
results determined for both the bacterial and avian lyases (0.9 and 0.8
per subunit(11) ). These observations are most simply explained
by the existence of an equivalent target in all three enzymes. Sequence
homology suggests that Cys-266 of the human lyase corresponds to
Cys-237 of the bacterial enzyme (Table 1).
Additional
rationale for testing the function of a cysteine within the
substrate-binding site stems from the pH dependence of the HMG-CoA
lyase reaction that has been reported for purified avian liver lyase (19) and for the partially purified bovine liver (18) and bacterial (23) enzymes. In all cases, the pH
optimum is distinctly alkaline, but no pK estimates were offered in those earlier reports nor were
details provided to indicate how closely the activity estimates
approximate V
. The pH dependence of V
for both C323S (Fig. 1A) and
wild-type (data not shown) human HMG-CoA lyases was found to
be identical. The curve shown fit to the data was calculated for
ionization of a single group with a pK
value of 7.99 ± 0.06. Such observations for C323S
lyase eliminate any possibility that the regulatory sulfhydryl group of
Cys-323 is responsible for the observed pH profile. However, it remains
possible that the observed pH dependence reflects the requirement to
form a thiolate anion from a different cysteine located in the active
site.
, measurements in 100 mM Tris-HCl, 0.1
mM EDTA;
, measurements in 100 mM glycine, 0.1
mM EDTA buffer. A, for C323S lyase, reaction rates
were measured by the enzymatic spectrophotometric coupled assay as
described under ``Experimental Procedures.'' The pH/rate
profile was fit using a curve calculated for ionization of a group with
a pK value of 7.99 ± 0.06. B, for C266A/C323S
HMG-CoA lyase, reaction rates were measured by the radioactive assay as
described under ``Experimental Procedures.'' Linear
regression analysis was used to generate the line shown fit to the
data.
The utility of a bacterial expression system for production of
recombinant human HMG-CoA lyase has already been demonstrated in
mutagenesis work that produced an active, oxidation-resistant C323S
lyase variant(10) . To test the hypothesis that Cys-266 plays
an important role in catalysis, the Cys-266 sulfhydryl was eliminated
by synthetic oligonucleotide cassette mutagenesis. Substitutions were
engineered in both pTrcHL-1 and pTrcHL-C323S expression plasmids. The
conservative substitutions correspond to replacement of the cysteine
sulfhydryl with either a serine hydroxyl (C266S, C266S/C323S) or the
C-3 proton of an alanine (C266A, C266A/C323S).
Kinetic Characterization of Human HMG-CoA Lyase
Mutants
Utilizing the purification system developed earlier for
human HMG-CoA lyase(10) , each of the Cys-266 mutant constructs
was expressed and purified. All Cys-266 variants of HMG-CoA lyase were
found in the soluble bacterial extracts at levels similar to those
observed upon expression of both wild-type and C323S enzymes. Each
mutant lyase purified identically to the wild-type enzyme; the elution
profiles for the anion-exchange, hydrophobic interaction, and gel
filtration chromatographic steps for each of the Cys-266 constructs
were identical to those observed with the wild-type enzyme, suggesting
that the overall conformation of each mutant is not drastically
altered. Each variant was purified to homogeneity, as determined by
SDS-polyacrylamide gel electrophoresis (data not shown). The apparent
subunit molecular weight (M = 34,000) was
identical to that observed for both wild-type and C323S enzymes.
-fold. In contrast to the
3-4 orders of magnitude difference in catalytic efficiency
between the serine or alanine mutants and the wild-type enzyme,
substantial differences in K
values for
HMG-CoA were not apparent (Table 2), suggesting that the active
site is not grossly altered when Cys-266 is replaced.
The pH
dependence of the C266A/C323S variant, which exhibits the greatest
diminution in catalytic function, was measured utilizing the
radioactive [C]HMG-CoA depletion assay.
Qualitatively, the data (Fig. 1B) indicate an increase
in rate as pH rises from values of
8 to
10. Despite the
increased sensitivity of this assay, signal-to-noise considerations (at
lower radioactive substrate levels) precluded a full substrate
concentration dependence study at pH <8.2. In such cases, the
observed rate was unchanged when measured at several elevated substrate
levels, suggesting that these rate estimates approach V
. The data for this mutant enzyme could not be
satisfactorily fit to a single ionization curve by the algorithm used
to generate the theoretical fit shown in Fig. 1A for
the catalytically functional C323S lyase. Interpretation is complicated
by the fact that, at low pH, no deviation from the linear relationship
between V and pH could be detected before activity diminished
to levels where signal-to-noise limitations precluded meaningful rate
estimates. At elevated pH, activity increased linearly with pH
increment until further estimates were precluded by enzyme denaturation
due to alkalinity of the assay mixture.
