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Originally published In Press as doi:10.1074/jbc.M910386199 on March 28, 2000
J. Biol. Chem., Vol. 275, Issue 22, 16746-16751, June 2, 2000
Kinetic Investigation of the Specificity of Porcine Brain
Thyrotropin-releasing Hormone-degrading Ectoenzyme for
Thyrotropin-releasing Hormone-like Peptides*
Julie A.
Kelly §,
Gillian R.
Slator,
Keith F.
Tipton,
Carvell H.
Williams¶, and
Karl
Bauer
From the Department of Biochemistry, Trinity College
Dublin, Dublin 2, Ireland, the ¶ School of Biology and
Biochemistry, Medical Biology Centre, Queen's University, Belfast
BT9 7BL, United Kingdom, and the Max-Planck-Institut
für experimentelle Endokrinologie,
D-30603 Hannover, Germany
Received for publication, December 23, 1999, and in revised form, March 6, 2000
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ABSTRACT |
Evidence indicates that neuronally released
thyrotropin-releasing hormone (TRH) is selectively inactivated by
TRH-degrading ectoenzyme (TRH-DE) (EC 3.4.19.6). TRH-DE inhibitors may
be used to enhance the therapeutic actions of TRH and to investigate the functions of TRH and TRH-DE in the central nervous system. Although
TRH-DE appears to exhibit a high degree of specificity toward TRH,
systematic specificity studies, which would facilitate inhibitor
design, have not been previously conducted for this enzyme. In this
paper we present the first description of TRH-DE specificity across a
directed peptide library in which the histidyl (P1')
residue of TRH was replaced by a series of amino acids. Peptides were
synthesized using standard solid phase chemistry. Kinetic parameters
were measured either by continuous or discontinuous fluorometric assays
or by quantitative high pressure liquid chromatography. The
P1' residue was found to influence significantly both the ability of the peptides to bind to TRH-DE, as measured by their Ki values, and the ability of TRH-DE to catalyze
their hydrolysis. Moderately bulky, uncharged P1' residues
were found to bind preferentially to TRH-DE. Results from this screen
provide valuable information for the development of TRH-DE inhibitors and have led to the identification of two potent, reversible TRH-DE inhibitors,
L-pyroglutamyl-L-asparaginyl-L-prolineamide
(Ki = 17.5 µM) and
Glp-Asn-Pro-7-amido-4-methyl coumarin (Ki = 0.97 µM).
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INTRODUCTION |
Thyrotropin-releasing hormone-degrading ectoenzyme
(TRH-DE)1 (EC 3.4.19.6) is a
type II cell surface peptidase located on synaptosomal membranes in the
central nervous system (1 4). TRH-DE catalyzes the hydrolysis of the
Glp-His bond in thyrotropin-releasing hormone (TRH), a tripeptide with
the amino acid sequence
L-pyroglutamyl-L-histidyl-L-prolineamide (Glp-His-ProNH2) (510). This enzyme is strategically
placed to play a significant role in extracellular inactivation of TRH, and current evidence strongly indicates that TRH-DE is the principal enzyme responsible for terminating the actions of neuronally released TRH (1114).
Although first recognized as a hypothalamic regulatory hormone, TRH is
now believed to function as a neurotransmitter and/or neuromodulator
within the central nervous system (15, 16) where it displays a broad
spectrum of stimulatory actions independent of its neuroendocrine
functions (1517). Based on its central nervous system effects, TRH
has been found to have potential use in the treatment of brain and
spinal injury (18, 19) and several central nervous system disorders,
including spinocerebellar degeneration, cognitive deficits, and spinal
cord pain transmission (16, 17). The mechanisms by which TRH improves
these conditions are not fully elucidated but appear to involve the
potentiation by TRH of other neurotransmitter systems. Despite its
promise, the use of TRH as a therapeutic agent is critically undermined
by its susceptibility to proteolytic degradation (20).
