From the Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029-6574
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ABSTRACT |
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Endopeptidase EC 3.4.24.15 (EP24.15) is a
zinc metalloendopeptidase that is broadly distributed within the
brain, pituitary, and gonads. Its substrate specificity includes a
number of physiologically important neuropeptides such as neurotensin,
bradykinin, and gonadotropin-releasing hormone, the principal
regulatory peptide for reproduction. In studying the structure and
function of EP24.15, we have employed in vitro mutagenesis
and subsequent protein expression to genetically dissect the enzyme and
allow us to glean insight into the mechanism of substrate binding and
catalysis. Comparison of the sequence of EP24.15 with bacterial
homologues previously solved by x-ray crystallography and used as
models for mammalian metalloendopeptidases, indicates conserved
residues. The active site of EP24.15 exhibits an HEXXH
motif, a common feature of zinc metalloenzymes. Mutations have
confirmed the importance, for binding and catalysis, of the residues
(His473, Glu474, and His477) within
this motif. A third putative metal ligand, presumed to coordinate
directly to the active site zinc ion in concert with His473
and His477, has been identified as Glu502.
Conservative alterations to these residues drastically reduces enzymatic activity against both a putative physiological substrate and
a synthetic quenched fluorescent substrate as well as binding of the
specific active site-directed inhibitor,
N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, the binding of which we have shown to be dependent upon the presence, and possibly coordination, of the active site zinc ion. These studies
contribute to a more complete understanding of the catalytic mechanism
of EP24.15 and will aid in rational design of inhibitors and
pharmacological agents for this class of enzymes.
Endopeptidase EC 3.4.24.15
(EP24.15)1 belongs to the
family of zinc metalloendopeptidases that includes among its members ACE (angiotensin-converting enzyme), EP24.11 (neutral endopeptidase), EP24.16 (neurolysin), and bacterial thermolysin (1-3). Also known as
thimet oligopeptidase (4), EP24.15 is a predominantly soluble, 77-kDa,
thiol-sensitive enzyme that preferentially cleaves peptide bonds on the
carboxyl side of hydrophobic amino acid residues. A hydrophobic or
bulky residue in the P2 and P3' positions
relative to the scissile P1-P1' bond
(nomenclature of Schecter and Berger (5)) further contributes to
substrate binding and catalytic efficiency (6, 7).
Most EP24.15 activity is known to be located in brain, pituitary, and
gonads (8). Within the central nervous system, EP24.15 exhibits a
widespread distribution with high levels observed in areas rich in
neuropeptide content such as anterior pituitary, cerebellum,
hippocampus, cortex, and hypothalamus (8, 9), thus suggesting a
potential role for EP24.15 in the metabolism of bioactive peptides. Of
particular interest to this laboratory is the decapeptide substrate
GnRH (gonadotropin-releasing hormone), the master regulatory peptide
for reproduction. Several studies have previously documented the
ability of EP24.15 to cleave GnRH at the
Tyr5-Gly6 bond in vitro (consistent
with its aforementioned substrate specificity) (6, 7, 10, 11). By using
the competitive, specific active site-directed inhibitor, cFP-AAF-pAB
(N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate) (12), workers (10, 13-15) have further demonstrated the potential importance of EP24.15 in the post-secretory regulation of GnRH signaling events in vivo. Other physiologically active
peptides such as neurotensin and bradykinin are also cleaved at sites
consistent with the substrate specificity of EP24.15 (6-8). This
enzyme has also been shown to generate Leu- and Met-enkephalin from
larger proenkephalin- and prodynorphin-derived precursors such as
dynorphin A1-8, The three-dimensional structure of EP24.15, or indeed any other
mammalian metalloendopeptidase of this class, is currently unknown.
Conversely, the structures of three related bacterial zinc
metalloendopeptidases, thermolysin (22), bacterial elastase (23), and
neutral protease (24), have previously been solved by x-ray
crystallographic analysis to a resolution of greater than 2 Å. The
mechanism of catalysis in these metalloenzymes centers upon an
active site zinc ion coordinated to three amino acid side chains and a
water molecule (1, 25-28). A zinc-binding nucleus is typically
provided by two ligands separated by a short spacer of 1-3 amino
acids. A third ligand, separated from the second by 20-120 amino
acids, completes the coordination sphere with a long polypeptide loop
that further aligns protein residues with the active site zinc. A
fourth residue, located between the first two ligands, subsequently
forms hydrogen bonds with a water molecule, thereby enabling the oxygen
to coordinate directly to the zinc ion. The hydrolytic efficiency of
zinc metalloenzymes is critically dependent on the nucleophilicity of
this activated water molecule, the lone electron pair from
oxygen serving to attack an approaching substrate carbonyl carbon.
