The Influence of Conserved Aromatic Residues in 3-Hydroxy-3-methylglutaryl-CoA Synthase* Ila ...

48 Pages·2003·0.18 MB·English

JBC Papers in Press. Published on May 12, 2003 as Manuscript M300244200
The Influence of Conserved Aromatic Residues in
3-Hydroxy-3-methylglutaryl-CoA Synthase*

Ila Misra, Chang-zeng Wang, and
Henry M. Miziorko
Department of Biochemistry
Medical College of Wisconsin, Milwaukee, WI 53226

Running title: Aromatic Residues in HMG-CoA Synthase
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*This work was supported in part by a grant from the National fro
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Institutes of Health (DK21491) ://w
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Address correspondence to: y
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Henry M. Miziorko n
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Biochemistry Department m
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Medical College of Wisconsin , 2
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Milwaukee, WI 53226
Phone: [phone]
Fax: [phone]
Email: [email protected]
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Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
SUMMARY
In order to evaluate the potential contribution of conserved
aromatic residues to the hydrophobic active site of 3-hydroxy-3-
methylglutaryl-CoA synthase, site directed mutagenesis was
employed to produce Y130L, Y163L, F204L, Y225L, Y346L, and Y376L
proteins. Each mutant protein was expressed at levels comparable
to wild-type enzyme and was isolated in highly purified form.
Initial kinetic characterization indicated that F204L exhibits a
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substantial (>300-fold) decrease in catalytic rate (kcat). ow
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Upon modification with the mechanism-based inhibitor, 3- de
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chloropropionyl-CoA, or in formation of a stable binary complex h
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with acetoacetyl-CoA, F204L exhibits binding stoichiometries w
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comparable to wild-type enzyme, suggesting substantial retention .org
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of active site integrity. Y130L and Y376L exhibit inflated u
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values (80-fold and 40-fold, respectively) for the K for acetyl- N
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CoA in the acetyl-CoA hydrolysis partial reaction; these mutants er 2
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also exhibit an order of magnitude decrease in kcat. Formation of 18
the acetyl-S-enzyme reaction intermediate by Y130L, F204L, and
Y376L proceeds slowly in comparison with wild-type enzyme.
However, solvent exchange into the thioester carbonyl oxygen of
these acetyl-S-enzyme intermediates is not slow in comparison
with previous observations for D159A and D203A mutants, which
also exhibit slow acetyl-S-enzyme formation. The magnitude of
the differential isotope shift upon exchange of H 18O into [13C]-
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acetyl-S-enzyme suggests a polarization of the thioester
carbonyl and a reduction in bond order. Such an effect may
substantially contribute to the upfield 13C NMR shift observed
for 13C-acetyl-S-enzyme. The influence on acetyl-S-enzyme
formation, as well as observed k (F204L) and K (Y130L; Y376L)
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effects, implicate these invariant residues as part of the
catalytic site. Substitution of phenylalanine (Y130F, Y376F)
instead of leucine at residues 130 and 376 diminishes the
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effects on catalytic rate and substrate affinity observed for ow
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Y130L and Y376L, underscoring the influence of aromatic side ed
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chains near the active site. http
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3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)1 synthase catalyzes a
committed step in the pathways for isoprenoid, cholesterol, and
ketone body production. The cytosolic isoform, involved in
isoprenoid/cholesterol biosynthesis is transcriptionally
regulated (1) by mechanisms distinct from those which mediate
transcriptional control of the mitochondrial isoform (2) that
supports ketogenesis.