Compounds that potently and selectively inhibit TRH-DE may be used to
enhance the therapeutic actions of TRH in the central nervous system by
either potentiating endogenous TRH and/or protecting exogenously
administered TRH or TRH analogs from degradation. TRH-DE inhibitors may
also be powerful tools for investigating the respective biological
roles of TRH-DE and TRH within the central nervous system (13, 20, 21,
22). TRH-DE appears to be an exceptional example of a
neuropeptide-specific peptidase (23) because no other ectopeptidase has
been shown to be capable of degrading TRH, and TRH-DE appears to
exhibit remarkable specificity for TRH (24). This peptidase does not
catalyze the hydrolysis of other, larger neuropeptides that also
contain an NH2-terminal Glp residue, such as luteinizing
hormone releasing hormone, neurotensin, bombesin, and gastrin (5, 810). With this unusual dual selectivity, TRH-DE is more similar to
acetylcholinesterase than to angiotensin-converting enzyme. Inhibition
of TRH-DE as a means of enhancing TRH signaling is attractive because
it should affect TRH signals exclusively, and consequently it may offer
significant investigative and therapeutic advantages. On the other
hand, the design of TRH-DE inhibitors is made difficult by the narrow
specificity of this enzyme (13).
Only a few inhibitors have been synthesized so far which exhibit a
significant effect on TRH-DE activity in vitro, none of which has been found to be sufficiently effective for pharmacological studies in vivo (13, 21, 22). With a Ki
of 8 µM, N-[1-carboxy-2-phenylethyl]N-imidazole benzyl
histidyl- -naphthylamide (CPHNA) appears to be the most potent of
these (21). CPHNA has been shown to increase the recovery of TRH
released from rat brain slices, indicating that TRH levels, and thus,
TRH neurotransmission, may be increased through TRH-DE inhibition
(21).
Protease inhibitors often incorporate structural features that enable
the inhibitor to interact with the enzyme's active site (20, 25, 26).
On the basis that TRH-DE has been shown to be a zinc metalloprotease
(27) and that it is weakly inhibited by Glp and His-ProNH2
(35), Bauer et al. (13) synthesized derivatives of Glp and
His-ProNH2 incorporating various functionalities, such as
hydroxamate, thiol, and phosphoamide groups known to inhibit other
metallopeptidases (26). All proved to be poor inhibitors of TRH-DE
(13).
The design of TRH-DE inhibitors is hampered by the fact that a
three-dimensional structure has not been ascertained for TRH-DE or the
aminopeptidases A and N, with which, according to cDNA sequence
analysis, TRH-DE shares approximately 30% homology (1). In the absence
of a target structure, however, structure-activity studies to identify
structural requirements for interaction with the enzyme's active site
can be used as a basis for the rational design of enzyme inhibitors
(25, 28, 29). No systematic structure-activity studies of TRH-DE have
been previously undertaken, and there is no active site model for
TRH-DE. Thus, to date, there has been insufficient information to
identify the structural features that need to be incorporated into
TRH-DE inhibitors.
Several investigators previously examined the ability of TRH
derivatives, with modifications to TRH's COOH- and/or
NH2-terminal residues, to act as substrates or inhibitors
of TRH-DE (6, 8, 10, 30). None was found to inhibit TRH-DE activity to any significant extent. Until recently, a histidine residue in the
P1' position was thought to be essential for TRH-DE
activity (5, 8, 22), yet it has now been shown that the naturally occurring TRH-like peptide Glp-Phe-ProNH2 is a substrate
for bovine brain TRH-DE (10, 31).
To investigate further the influence of P1' residues on
ligand binding and catalytic activity of TRH-DE and to facilitate the
rational design of TRH-DE inhibitors, we have conducted kinetic studies
on a directed peptide library in which the central histidyl residue of
TRH was replaced by a series of amino acids. New and improved TRH-DE
assays (32) were employed to investigate the kinetic properties of the
peptide library rigorously. The data presented here provide the first
illustration of TRH-DE specificity across a library of TRH-like
peptides. Furthermore, two potent, reversible TRH-DE inhibitors were
identified from the screen of this library.
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EXPERIMENTAL PROCEDURES |
Pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (TRHAMC) and
7-amino-4-methyl coumarin (AMC) were purchased from Bachem U. K. Ltd.