Sequence comparison reveals that a number of key amino acid residues
known to be involved in this catalytic mechanism in the aforementioned
bacterial enzymes are conserved within a similar sequential context
within the carboxyl half of EP24.15 and other mammalian homologues
(Fig. 1). These include an HEXXH motif (the histidines
representing the first and second ligands, respectively, and the
glutamate providing the activated water molecule) in
addition to a glutamate residue 20 amino acids carboxyl to this motif
(possibly representing the third zinc
ligand).2 A similar ligand
configuration is also evident in the primary structures of other
mammalian zinc metalloenzymes such as EP24.11 (29, 30), EP24.16
(neurolysin) (3), and ACE (31). To assess further the functional
importance of these residues in EP24.15, we have employed site-directed
mutagenesis of the cDNA encoding rat EP24.15 (32) to prepare
mutants in which residues comprising the HEXXH motif
(His473, Glu474, and His477) and
residues postulated to function as the third zinc ligand (Glu497, Glu502, and Glu537) have
been genetically substituted. Alterations in the kinetic parameters of
EP24.15 mutants compared with wild type were quantitated using both a
physiologic substrate, GnRH1-9 (11, 33), and a model
quenched fluorescent substrate, QFS
(7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-D-Lys-(2,4-dinitrophenyl)). Furthermore, the competitive EP24.15-specific active site-directed inhibitor, cFP-AAY-pAB (12), was employed as a probe to assess the
three-dimensional integrity of the catalytic site in both wild type and
mutant enzymes. These studies have been conducted in parallel with the
ongoing structural determination of EP24.15 and are crucial in
assessing its catalytic mechanism and role with respect to bioactive
neuropeptide substrates. These are among the first such studies to
address the structural organization of this enzyme and to indicate the
important differences that exist within this class of peptidases.
Materials--
Bradford protein assay reagent and Bio-Gel HT
hydroxyapatite were purchased from Bio-Rad; BSA was obtained from
Pierce; QFS was obtained from Auspep (Victoria, Australia); Mca-Pro-Leu
standard was from Calbiochem; DTT (dithiothreitol) was purchased from
Roche Molecular Biochemicals; Chameleon mutagenesis kit was purchased from Stratagene (La Jolla, CA); all reaction buffers, restriction and
modification enzymes, were purchased from New England Biolabs (Beverly,
MA); T7 Sequenase was purchased from Amersham Pharmacia Biotech;
QIAprep spin plasmid preparation kit was from Qiagen (Chatsworth, CA);
unless otherwise indicated, all other chemicals were purchased from Sigma.
Sequence Alignments--
Utilizing the computer
algorithms, CLUSTAL (34) and BESTFIT (35), the protein sequences of
EP24.15 and the bacterial homologues, thermolysin, bacterial elastase,
and neutral protease, were compared as described
previously2 to obtain homology between functional domains
and key conserved catalytic residues.
Mutagenesis and Protein Expression--
Double-stranded
site-directed mutagenesis of rat EP24.15 was performed on the EP24.15
expression vector pGEX-24.15, modified for rapid screening of mutations
by addition of a unique restriction endonuclease site
(EcoRI) that replaces the vectors sole ApaI restriction site (36). Oligonucleotide primers were synthesized with
mismatches coding for the appropriate amino acid change
(Table I) but following prokaryotic codon
usage rules to obviate the use of rare codons that may hinder
subsequent protein expression. Mutant and selection primers,
phosphorylated at the 5'-end, were annealed to the double-stranded
expression vector before being extended and ligated in a single
reaction. The resulting plasmid DNA was selected for the
ApaI to EcoRI mutation, transformed into repair-deficient XLMutS bacterial cells, and selected for
ampicillin resistance. Plasmid DNA was subsequently purified (QIAprep
spin plasmid kit) and subjected to a further round of selection for the
ApaI to EcoRI mutation before being transformed
into XL1-Blue bacterial cells and screened for ampicillin resistance.
Purified plasmid DNA (37) was cleaved with EcoRI to screen
for desired mutations. Putative positives were confirmed by
double-stranded template dideoxy sequencing (38). Expression and
purification of the mutant proteins for biochemical characterization
were as described previously (36), with all enzymes stored at
Native Gel Electrophoresis--
Assessment of purification to
homogeneity, yield, and proper folding of expressed proteins was by
native polyacrylamide gel electrophoresis (PAGE) on an 8% gel under
reducing conditions as described previously (39). Yields of expressed
protein were similar for all of the mutations.