The condensation of substrates acetyl-CoA and acetoacetyl-CoA
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(AcAc-CoA) to form HMG-CoA may be viewed as a three step process lo
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(3) involving formation of an acetyl-S-enzyme intermediate, fro
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condensation to form a transient enzyme-S-HMG-CoA intermediate, ://w
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and hydrolysis to release product HMG-CoA. .jbc
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(1) Acetylation ue
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CoASH t o
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H O O H ov
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Enz-S + H C C S CoA Enz-S C C H be
H H r 21, 2
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(2) Condensation
A H
O H O H H O H O
Enz-S C C H Enz-S C C H + H C C C C S CoA
H H H
B:
(3) Hydrolysis
H O
2
O OH O O OH O
Enz-S C CH2 C CH2 C S CoA OH C CH2 C CH2 C S CoA
CH3 CH3
+
4 Enz S H
Early mechanistic and protein chemistry studies led to selective
modification (4) and mapping (5) of the cysteine that forms the
acetyl-S-enzyme intermediate. Development of a recombinant form
of the avian cytosolic enzyme (6)allowed the demonstration of
the strict requirement for cysteine in formation of this
intermediate. Evaluation of invariant residues implicated as
active site residues not only a histidine (7) but also several
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acidic residues that influence either formation of the acetyl-S- lo
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enzyme intermediate (8) or condensation of this intermediate fro
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with the second substrate (9). ://w
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Recent work on the acetyl-S-enzyme intermediate (9,10) has rg
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suggested that the active site has substantial hydrophobic e
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character. A report on acetoacetyl-CoA binding to acyl-CoA o
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dehydrogenase (11) documented large NMR shifts for the bound r 2
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metabolite that are comparable in magnitude to those observed 8
for HMG-CoA synthase’s acetyl-S-enzyme intermediate (10,12).
Model studies and molecular orbital calculations (11) suggested
that the effect was potentially attributable to stacking of
metabolite with the heterocyclic flavin cofactor of acyl-CoA
dehydrogenase. These observations suggested the potential
influence of hydrophobic, aromatic residues at the active site
of HMG-CoA synthase and prompted evaluation of the importance of
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aromatic residues that are conserved in this protein. This
report documents the characterization of mutant enzymes in which
aromatic side chains have been replaced and indicates that
several conserved aromatic residues influence either the
catalytic rate or enzyme-substrate interactions.

EXPERIMENTAL PROCEDURES

Escherichia coli BL21 (DE3) and the expression vector pET-
3d were purchased from Novagen (Madison, WI).
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Deoxyoligonucleotides were purchased from Operon Technologies lo
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(Alameda, CA). QuickChange site-directed mutagenesis kit was fro
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obtained from Stratagene (LaJolla, CA). Qiagen (Chatsworth, CA) ://w
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plasmid kits were used to isolate plasmid DNA from bacterial .jbc
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cultures. The restriction enzymes and T4 DNA ligase were y
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purchased form New England Biolabs (Beverly, MA) and Amersham n
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Pharmacia Biotech, Inc (Piscataway, NJ). DNA sequencing was m
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performed on an ABI 3100 Genetic Analyzer at the Protein/Nucleic , 2
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Acid Facility of the Medical College of Wisconsin. Ampicillin
and isopropylthiogalactoside were purchased from United States
Biochemical (Cleveland, OH). [1-14C] acetyl-CoA and ethyl[3-14C]-
acetoacetate were products of Moravek Biochemicals (Brea, CA).
3-chloro-[1-14C]-propionic acid was purchased from American
Radiolabeled Chemicals (St. Louis MO). [1,2-13C] Acetic
anhydride, used for the production of [1,2-13C] acetyl-CoA (13),
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was a product of Isotech (Miamisburg, OH). All other reagents
were purchased from Sigma (St. Louis, OH), Aldrich (Milwaukee,
WI), or Pharmacia Biotech, Inc. (Piscataway, NJ).

Methods

Sequence homology analysis. All sequences used in the alignment
analysis are defined in the published databases as HMG-CoA
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synthases. Only sequences encoding full length proteins were o
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included in the analysis. Amino acid sequences were aligned de
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using the Pileup program in the Genetics Computer Group - h
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Wisconsin Sequence Analysis Package (Genetics Computer Group, w
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Inc., Madison, WI). .org
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Synthesis of radiolabeled 3-chloropropionyl-CoA. Synthesis was N
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performed according to the procedures of Miziorko and Behnke er 2
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(4) except that ethylchlorofomate was used to activate the 3- 1
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chloro-[1-14C]-propionic acid to the mixed anhydride and this
intermediate was used to thioesterify CoASH. The product was
precipitated from cold methanol/acetone (1:4). The purified
product was assessed for concentration and purity by UV
spectroscopy and reverse-phase HPLC.