Glp-Asn-ProAMC was custom synthesized by the American Peptide Company
(Sunnyvale, CA). All other chemicals, except those specified below,
were of analytical grade and obtained from either Sigma-Aldrich
(Ireland) or Merck (Germany).
Peptide Library--
Glp-His-ProNH2,
Glp-Glu-ProNH2, and Glp-His-ProOH were obtained from
Sigma-Aldrich. Glp-Gln-ProNH2 and
Glp-Phe-ProNH2 were purchased from Peninsula Laboratories
Inc. (U. K). Glp-Asn-ProNH2, Glp-Tyr-ProNH2,
and Glp-D-Asn-ProNH2 were obtained from the
American Peptide Company in addition to being synthesized by the method described below. All other TRH-like peptides in the library were synthesized either manually using a bubbler system (for details, see
Ref. 33) or using a Synergy Peptide Synthesizer (Applied Biosystems,
U. K.). In both cases, standard solid-phase Fmoc chemistry was
employed (33). Pyroglutamic acid and Fmoc amino acid derivatives were
purchased from Calbiochem-Novabiochem U. K. Ltd. Trifunctional amino
acids were obtained with side chain-protecting groups as follows:
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Asn(Trt)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-His(Trt)-OH, and Fmoc-Cys(Trt)-OH.
Synthesis was carried out on Rink amide 4-methylbenzylhydrylamine resin
(Calbiochem-Novabiochem U. K. Ltd.) with a loading capacity 0.64 mmol
g 1. The resin was swollen using
N,N-dimethylformamide and deprotected with
20% piperidine in N,N-dimethylformamide.
Coupling was performed twice for each amino acid using the Synergy
Peptide Synthesizer and once when the bubbler system was employed.
Three equivalents (i.e. 3-fold excess over the resin loading
capacity) of each amino acid were coupled onto the resin with
HBTU/HOBt/DIPEA (1:1:2 equivalents) at each step. No deprotection step
was necessary after the coupling of pyroglutamic acid. On completion of
peptide assembly, the resin was washed thoroughly with dichloromethane
followed by methanol and allowed to dry overnight. In the absence of
labile amino acids and side protection groups, cleavage of the peptide
from the resin was achieved by placing the dry resin in a round
bottomed flask and adding 95% (v/v) trifluoroacetic acid in water (10 ml/g of dry resin). This reaction mixture was stirred at room
temperature for approximately 1 h before filtering the suspension
through a sintered glass funnel. Sequences containing Asn were
deprotected and cleaved using a trifluoroacetic acid solution
containing 95% trifluoroacetic acid, 2.5% water, and 2.5%
triisopropylsilane (v/v/v). For sequences containing Ser, Trp, or Arg,
cleavage/deprotection was achieved using reagent K (82.5%
trifluoroacetic acid, 5% water, 5% thioanisole, 5% phenol, 2.5%
1,2-ethanedithiol, v/v/v/v/v) in place of trifluoroacetic acid (33,
34).
These small peptides proved difficult to precipitate directly from the
filtrate using diethyl ether, so the trifluoroacetic acid and
scavengers were first removed by rotary evaporation under vacuum. The
residue was washed with petroleum ether. Diethyl ether was then added
to the semisolid residue to crystallize the peptide. Because of the
hygroscopic nature of these peptides during isolation, after
decantation of the bulk of the solvent, a steady stream of nitrogen was
used to evaporate the diethyl ether and to dry the peptide pellet
simultaneously. The peptide pellets were dried thoroughly under
nitrogen before transferring the dried material to preweighed glass
containers for storage in a desiccator. After weighing the dried
material, the peptides were stored at 20 °C. Stock solutions were
prepared from this material.
Peptides were analyzed and judged to be homogeneous by HPLC. HPLC
analysis was conducted using a Thermo Separation Products Inc. Spectra
System HPLC. Standard 1 mM solutions of each peptide in 20 mM potassium phosphate buffer, pH 7.5, were analyzed on a
C-18 reverse phase column (Hypersil U. K.) using a linear gradient of
0-70% acetonitrile in 0.08% trifluoroacetic acid as described previously (31).