Circular Dichroism (CD)--
To determine if gross structural
alterations occurred during mutagenesis and subsequent protein
expression, selected mutants displaying a substantial decrease in
catalytic and inhibitor binding capacity were examined by CD
spectroscopy. CD spectra were collected in the wavelength range of 300 to 185 nm at 0.2-nm intervals with an AVIV 60DS spectropolarimeter
(AVIV Instruments, Lakewood, NJ). The instrument wavelength was checked
with benzene vapor. Optical rotation was calibrated by measuring the
ellipticity of D10-camphorsulfonic acid at
192.5 and 290 nm. Measurements of optical ellipticity were made at
25 °C using a 0.1-cm path length quartz cell. At least eight
reproducible scans were collected for each sample. Buffer alone was
used for a control blank in these experiments. Secondary structure
estimation of the proteins was performed using data in the wavelength
range of 240 to 190 nm by an unconstrained linear least squares fitting
procedure (40) and using a reference data base from 15 water soluble
proteins of known secondary structure, including thermolysin (41).
GnRH1-9 Assay--
Activity of EP24.15 wild type
and mutants was determined in duplicate in a high pressure liquid
chromatography-based single point assay using as substrate the
deamidated GnRH metabolite, GnRH1-9, proposed to be a
physiologic substrate for EP24.15 (11, 33) (cleaved to yield
GnRH1-5 and GnRH6-9). Substrate and product
peaks were previously identified by coelution with known standards.
Briefly, 2-5 ng of recombinant EP24.15 was incubated at 37 °C with
a GnRH1-9 concentration range of 10-100 µM
(begins above the average Km of 5.1 µM
observed for this substrate with EP24.15 wild type) in reaction buffer (25 mM Tris/HCl, 125 mM NaCl, 0.3 mM DTT, pH 7.5) in a final volume of 150 µl. Reactions
were terminated after 15 min with the addition of 150 µl of methanol
containing 1% trifluoroacetic acid. GnRH1-5 product was
measured using a Waters high pressure liquid chromatography system
(Milford, CT) with absorbance detection at 214 nm. Solvents were
filtered and degassed before use (solvent A, 0.08% trifluoroacetic acid; solvent B, 0.08% trifluoroacetic acid + 70% acetonitrile). Samples were eluted at 1 ml/min from a Delta-Pak C-18 reversed-phase column (Waters) by a linear gradient from 3 to 85% solvent B over 15 min with flow being maintained at 85% solvent B for a further 2 min.
Enzyme activities were determined from a GnRH1-5 standard
curve prepared under identical assay conditions. Suitable negative
controls were included in each assay. Total substrate hydrolysis was
less than 10%. Kinetic parameters (Km, Vmax, kcat, and
kcat/Km) were subsequently
evaluated using the double-reciprocal plot method of Lineweaver and
Burk (42).
QFS Assay--
Activity of EP24.15 wild type and mutants was
determined in duplicate by continuous assay using the quenched
fluorescent substrate, QFS. Briefly, 50-75 ng of recombinant EP24.15
were incubated at 37 °C with 1.1 µM QFS in reaction
buffer (25 mM Tris/HCl, 125 mM NaCl, 0.3 mM DTT, pH 7.5) in a final volume of 1.27 ml. By employing
a starting substrate concentration substantially below the estimated
Km for QFS, substrate hydrolysis obeys a first-order
process and the Michaelis-Menten equation subsequently integrates to
give the expression ln[S] = Active Site-directed Inhibitor Binding Assay--
Binding of the
competitive, specific active site-directed inhibitor, cFP-AAY-pAB (12),
was quantitatively determined in triplicate for all mutant and wild
type enzymes using a modification of the method of Shrimpton et
al. (44). Briefly, 2 µg of enzyme was incubated for 30 min at
37 °C with 10 nM cFP-AAF-pAB containing 20,000 cpm of
cFP-AA-125I-Y-pAB in a final buffer volume of 200 µl (0.3 mM DTT, 5 mM Sørensen's buffer, pH 7.2).
Iodinated inhibitor was prepared via the IODO-GEN method (45).
Following incubation, an equal volume of hydroxyapatite resin, prepared
and equilibrated as described previously (44), was added to the
reaction mixture and then placed immediately on ice for 30-45 min with
frequent mixing to facilitate maximal protein binding to the resin.
Each sample was subsequently centrifuged at 6500 × g
for 5 min, the supernatant decanted, and the pellet washed in 350 µl
of the above buffer. This process was repeated a further two times.