Characterization of the Structural Integrity of HMG-CoA
Lyase Mutants
To test whether the marked diminution in catalytic
activity observed for Cys-266 mutants may be the consequence of a
structure that is seriously perturbed in comparison with that of active
wild-type or C323S HMG-CoA lyase, biophysical characterization of these
mutants was performed. CD spectroscopy (data not shown) did not reveal
significant differences between catalytically active wild-type or C323S
lyase and the Cys-266 mutant proteins, suggesting that secondary
structure is not substantially different in these proteins. However, it
remained possible that more subtle perturbations than can be detected
by this optical spectroscopy approach could account for the observed
diminution in activity. This concern prompted development of a more
refined approach to test this issue.
values for
Mg
that are
100-fold weaker than reported for
the K
values for
Mn
(10) . When the cation concentration
dependence data, measured under conditions of saturating HMG-CoA, are
extrapolated for estimation of V
, equivalent
values are observed using either Mg
or
Mn
, underscoring the efficacy of Mn
as an activator of the human enzyme. The observation that
Mn
, which is spectroscopically active due to its five
unpaired d electrons, binds with such high affinity in comparison with
the spectroscopically silent cation Mg
suggested the
utility of this cation as a structural probe for the enzyme and its
variants. For this reason, the binding of manganese to the enzyme was
investigated by both kinetic and ESR spectroscopic methods.
value previously reported (10) for Mn
(0.34 µM) was
determined under saturating HMG-CoA conditions. To determine the
activator constant (K
) for
Mn
, the activity of HMG-CoA lyase was determined
using variable concentrations of both Mn
and HMG-CoA; Fig. 2shows a double-reciprocal plot of these data. Based on
the intersection point of the lines(25) , an activator
constant, which represents an estimate of an equilibrium dissociation
constant (26) for the binary Mn
-lyase
complex, was calculated for human HMG-CoA lyase (K
= 0.5 µM).
concentration. Concentrations of the substrate, HMG-CoA, were 5
(
), 10 (
), 25 (
), 50 (
), and 100 (
)
µM. The activator constant (K) was calculated
from the intersection point.
The binding of activator
Mn to human HMG-CoA lyase was directly measured by
ESR spectroscopy. Before conducting these measurements, the stability
of HMG-CoA lyase in the absence of glycerol had to be determined. The
viscosity that results from inclusion of a significant concentration of
glycerol in the sample can lead to broadening of the Mn
signal, complicating interpretation of the resulting data. This
constraint seemed potentially serious since bacterial HMG-CoA
lyase loses activity rapidly in the absence of glycerol(9) . To
evaluate the consequences for the human enzyme, glycerol was
rapidly removed from both the wild-type and C323S lyases by centrifugal
gel filtration. Both glycerol-free samples were incubated at 4 °C,
and the activity was periodically measured under standard assay
conditions. The half-life of the wild-type enzyme was determined to be
>12 h, which is sufficient for execution of extensive ESR
experiments prior to any significant decrease in activity.
Unfortunately, the half-life of C323S was <4 h. In this respect,
C323S human lyase was very similar to bacterial HMG-CoA lyase, which
also lacks the C-terminal region cysteine residue.
ESR experiments were performed
with either the wild-type enzyme or the Cys-266 mutants that, like
wild-type lyase, contained Cys-323. The spectrum of free Mn
(90 µM) is shown as the larger amplitude trace in
the inset of Fig. 3. When wild-type HMG-CoA lyase (100
µM sites) was added, the free Mn
was
diminished upon binding of this activator cation by the enzyme, and the
amplitude of the signal was correspondingly decreased (Fig. 3, inset). By comparing the peak-to-peak amplitudes of
corresponding lines of Mn
spectra measured in the
absence and presence of lyase, the amount of bound manganese was
determined. While signal-to-noise considerations limit accurate
measurements to mixtures with total Mn
>25
µM, the data are adequate for Scatchard analysis (Fig. 3), which provides a good estimate of the binding
stoichiometry (n = 0.7). Linear regression analysis of
the data also provides a reasonable slope estimate, used for
calculation of a dissociation constant (K
= 1.5 µM) for the binary E
M complex (Table 2). The K
value determined by these physical measurements is in good
agreement with the kinetically determined activator constant (K
= 0.5 µM). When
similar experiments were performed using C266S and C266A enzymes,
Scatchard analyses indicated K
values of
7.9 and 22.6 µM, respectively (Table 2). While these
values indicate slightly weaker binding than measured with wild-type
lyase, differences are modest in comparison with the observed contrasts
in catalytic efficiency. Significantly, the stoichiometry of activator
Mn
binding, which can be well determined in the
samples due to the high Mn
site occupancy, is
comparable for both the wild-type enzyme and the Cys-266 variants (Table 2), indicating that these mutants possess a full
complement of functional binding sites. Therefore, these data confirm
that the active-site structure of the Cys-266 variants is not grossly
altered by the conservative substitution of serine or alanine.
binding to HMG-CoA
lyase. Enzyme was freed of glycerol as described under
``Experimental Procedures'' and maintained on ice until used
for sample preparation. Approximately 10 min were required for sample
equilibration at 22 °C and subsequent ESR measurement; no
significant loss of enzyme activity occurs in this time period. Inset, spectra of Mn
(90 µM) in
the absence or presence of HMG-CoA lyase (100 µM). Each xaxis division corresponds to 100 G. Instrumental
conditions were as follows: modulation amplitude, 10 G; modulation
frequency, 100 KHz; microwave power, 60 milliwatts; center field, 3000
G; field sweep, 1000 G; scan time, 4 min; and time constant, 0.25 s. Main panel, Scatchard plot of the ESR data for Mn
binding to wild-type human HMG-CoA lyase. By comparing the
amplitudes of the Mn
ESR spectra in the absence and
presence of HMG-CoA lyase, the amount of bound Mn
was
determined. Linear regression analysis was used to calculate the line
shown fit to the data.