Enzyme Purification--
TRH-DE was purified almost 20,000-fold
from porcine brain as described previously (32, 35). A
Coomassie-stained gel of the purified enzyme after SDS-polyacrylamide
gel electrophoresis showed one major band, the molecular mass of which
was consistent with that of 116 kDa reported previously for TRH-DE
(35). This highly purified TRH-DE preparation did not contain any of
the various peptidase activities tested, including aminopeptidases, carboxypeptidases, and dipeptidyl aminopeptidases, and it was completely devoid of other TRH-degrading enzymes, including
pyroglutamyl aminopeptidase I (EC 3.4.21.26) and prolyl oligopeptidase
(EC 3.4.19.3) (32). The preparation was found to have (i) a protein concentration of 0.8 mg ml1 using a modification of the
Lowry method (36) with bovine serum albumin as a standard and (ii) a
specific activity of 0.17 unit mg1 with TRHAMC as
substrate under standard conditions of a continuous assay described
previously (32) and outlined below.
Dipetidyl peptidase IV (DPP-IV) (EC 3.4.14.5) was purified 370-fold
from bovine kidney (37) and was found to have a protein concentration
of 7.7 mg ml1 using the method of Markwell et
al. (38). This preparation did not contain any measurable amount
of either dipeptidase or oligopeptidase activity. The specific activity
was 17.5 units mg1 with Gly-ProAMC as substrate (32).
1 unit of enzyme activity was defined as that amount catalyzing the
formation of 1 µmol of product in 1 min under the standard conditions
employed. All incubations in the assays described below were carried
out in 20 mM potassium phosphate buffer, pH 7.5, at
37 °C. Fluorescence measurements were made using a Perkin-Elmer LS
50B luminescence spectrometer fitted with a thermostatted cell holder.
Wavelengths for excitation and emission were set at 370 and 440 nm,
respectively, with slit widths of 10 and 5 nm, respectively.
Kinetic Analysis of the Peptide Library Using HPLC--
The
ability of each peptide in the library to act as a TRH-DE substrate was
assessed using HPLC. As an initial screen, 1 mM peptide was
incubated with 0.8 µg of TRH-DE in a total volume of 1 ml for 18 h at 37 °C. Control samples were included in which peptide (1 mM) or Glp (0.2-0.8 mM) was incubated under
identical conditions in the absence of TRH-DE. TRH-DE activity was
terminated by the addition of trifluoroacetic acid (0.15%, v/v), and
samples were then analyzed using HPLC as described previously (31). Products resulting from TRH-DE hydrolysis were separated on a C-18
reverse phase column using a linear gradient of 0-70% acetonitrile in
0.08% (v/v) trifluoroacetic acid. The concentration of Glp formed by
the action of TRH-DE on the peptide library was measured by UV
absorbance at 206 nm with a quantitative detection limit of 0.1 mM for a 40-µl injection volume, employing a signal to noise ratio of 10. Following the incubation of each peptide with TRH-DE, evidence of Glp in the sample was taken to indicate that the
peptide was a TRH-DE substrate.
The rates of hydrolysis for each of those peptides identified as
substrates were then compared by measuring Glp production using
microassays with shorter incubation times (39). In these assays,
peptide (1 mM) was incubated at 37 °C in a total volume of 100 µl, and incubation times and TRH-DE concentration were adjusted to produce a detectable amount of Glp while remaining within
the initial rate period of the reaction. Typically, 0.8-3.2 µg of
TRH-DE and incubations times of 5 min to 18 h were used. Measurements were made in triplicate. The rate of TRH-OH hydrolysis was
also measured by this method.
Determination of Inhibitor Constants for Selected TRH-DE
Substrates--
The HPLC assay lacked sufficient sensitivity for
determining kinetic constants. Therefore, those peptides undergoing
significant hydrolysis (i.e. those that exhibited rates of
hydrolysis  0.5 unit mg1) were examined as
competitive substrates of TRH-DE using a recently published
discontinuous fluorometric TRH-DE assay (32). This assay employs TRHAMC
as the substrate and depends on the measurement of the fluorescence of
AMC produced as shown in Reactions 1 and 2.