Total radioactive counts were determined in both the pellet and
supernatants (3 supernatants) collected for each sample using a Packard
Multi-Prias 4 gamma counter (Meriden, CT). Percentage inhibitor binding
was calculated as
(cpmpellet/cpmpellet+supernatants) × 100. Nonspecific inhibitor binding was estimated by replacing EP24.15 in the
binding assay with a molar equivalent of BSA (bovine serum albumin) and
was found to be less than 2%.
Protein Determination--
Protein was quantitated according to
the method of Bradford (46) with BSA as standard.
Sequence Alignment with Bacterial Homologues--
Computer-aided
homology searching between functional domains of EP24.15 with those of
thermolysin, bacterial elastase, and neutral protease, bacterial
homologues previously solved by x-ray crystallography, reveals
conserved structural and catalytic elements. These include an
HEXXH motif conserved within an active site Site-directed Mutagenesis and EP24.15 Expression--
Following
expression and affinity purification, wild type and all mutants were
subjected to native PAGE under reducing conditions (Fig.
2, panel A). Results indicate
purification to homogeneity and identical native molecular mass (77 kDa) for all enzymes studied. This latter result, coupled with the
equivalently high expression yields observed for all proteins examined,
would argue against errant protein folding and stability during
expression. EP24.15 mutants displaying a substantial ablation in
catalytic and inhibitor binding capacity (i.e. substitutions
to His473, Glu474, His477, and
Glu502) were further subjected to analysis by circular
dichroism spectroscopy, a technique that is sensitive to detecting net
changes (~2%) in secondary structure (47). No significant alteration
in the CD spectra of any of these mutants relative to that of EP24.15
wild type was observed (Fig. 2, panel B).
Kinetic Analyses of EP24.15 Wild Type and Mutants--
The effects
of individual mutations on EP24.15 catalytic activity were assessed
using the substrates GnRH1-9 and QFS (Table II). Complete ablation of EP24.15
activity was observed with both substrates when different substitutions
were made for His473, Glu474, and
His477. In addition, of the three glutamates
(Glu497, Glu502, and Glu537)
postulated to function as the third zinc ligand, only
Glu502 demonstrated a complete ablation of activity when
substituted with either aspartate or glutamine. By contrast, similar
substitutions for Glu497, only five residues away,
substantially increased the catalytic efficiency of the enzyme relative
to wild type for QFS but not GnRH1-9. Substitution of
Glu537 for a glutamine residue demonstrated some activity
losses with both substrates.
Effect of Metal-chelating Agents on EP24.15 Wild Type Binding to
cFP-AAY-pAB--
The highly potent, specific active site-directed
inhibitor, cFP-AAY-pAB, was employed in this study to probe the
three-dimensional integrity of the catalytic site in EP24.15 wild type
and mutants. Orlowski and co-workers (12) have previously shown that
the iodinated AAY inhibitor displays very similar inhibition kinetics (Ki = 24 nm) with the non-iodinated AAF and AAY
species (Ki = 27 and 16 nm, respectively). These
researchers have also demonstrated that the carboxyl group of the
N-(1-carboxy-3-phenylpropyl) moiety of this inhibitor
specifically coordinates with the active site zinc of EP24.15 (12).
Based on this information, we specifically addressed the requirement of
an active site zinc in EP24.15 for inhibitor binding. Assays were
subsequently performed in which EP24.15 wild type was preincubated for
30 min with various metal-chelating agents to remove active site zinc.
Results indicate a drastic, dose-dependent decrease of
inhibitor binding following enzyme pretreatment with 1 and 2 mM 1,10-phenanthroline. Pretreatment with 10 mM
EDTA also reduced inhibitor binding by over 85% (Fig. 3). These effects could be reversed
following reincubation of EP24.15 with 1 mM
ZnCl2. These results confirm the dependence of inhibitor
binding on the presence of an active site zinc as previously suggested
(12). Consequently, this binding assay serves as a useful tool for
assessing the effects of specific point mutations postulated to alter
the ability of EP24.15 to coordinate properly the catalytically
essential active site zinc.
Comparison of EP24.15 Wild Type and Mutant Binding to
cFP-AAY-pAB--
The importance of both the HEXXH motif and
the third zinc-coordinating ligand to inhibitor binding is clearly
evident (Table III). Substitution of
His473 and His477 for either alanine or
glutamine completely eliminates inhibitor binding. Similarly,
replacement of Glu474 with either aspartate or glutamine
reduces inhibitor binding by 100 and 80%, respectively. A complete
loss of binding was also noted when Glu502 was substituted
for either aspartate or glutamine, whereas substitution of
Glu537 (equivalent to Glu646 in EP24.11) for a
glutamine residue had virtually no effect, despite reduced activity
with this mutation. Of particular interest was the observation of
enhanced inhibitor binding when Glu497 was substituted for
either aspartate or glutamine (154 and 197%, respectively), a finding
that mirrors the increased QFS hydrolyzing activity noted above with
mutations at this position.