CoA
(carboxy-PROXYL-CoA(28) ) to be a useful probe in ESR
characterization of wild-type and mutant forms of these enzymes. This
analog not only proves to be a competitive inhibitor with respect to
acyl-CoA substrate, but also can be shown by physical measurements to
bind to the active site of both enzymes with K
values comparable to the kinetically determined K
estimates. When the utility of this
spin probe was evaluated using human HMG-CoA lyase, inhibition (K
> 500 µM) was observed
at levels of R
CoA that are severalfold higher than used with the
bacterial lyase (K
= 98
µM), and therefore, this analog is unattractive for ESR
spectroscopy studies. Differences between bacterial and human enzymes
with respect to R
CoA binding are not surprising since binding of
2-butynoyl-CoA to the avian enzyme was 5-fold weaker than to the
bacterial enzyme(11) . On the basis of these results, the
dissociable divalent cation activator was considered for use as a
physical probe.
binding required elimination of glycerol from the
enzyme buffer restricted such measurements to wild-type, C266S, and
C266A lyases, the value of Mn
as a structural probe
has been amply demonstrated. The observation that the mutant lyases
possess a full complement of high affinity binding sites for activator
cation represents a stringent criterion of structural integrity that
complements secondary structure comparisons. An additional benefit of
the ESR approach to quantitate cation affinity involves the consequent
refinement of our understanding of the mechanism of cation activation
of HMG-CoA cleavage. Demonstration by the biophysical approach that a
binary Mn
-enzyme complex forms with a K
value comparable to the kinetically
determined K
value argues that lyase
functions as a metal-enzyme complex. This direct measurement allows us
to refine earlier speculation, based on indirect kinetic data generated
using an alternative substrate(24) , by now suggesting that
formation of a binary enzyme-cation species is a prelude to
production of a catalytically active ternary enzyme-substrate-metal
complex.
value of
8.0 coincides with
formation of a catalytically functional enzyme. Is it plausible that
the data reflect ionization of Cys-266 and that this residue functions
as the general base? Replacement of the Cys-266 sulfhydryl with a
higher pK
serine hydroxyl lowers activity
by 3 orders of magnitude. The effect increases to 4 orders of magnitude
in the C266A and C266A/C323S mutants, which is compatible with the
elimination of any nucleophile at this position. The magnitude of these
effects precludes performing rate measurements at pH
7 just as
alkaline lability of the protein precludes measurements at pH
10.
With these constraints, it is unclear whether the increase in rate for
C266A as pH rises reflects titration of an amino acid side chain other
than the Cys-266 sulfhydryl or whether specific base catalysis by
solvent-derived hydroxyl ion accounts for the effect. In the related
lyase reaction catalyzed by Escherichia coli phospho-2-keto-3-deoxyheptulonate aldolase (EC 4.1.2.15;
3-deoxyarabinoheptulosonate-7-phosphate synthase), mutagenic
replacement of the Cys-61 sulfhydryl resulted in diminution of activity
by
10
-fold(30) , an effect comparable in
magnitude to our observation with C266A lyase mutants. The cysteine
residue was viewed as critical to catalysis, but was assigned a role in
metal binding. In the case of rabbit muscle fructose-1,6-bisphosphate
aldolase (EC 4.1.2.13), a more exaggerated effect (10
-fold
diminution in activity) results when the
-amino group of Lys-146
is eliminated(31) . Only a 10
-fold drop in activity
is observed when histidine is substituted for lysine at this position.
Potential roles proposed for this lysine include stabilization of a
carbanionic reaction intermediate, electrostatic interaction with an
acidic active-site residue to enhance that residue's function as
a catalytic base, and a direct general base function for lysine's
-amino group in accepting a proton from the substrate's C-4
hydroxyl group. This latter possibility would appear analogous to the
requirement for a general base to accept a proton from the C-3 hydroxyl
of the substrate in the HMG-CoA lyase reaction. The retention of
measurable activity in the Cys-266 HMG-CoA lyase mutants suggests that
an unambiguous assignment of this residue as a catalytic base cannot
yet be made. As in the case of aldolase, an alternative role in
enhancing the reactivity of another active-site amino acid that
participates more directly in reaction chemistry remains to be
excluded. Nonetheless, the 10
-fold effect observed upon
eliminating the Cys-266 sulfhydryl of HMG-CoA lyase certainly qualifies
Cys-266 as a catalytic residue, validating the active-site assignment
made on the basis of protein chemistry studies(11) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.