We have shown that the amount of AMC formed under these
conditions is a quantitative measure of TRHAMC cleavage (32). Initial rates for the hydrolysis of TRHAMC by TRH-DE were determined in triplicate at five different substrate concentrations, both in the
absence and presence of at least three concentrations of peptide. Ki values were obtained by nonlinear regression
analysis of the data collected. When determined in this way
(i.e. by treating the peptide substrates as inhibitors of
TRHAMC hydrolysis), these Ki values correspond to
the Michaelis constants for those library peptides hydrolyzed by TRH-DE
(40).
Kinetic Analysis of Peptides That Are Not Hydrolyzed by
TRH-DE--
A recently developed continuous coupled fluorometric assay
(32) was used to investigate the ability of those library peptides that
were not hydrolyzed by TRH-DE to inhibit TRHAMC degradation by TRH-DE.
Glp-Asn-ProAMC was also included in this study. In this assay, TRHAMC
is the substrate, and DPP-IV is the coupling enzyme. The sequence is
shown in Reaction 3.
The reaction was monitored continuously by measuring the
increase in AMC fluorescence. A linear progress curve with no
discernible lag period was observed when the reaction was monitored
over a period of 10 min. Nevertheless, data sampling was not commenced until 100 s from the start of the reaction to ensure that
measurements were taken after a steady state had been reached (see Ref.
32). This continuous assay permits accurate assessment of nonlinear progress curves that may arise in the presence of tight binding inhibitors.
In a preliminary experiment, the peptides were screened for their
ability to inhibit TRH-DE activity. The initial rate of TRHAMC
hydrolysis by TRH-DE was measured by incubating 5 µM
TRHAMC with 1.23 µg of DPP-IV and 0.32 µg of TRH-DE, both in the
absence and presence of each library peptide (1 mM final
concentration) under standard assay conditions (32).
Peptides showing < 20% inhibition (Ki > 1 mM) were not examined further. The Ki
values for the remainder of the peptides were determined by measuring
their effects on TRHAMC degradation by TRH-DE using the continuous
assay. Data were collected in duplicate at five different substrate
concentrations and at least three different concentrations of each peptide.
All peptides that were observed to inhibit AMC production in the
continuous coupled assay were assessed for their ability to inhibit the
coupling enzyme, DPP-IV, using a direct continuous assay for DPP-IV
which employed Gly-ProAMC as the substrate (32). Peptide concentrations
similar to those used to determine the Ki values
above were employed in the DPP-IV assay. None of these peptides was
found to inhibit DPP-IV hydrolysis of Gly-ProAMC. Thus, the effects
produced by the peptides can be attributed solely to their inhibition
of TRH-DE.
Reversibility and time dependence of the inhibition produced by
Glp-Gln-ProNH2, Glp-Asn-ProNH2, and
Glp-Asn-ProAMC were examined by initially preincubating TRH-DE with
each peptide at 37 °C for various periods up to 75 min. To
investigate time dependence, the enzyme-peptide solution was
subsequently added to a reaction mixture containing TRHAMC, DPP-IV,
buffer, and peptide, and TRH-DE activity was measured using the
continuous assay. The final concentration of
Glp-Gln-ProNH2, Glp-Asn-ProNH2, and
Glp-Asn-ProAMC used was 400, 160, and 1 µM, respectively.
To test for reversibility, the enzyme-peptide solution was added to a
reaction mixture that did not contain peptide. TRH-DE activity was not
affected by preincubation at 37 °C.
Analyses of Results--
All kinetic parameters were determined
by nonlinear regression analysis using the computer program Prism
(Graph Pad Software Inc.). Linear regression analysis employing
proportional weighting was used to fit data to linear plots for display
purposes only. Unless otherwise stated, all values are shown as the
mean ± S.D.
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RESULTS |
HPLC Analysis of the Peptide Library--
HPLC analysis revealed
that TRH-DE catalyzed the removal of the NH2-terminal Glp
residue from 15 out of the 25 members of the peptide library, including
TRH. It can be seen from the representative HPLC traces shown in Fig.