Effect of ZnCl2 on EP24.15 Wild Type and
Mutants--
The possibility that some of the mutations examined may
lower the affinity of the active site for the catalytically essential zinc was investigated. EP24.15 wild type and mutants displaying a
substantial decrease in catalytic and inhibitor binding capacity (i.e. substitutions to His473,
Glu474, His477, and Glu502) were
further examined by addition of ZnCl2 over a broad
concentration range (0.001-10 mM), via a discontinuous QFS
assay.3 Results demonstrated
that activity of the wild type enzyme was unaffected after zinc was
added back. Concentrations Coordination of a zinc ion within the catalytic site of
metalloenzymes is typically affected by an HEXXH motif,
a third zinc ligand distantly located carboxyl to this motif, and a
water molecule. Previous studies with zinc metalloendopeptidases
isolated from bacterial (1, 25-28, 48, 58, 59) and mammalian (29, 49)
species have all identified a similar role for the histidine residues
within this motif, to coordinate directly to the active site zinc ion,
whereas the HEXXH glutamate has been shown to coordinate weakly to zinc via an activated water molecule, thus facilitating the
acid-base catalytic mechanism. The absolute requirement for EP24.15
function of both an HEXXH motif and a glutamate 25 residues distant is evident from these investigations. Consequently, we propose
similar roles for His473, Glu474, and
His477 in EP24.15. We further propose a role for
Glu502 as the third zinc ligand in EP24.15. This differs
from the expected candidate, Glu497, which is highly
conserved in the bacterial metalloendopeptidases thermolysin, bacterial
elastase, and neutral protease.
In support of this hypothesis is the following experimental evidence.
(i) Replacement of any one of the aforementioned residues (see Table I)
results in ablation of enzyme activity with both GnRH1-9
and QFS utilized as substrates. (ii) These substitutions are also
characterized by a drastic reduction in the ability of EP24.15 to bind
the active site-directed inhibitor, cFP-AAY-pAB. We have demonstrated
in this study that binding of this inhibitor is dependent on the
presence of zinc within the catalytic site of EP24.15, in agreement
with previous observations by Orlowski and co-workers (12). The loss of
inhibitor binding in these instances, however, may also reflect a
distortion of the zinc coordination sphere following substitution of a
metal ligand. Previous studies with the ACE inhibitors, enalaprilat and
trandolaprilat, for example (also cFP-related inhibitors), demonstrate
the importance of both zinc presence and coordination geometry to
site-directed inhibitor binding (31, 50, 51, 57). In this regard,
Lesburg et al. (56) have also clearly demonstrated how
substitution of a zinc-coordinating ligand in the metalloenzyme
carbonic anhydrase II alters zinc coordination geometry with subsequent
detrimental effects on catalytic and inhibitor binding properties.
(iii) The results obtained were similar for both substitutions employed (Glu to Asp/Gln, His to Ala/Gln). Amino acid substitutions were chosen
which attempted to preserve side chain charge and/or space-filling conformation in order to minimize substantial three-dimensional changes
to the mutant protein. Glutamates were therefore substituted for either
aspartate (side chain length reduction by ~1.5 Å) or glutamine (loss
of the negative charge but retaining space-filling geometry).
Histidines were substituted for either an alanine or glutamine residue.
Replacement of histidine for alanine is frequently employed in scanning
mutagenesis to assess the putative function of histidine residues
within a protein (52), whereas replacement with glutamine is a common
occurrence in nature, reflecting the similarity of the two residues in
terms of hydrophilic and hydrophobic properties as well as partial
specific volume and accessible surface area (53).
With respect to the proposed third zinc ligand, one notes that
Glu502 is located further carboxyl to the HEXXH
motif in EP24.15 (25 residues) than its counterpart in thermolysin (20 residues) or, indeed, the other bacterial homologues (see Fig. 1).
However, the sequences of EP24.15 and the bacterial
metalloendopeptidases also reveal a highly conserved
DXXXH motif, located equidistant to the glutamate third zinc
ligand (Asp561-XXX-His565 in EP24.15
numbering). In thermolysin, these residues have been shown to form
hydrogen bonds with the incoming substrate, thereby stabilizing the
hydrated peptide in the transition state (54). Consequently, one would
expect the spatial position of these two residues relative to the third
zinc ligand to be crucial to catalysis. Assuming a similar function for
these residues in EP24.15 (currently under investigation), it is
significant, therefore, that the amino acid spacing between
Glu502 and Asp561/His565 in EP24.15
is virtually identical to that observed
(Glu166 and
Asp226/His231) in thermolysin.