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Synthesis of radiolabeled acetoacetyl-CoA. [3-14C] Acetoacetyl-
CoA was prepared according to the method of Hersh and Jencks
(14), as modified by Miziorko and Lane (3). Ethyl [3-14C]
acetoacetate (3.7 umol; 54 mCi/mmol) was hydrolyzed in 80 mM
LiOH at 22 C for 7 h. After hydrolysis, pH was adjusted to 7.2.
Unlabeled acetoacetyl-CoA (80 umol,Li+ salt), and porcine heart
succinyl-CoA transferase (2 units) were added to the hydrolyzed
product. The exchange was allowed to proceed for 30 min at 30 C.
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The reaction was terminated by cooling of the incubation mixture ow
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on ice and adjustment to pH 2.5 with HCl. The mixture was ed
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extracted with cold ethyl ether to remove free acetoacetate. The http
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aqueous layer was evaporated, dissolved in cold methanol, and 4 w
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volumes of cold acetone were added to precipitate [3-14C] rg
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acetoacetyl-CoA as the Li+ salt. ue
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Construction of mutant HMG-CoA synthases. Mutagenesis was r 2
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performed using Stratagene’s “Quick Change” mutagenesis kit and 8
a pair of complementary mutagenic primers appropriate for
substitution of the aromatic residue encoding codon by a leucine
encoding codon. Sequences of the forward primer for each
mutation with substitutions indicated in italicized characters
are:Y130L,5’-GAC ACA ACC AAT GCG TGC TTA GGA GGC ACT GCT GCT-3’;
Y130F, 5’-GAC ACA ACC AAT GCG TGC TTT GGA GGC ACT GCT GCT-3’;
Y163L,5’-T GGA GAC ATT GCT GTG TTG GCC ACT GGA AAT GCC A-3’;
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F204L, 5’-G CAT GCT TAT GAC TTG TAT AAA CCA GAT ATG G -3’;
Y225L, 5’- CTG TCT ATA CAG TGC TTG CTC AGT GCA TTA GAC C-3’;
Y346L, 5’-CAG AAT GGA AAC ATG TTG ACG CCT TCA GTC TAC GGT-3’;
Y376L, 5’-GA ATC AGT GTG TTC TCA TTG GGC TCT GGT TTT GCT G-3’;
Y376F, 5’-GA ATC AGT GTG TTC TCA TTT GGC TCT GGT TTT GCT G-3’.
The plasmids containing various mutants were used to transform
competent XL1-Blue cells. Mutagenic plasmid DNA was isolated
from selected transformants and analyzed by restriction mapping
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and DNA sequencing. The verified mutant clones were transformed ow
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into competent BL21(DE3) cells for expression and isolation as ed
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previously described for wild type HMG-CoA synthase (6). http
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Isolation of HMG-CoA synthase. The procedure developed for rg
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purification of the wild-type enzyme (6) was followed for e
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isolation of the aromatic substituted enzymes from IPTG induced o
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bacterial cultures. Protein content of the purified enzymes was r 2
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estimated by the Bradford assay (15), using bovine serum albumin 8
as the standard. The purity of the enzymes was assessed by SDS-
polyacrylamide gel electrophoresis.

Activity assay and enzymological characterization of HMG-CoA
synthase mutants. Either the standard spectrophotometric assay
(6, 16) or the more sensitive radioisotopic assay (16) was used
to measure activity. In the spectrophotometric assay, the
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reaction mix included 100 mM Tris-Cl, pH 8.2, 100 uM EDTA,
appropriate amounts of HMG-CoA synthase (approximately 6 ug for
wild-type enzyme), 20 uM acetoacetyl-CoA and 200 uM acetyl-CoA
(unless varied concentrations are used for K determinations).
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The reaction was performed at 30 C and acetyl-CoA dependent loss
of acetoacetyl-CoA was measured as a decrease in 300 nm
absorbance, using a millimolar extinction coefficient of 3.6.
For improved sensitivity, the spectrophotometric assay can be
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performed in the presence of 40 mM MgCl2; in such cases, the own
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millimolar extinction coefficient of 20.0 is used for ed
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acetoacetyl-CoA. http
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For the radioisotopic assay, the reaction mixture included 100 rg
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mM Tris-HCl, pH 8.2, 100 uM EDTA, 20 uM acetoacetyl-CoA, 200 uM e
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[14C]-acetyl-CoA,8,600-10,000 dpm/nmol(unless reagent o
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concentration is varied for Km determinations), and appropriate r 21
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amounts of wild-type or mutant HMG-CoA synthase. The reaction 8
was initiated by addition of radiolabeled acetyl-CoA to the
assay mixture containing the rest of the components at 30 C. At
specified time intervals, 40 ul aliquots were removed from the
incubation mixture and acidified with 6 N HCl. The mixture was
heated to dryness and acid-stable radioactivity due to [14C]-HMG-
CoA was measured by liquid scintillation counting.

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