1 that 1 mM TRH was fully
degraded after overnight incubation with TRH-DE. No detectable Glp was
released from those peptides where the P1' position was
occupied by D-Asn, Gly, or the L-amino acids
Asn, Gln, Trp, L- -phenylglycine, homoproline, Glu, Asp,
and Pro. The HPLC-based assay also showed that there was no detectable
cleavage of Glp-Cys-ProNH2 by TRH-DE. This peptide was
observed, however, to undergo oxidation with disulfide bond formation
during incubation and was not examined further. Table I shows the rates of hydrolysis of those
peptides that were found to be substrates for TRH-DE.
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Fig. 1.
Representative HPLC traces obtained after the
incubation (18 h) of TRH with TRH-DE. The trace in the
foreground represents a control sample for TRH. The trace in
the background shows the products formed from TRH by the
action of TRH-DE. TRH was degraded completely to give Glp and
His-ProNH2. Consistent with previously published results
(31, 32, 39), His-ProNH2 was found to undergo spontaneous
intramolecular cyclization slowly to form His-prodiketopiperazine
(His-ProDKP).
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Table I
Comparison of hydrolysis rates for library peptides (1 mM) found to be TRH-DE substrates
Rates of hydrolysis were determined as outlined under "Experimental
Procedures." Each value represents the mean ± S.D. The number
of determinations is indicated in parentheses.
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Inhibitor Constants for Selected TRH-DE Substrates--
Because of
the difficulty in obtaining reliable Km values for
library peptides acting as substrates, we measured instead their
Ki values as competitive substrates.
Ki values for those library peptides that were
significantly hydrolyzed by TRH-DE are shown in Table
II. All of these peptides were found to
act as simple competitive inhibitors of the degradation of TRHAMC by
TRH-DE (example shown in Fig. 2).
Nonlinear regression analysis of the data collected in this study gave
a Km value for TRHAMC of 3.1 ± 0.5 µM (n = 8). This compared with the value
of 3.4 ± 0.7 µM (n = 5) which we
published recently for the discontinuous fluorometric assay (32). The
observed hydrolysis and Ki value obtained for
Glp-Phe-ProNH2 are consistent with previous reports for
TRH-DE purified from bovine brain (10, 31).
Vmax/Km values (Table II)
were calculated assuming that the Ki values for
these peptides correspond to Michaelis constants (40). It can be seen
from Table II that TRH is the most favorable substrate of those
tested.
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Table II
Comparison of kinetic parameters for selected library peptides
hydrolyzed by TRH-DE
The Michaelis constant for Glp-His-ProAMC (TRHAMC) was measured
directly, whereas the Km values for the library
peptides were measured indirectly by treating them as competitive
inhibitors of TRHAMC hydrolysis by TRH-DE. The Km
values were all determined by nonlinear regression analysis of data
obtained from the discontinuous fluorometric TRH-DE assay and represent
the mean ± S.D. The number of determinations is shown in
parentheses. Included for comparison are the corresponding
Km values for TRH and TRH-OH, which we published
recently (32). Vmax values were estimated from rates
of hydrolysis measured by HPLC using the relationship
Vmax = v0 ((Km + [S])/[S]).
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Fig. 2.
Lineweaver-Burk plot for TRHAMC degradation
by TRH-DE in the presence of increasing concentrations of
Glp-Thi-ProNH2. Data were obtained using the
discontinuous fluorometric assay and represent the mean ± S.E.
(n = 3). Error bars can be seen where the
S.E. is greater than the size of the symbol.
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Kinetic Analysis of Peptides Not Hydrolyzed by TRH-DE--
Table
III shows the percent inhibition of
TRH-DE activity produced by those peptides not hydrolyzed by TRH-DE.
Presented also are the Ki values obtained for those
peptides exhibiting greater than 20% inhibition in the initial
screening. The latter peptides were all found to act in a simple
competitive manner as illustrated by a Lineweaver-Burk plot of data
obtained for Glp-Asn-ProNH2 (Fig.