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- and
-neoendorphin,
metorphamide, and Leu-enkephalin-Arg-Gly-Leu (8, 16, 17) and
to cleave the endogenous opioid receptor-like ligand,
nociceptin/orphanin FQ, a recently identified heptadecapeptide structurally resembling dynorphin A (18). EP24.15 has further been
implicated in the secretase processing of the wild type and mutant
forms of
-amyloid precursor protein in Alzheimer's disease (19-21). This latter implication is unusual given its general
preference for oligopeptides.
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80 °C for subsequent analysis. The reproducibility of the
mutagenesis procedure was also addressed in this study. To this end,
EP24.15 wild type and a selection of mutants were re-prepared from
bacterial glycerol stocks and assayed (as described below), yielding
very similar kinetic trends.
Oligonucleotide primers for mutagenesis
(kobs)t + ln[S0]
(where kobs = Vmax/Km). By monitoring
substrate hydrolysis continuously, a plot of ln[S] versus
time will generate a straight line, the slope of which equals
kobs. When divided by the enzyme concentration, this yields the specificity constant
(kcat/Km). This method
provides a rapid means for direct quantitation of the specificity constant for a fluorimetric substrate by continuous assay to monitor substrate hydrolysis (43, 62). QFS hydrolysis was continuously monitored with the aid of a Perkin-Elmer LS-5B fluorescence
spectrophotometer (South Plainfield, NJ) at excitation and emission
wavelengths of 328 and 393 nm, respectively. Product formation was
determined from an Mca-Pro-Leu standard curve prepared under identical
assay conditions. Suitable negative controls were included in each
assay. Total substrate hydrolysis was less than 10%.
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-helix (His473, Glu474, and His477 in
EP24.15, equivalent to His142, Glu143, and
His146 in thermolysin), and a glutamate residue, 20 amino
acids carboxyl to this motif (Glu497 in EP24.15, equivalent
to Glu166 in thermolysin) (Fig.
1). A C-terminal four-helix bundle is
also conserved among these metalloenzymes.2
Site-directed mutagenesis of the cDNA encoding rat EP24.15 (32) was
subsequently employed to prepare mutants in which the above histidine
and glutamate residues were genetically substituted. Two additional
glutamate residues carboxyl to the HEXXH motif, Glu502 and Glu537, were also mutated. The close
proximity of Glu502 to Glu497, coupled with the
earlier identification of Glu537 as the third zinc ligand
in EP24.11 (equivalent to Glu646 in EP24.11) (30),
warranted their investigation as putative third zinc ligands.
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Fig. 1.
Sequence alignment of EP24.15 with bacterial
homologues. A comparison of specific residues within the catalytic
site region of EP24.15 (rat) with those of thermolysin (Bacillus
thermoproteolyticus), bacterial elastase (Pseudomonas
aeruginosa), and neutral protease (Bacillus cereus),
bacterial homologues of mammalian metalloendopeptidases that have been
solved to atomic resolution. The sequences of mammalian EP24.11 and ACE
have also been included for comparison. Numbers in
superscript indicate residue positioning relative to each protein
sequence. Distances between amino acids are indicated in
parentheses.
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Fig. 2.
Structural integrity of recombinant
enzymes. Panel A, native polyacrylamide gel
electrophoresis (8%, reduced) of bacterially expressed, affinity
purified EP24.15 wild type and mutants (1.5 µg/lane). Gel was stained
with Coomassie Blue. Panel B, CD spectra for selected
mutations (H473A/H473Q, E474D/E474Q, H477A/H477Q, and
E502D/E502Q) and wild type as described under "Experimental
Procedures."
Kinetic analysis of EP24.15 wild type and mutants
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Fig. 3.
Effect of chelating agents on EP24.15 wild
type binding to the active site-directed inhibitor, cFP-AAY-pAB.
The binding assay is outlined under "Experimental Procedures."
Results for each chelator concentration are expressed relative to
untreated wild type enzyme (100% binding) and have been corrected for
nonspecific binding. Data are the mean of triplicate binding
experiments (± S.E.).