3). Inhibition by
Glp-Asn-ProNH2, Glp-Gln-ProNH2, and
Glp-Asn-ProAMC was found to be fully reversible and not time dependent.
Nonlinear regression analysis of data from the continuous assay gave a
Km value of 3.5 ± 0.4 µM
(n = 6) for TRHAMC. This is comparable to the value
estimated by the discontinuous assay above and to previously published
values (32).
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Table III
Inhibitory effects of peptides that were not hydrolyzed by TRH-DE
Data were obtained using TRHAMC as substrate in the continuous coupled
assay. The percent inhibition produced by each library peptide and
Glp-Asn-ProAMC was determined in the presence of 5 µM
substrate. Ki values (mean ± S.D.
(n = 3)) were measured by the continuous coupled assay
at five different TRHAMC concentrations and at least three different
concentrations of peptide. Ki values for peptides
displaying < 20% inhibition were estimated to be > 1 mM. The Ki for Glp-Asn-ProNH2
represents the mean ± S.D. of three separate experiments using
three different batches of Glp-Asn-ProNH2; one batch was
obtained from the American Peptide Company, and the other two batches
were synthesized as described under "Experimental
Procedures." No significant difference was found in the
Ki among the three different batches.
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Fig. 3.
Inhibition of TRH-DE hydrolysis of TRHAMC by
Glp-Asn-ProNH2. Initial rates were determined using
the continuous fluorometric assay. Data are shown as a Lineweaver-Burk
plot for illustrative purposes and represent the mean ± S.E.
(n = 3). Error bars can be seen where the S.E. is
greater than the size of the symbol.
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DISCUSSION |
The results from this study show that alteration of the
P1' residue in the tripeptide structure of TRH
significantly affects both the ability of the resulting peptides to
bind to TRH-DE, as measured by their Ki values, and
the ability of TRH-DE to catalyze their hydrolysis.
The low turnover rates observed for library peptides containing Ala,
norvaline, Ser, Thr, Ile, Leu, Val, or Gly in place of His indicate
that they lack a critical stereochemical element required for efficient
hydrolysis, although they are clearly not sterically excluded from the
binding site of TRH-DE.
Kinetic data obtained for library peptides in which His was replaced by
Asp or Glu indicate that a negatively charged residue at the
P1' site is not favorable for binding or catalysis. The poor rates of hydrolysis observed for peptides containing Arg or Lys in
the P1' position compared with those containing
thienylalanine and Phe suggest that although the S1' site
on the enzyme is capable of accepting a positively charged residue, a
neutral, aromatic residue is preferred for catalysis (Fig.
4). These results imply that the
imidazole group in TRH may not be protonated during the binding of TRH
to TRH-DE. Further support for this is given by the observation that
substitution of histidine by either of the non-basic isosteres,
L-Asn and L-Gln, does not lead to significant loss of apparent binding affinity.
On the whole, occupation of the P1' site by residues with
aromatic character appears to be particularly favorable for catalysis. Factors other than the nature of the residue, such as the size of the
side chain, also seem to be influential because elongation of the side
chain of Phe by one methylene group produced a significant loss in
activity, and reduction in the length of the side chain by the same
amount resulted in a peptide that exhibited a reduced affinity for
TRH-DE and was not hydrolyzed by the enzyme. Moreover, the large indole
ring of Trp appeared to be accommodated somewhat by the S1'
subsite (Ki = 232 µM), but
Glp-Trp-ProNH2 was not hydrolyzed.
Although the presence of aromatic character in the P1'
residue appears to be advantageous for substrates, it is clearly not essential for ligand binding because both Glp-Asn-ProNH2
and Glp-Gln-ProNH2 were able to bind to TRH-DE, with an
apparent affinity similar to that of TRH, but they were not hydrolyzed.