Active site-directed inhibitor binding analysis
0.5 mM ZnCl2, however, tended to inhibit the enzyme, a finding previously reported by
Orlowski and co-workers (7) while studying the native rat testes
enzyme. Furthermore, the mutants tested demonstrated no significant
activation following incubation with zinc over this concentration
range. Any direct effect of ZnCl2 on product (Mca-Pro-Leu) fluorescence was also tested in these experiments and found to be
non-existent.
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Fig. 4.
Model of zinc-coordinating residues within
the EP24.15 active site. A view of conserved residues involved in
zinc coordination and substrate catalysis. For clarity, only the side
chains and C- ' carbons of the coordinating residues as well as the
zinc atom and the activated water molecule are shown. Atoms proposed to
coordinate with zinc are connected by a thin white line. The
graphical representations were depicted with MOLSCRIPT (60)
with subsequent renderings using RASTER 3D (61).
A previous report by Le Moual and co-workers (30) shows that mutation of the glutamate third zinc ligand in EP24.11 (Glu646) to an aspartate residue results in a marked but incomplete decrease in catalytic activity. The present study however, demonstrates a complete ablation of activity following an identical substitution of the proposed glutamate third zinc ligand in EP24.15 (Glu502). The greater effect on the catalytic activity of this mutation in EP24.15 compared with EP24.11 can most likely be explained in terms of the distance between the second and third zinc ligands. In EP24.15, this distance is 25 amino acid residues compared with 59 in EP24.11. It has been postulated that the length of this spacer sequence is directly related to the flexibility of the zinc coordination sphere (55). Based on this hypothesis, therefore, one would expect the coordination sphere to be less flexible in EP24.15 compared with EP24.11, subsequently reflecting more rigid requirements for the spatial positioning of the active site zinc. Similar observations have also been reported for human ACE by Williams and co-workers (31). This latter research group reports that substitution of the glutamate third zinc ligand in the ACE carboxyl domain (Glu987 to Asp987) virtually eliminates enzyme activity. Interestingly, Glu987 in ACE is located a distance of 24 residues carboxyl to the HEXXH motif (compared with the 25 amino acid spacing between Glu502 and His477 in EP24.15).
The possibility that the results observed with any of the substitutions examined stems from global alterations to the protein folding pattern is unlikely. (i) All of the proteins examined were expressed to an equivalently high level and exhibit an identical native PAGE migration profile, indicative of normal protein folding. (ii) CD analysis of selected mutants displaying substantial alterations in catalytic and inhibitor binding properties (i.e. substitutions to His473, Glu474, His477, and Glu502) indicated no significant change in their global secondary structure relative to EP24.15 wild type. (iii) Other mutations introduced in the same area of the EP24.15 molecule, such as the substitution of Val468 for either alanine or leucine, had no significant effect on either the enzymes Km for GnRH1-9 or its ability to bind to cFP-AAY-pAB.4 (iv) Substitution of Glu497 generates EP24.15 mutants (E497D, E497Q) displaying increased QFS-hydrolyzing efficiency and inhibitor binding capacity.
The possibility that mutations causing a decrease in catalytic and inhibitor binding capacity may lower the affinity of the active site for the catalytically essential zinc was also investigated in these studies. Both EP24.15 wild type and mutants with substitutions to His473, Glu474, His477, and Glu502, amino acids that comprise the proposed zinc coordination model, were subsequently examined by addition of ZnCl2 over a broad concentration range (0.001-10 mM) via QFS assay. Results demonstrated that activity of the wild type enzyme was unaffected after zinc was added back, indicating no significant metal loss during the expression/purification process. Furthermore, the mutants tested demonstrated no significant activation following incubation with zinc over this concentration range, suggesting a reduction in affinity of the metal for the active site. These observations are consistent with a previous study by Lesburg et al. (56) who demonstrate that substitution of any one of the zinc-coordinating ligands in the metalloenzyme carbonic anhydrase II drastically reduces catalytic and inhibitor binding capacity, in parallel with reduced affinity of the enzyme active site for zinc. Similar findings have also been reported by Le Moual and co-workers (30) while identifying the third zinc ligand in the metalloenzyme, EP24.11. One also notes that in both of these latter studies, zinc is still bound within the active site, although improperly coordinated.
As mentioned previously, mutation of Glu497 to either glutamine or aspartate increases both QFS-hydrolyzing efficiency and inhibitor-binding capacity. By contrast, these mutations had relatively minor effects on GnRH1-9 catalysis. Given the close proximity of Glu497 to Glu502, these results may possibly be explained by a small distortion in the geometry of the zinc-coordinating residues (or possibly a loop of undetermined secondary structure), subsequently improving accessibility of the catalytic zinc and/or activated water molecule to an incoming substrate (QFS) or inhibitor (cFP-AAY-pAB) moiety. The lack of significantly increased GnRH1-9 hydrolysis in this instance may reflect subtle differences in its interaction with catalytic subsites. Minor alterations to the active site tertiary structure may also explain the reduced activity observed when Glu537 was mutated to glutamine. The normal levels of inhibitor binding observed with this substitution suggest that zinc coordination is unaffected. It is possible, however, that this mutation induces a conformational change that alters the distance between the bound substrate and the aforementioned Asp561/His565 residues (postulated to stabilize the transition state complex by forming hydrogen bonds with a carbonyl oxygen), thus reducing the hydrolytic efficiency of the enzyme.
In conclusion, the spatial organization of the proposed zinc-coordinating ligands in EP24.15 (Fig. 4) is consistent with observations for other zinc metalloenzymes (1) that a zinc-binding nucleus is provided by two ligands (His473 and His477) separated by a 1-3-amino acid spacer. A third zinc ligand (Glu502), separated from the second by a distance of 20-120 amino acids, completes the coordination sphere. A fourth residue (Glu474), located between the first and second ligands, subsequently provides a hydrogen-bonded water molecule that weakly coordinates via oxygen to the active site zinc ion, thereby creating a nucleophilic center for catalysis. The observations made in this investigation are indicative of the absolute requirement of these residues to binding and catalysis, although definitive interpretations await the availability of the solution of the enzyme structure by x-ray diffraction.
EP24.15 is one of the most extensively characterized soluble
endopeptidases with respect to physiological regulation and the neural
environment. We report here an integrated approach of structural, genetic, and biochemical methodologies to assess the effects of specific point mutations on EP24.15 function. Unlike similar
investigations with other mammalian zinc metalloendopeptidases
that address the functionality of one or two residues, this study
details the comprehensive examination of six residues postulated to
function in EP24.15 active site zinc coordination. Each residue under
investigation has been examined via two appropriate amino acid
substitutions, the structural integrity of all enzymes subsequently
assessed by native PAGE and CD spectroscopy, and their catalytic
efficiencies determined with multiple substrates. Furthermore, the
radioiodinated, site-directed inhibitor, cFP-AAY-pAB, serves as a
useful, zinc-sensitive probe to evaluate the binding efficiency of each
mutant. Using this strategy, further experimentation is currently
underway to identify other functional residues within the active site
of EP24.15. This will be coupled with information gleaned from ongoing
experiments to determine the three-dimensional structure of the enzyme.
A detailed understanding of the molecular architecture of EP24.15 will
lead to a more complete understanding of the mechanism of substrate
binding and the action of site-directed inhibitors as well as providing
a useful template for the rational design of pharmacological agents for
this prototypical member of the mammalian family of zinc metalloendopeptidases.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the continued valuable advice of Prof. James L. Roberts. We also thank Drs. T. John Wu, and John W. Tullai for critical comments. Many thanks to Dr. Ian Smith (Baker Medical Research Institute, Victoria, Australia) for the provision of cFP-AAF-pAB and cFP-AA-125I-Y-pAB, to Dr. Paul Schober (PepTech, Dee Why, Australia) for GnRH1-9 and GnRH1-5, and we are indebted to Dr. Michael Cascio (University of Pittsburgh, PA) for expert consultation with CD spectroscopy and model rendering.
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FOOTNOTES |
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* This work was supported by U. S. Public Health Service Grant 2T32-DA7135-16 (to P. M. C.) and National Institutes of Health Grant DK45493 (to M. J. G.).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.
Current address: Dept. of Human Genetics, University of Michigan
Medical School, Ann Arbor, MI 48109-0168.
§ To whom correspondence should be addressed: Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, Box 1065, 1425 Madison Ave., New York, NY 10029-6574. Tel.: 212-659-5973; Fax: 212-996-9785; E-mail: glux{at}msvax.mssm.edu.
2 M. J. Glucksman, M. Cascio, and J. L. Roberts, submitted for publication.
3 P. J. Crack, T. J. Wu, P. M. Cummins, J. W. Tullai, E. S. Ferro, M. J. Glucksman, and J. L. Roberts, submitted for publication.
4 P. M. Cummins, A. Pabon, and M. J. Glucksman, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: EP24.15, endopeptidase EC 3.4.24.15; ACE, angiotensin-converting enzyme; cFP-AAF-pAB, N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate; GnRH, gonadotropin-releasing hormone; QFS, 7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-D-Lys-(2,4-dinitrophenyl); BSA, bovine serum albumin; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MCA, 7-methoxycoumarin-4-acetyl.
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REFERENCES |
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