It is not obvious why Glp-Asn-ProNH2 and
Glp-Gln-ProNH2 do not act as substrates, but it might be
postulated that binding of these peptides to the enzyme is distorted,
thus preventing catalysis. Lowe et al. (41) noted that it is
possible to superimpose, two-dimensionally, the side chains of
L-Asn and L-Gln onto that of L-His
such that the amide nitrogen of L-Asn overlaps with the
 N of L-His and that the amide nitrogen of
L-Gln coincides with the  N of L-His. Because the TRH-like peptides that contain Asn, Gln, and His in the
P1' position all bind relatively well to TRH-DE, it might
be suggested that the nitrogen atoms in these side chains represent
recognition moieties for binding to the enzyme's S1'
subsite. The more favorable inhibitory properties of
Glp-Asn-ProNH2 compared with those of Glp-Gln-ProNH2 may be related to the position of the
nitrogen in the side chain and to the position of the amide carbonyl
group relative to the S1' subsite on the enzyme. Because
Glp-D-Asn-ProNH2 displays poor affinity for
TRH-DE, the interaction of Asn with the S1' subsite on the
enzyme appears to be stereospecific.
We were able to achieve significant improvement of the apparent binding
affinity of Glp-Asn-ProNH2 by substituting the amide group
of the COOH terminus with AMC. The resulting compound, Glp-Asn-ProAMC, was found to have a Ki of 0.97 ± 0.08 µM and is the most potent, competitive TRH-DE inhibitor
described to date. Previously, the most potent TRH-DE inhibitor to be
reported was CPHNA (21). This compound, though substantially
structurally different from TRH and Glp-Asn-ProAMC, was found to
inhibit TRH-DE in a competitive manner with a Ki of
8 µM (21). The enhanced binding of the coumarin
derivative of Glp-Asn-ProNH2 is most likely caused by more
favorable hydrophobic interactions occurring between the COOH terminus
of the peptide and the enzyme. Indications that this substitution would
improve inhibitor binding were deduced from comparison of the kinetic
parameters obtained for TRH, TRHAMC, and TRH-OH (Table II). The
respective Km values for these peptides were
consistent with those reported previously (10). Based on the value of
Km for TRHAMC, Gallagher and O'Connor (10)
suggested that TRH-DE has a preference for large hydrophobic groups,
such as AMC, at the carboxyl terminus of substrates. The results
presented in Table II indicate that the addition of AMC to the COOH
terminus of TRH causes a reduction not only in the Km, as reported previously, but also in the
catalytic rate. It was concluded, therefore, that this may be a useful
feature to incorporate into an inhibitor. Lead compounds such as
Glp-Asn-ProNH2 and Glp-Asn-ProAMC can contribute to the
understanding of pharmacophores that are recognized by binding sites on
the enzyme and may provide a basis for the development of
peptidomimetic inhibitors (29).
The results presented here raise the possibility that TRH-DE may be
involved in the metabolism of endogenous peptides that are structurally
analogous to TRH. Three library members, Glp-Glu-ProNH2, Glp-Phe-ProNH2, and Glp-Gln-ProNH2, have
recently been found to occur in a variety of mammalian tissues
(4248). However, the apparent absence in brain tissue of TRH-like
peptides, other than TRH, (43, 44) is in keeping with the hypothesis
that brain TRH-DE is uniquely involved in terminating TRH signals in
the central nervous system.
In conclusion, the structure-activity relationships determined from
this study provide a detailed picture of the stereochemical requirements at the S1' subsite of TRH-DE for ligand
binding and hydrolysis. Overall, the results suggest that the
S1' subsite of TRH-DE preferentially binds moderately
bulky, uncharged residues. Significantly, the systematic screening of
the directed peptide library has led to the development of two potent,
reversible, competitive TRH-DE inhibitors, one of which is the most
potent described to date. The data presented contribute to the
identification of structural features critical to the design of TRH-DE
inhibitors and to a description of TRH-DE's active site.
Together, these provide a solid basis for optimizing the design of
potent and selective TRH-DE inhibitors, which may have therapeutic
application and may be used to investigate the biological functions of
this important neuropeptidase and the role of TRH in the central
nervous system.
| |
ACKNOWLEDGEMENTS |
We are indebted to Brian Walker and Pat
Harriott for expert help in synthesizing the peptide library. We thank
Janet Brownlees for the preparation of DPP-IV and Petra Affeldt for
assistance in purifying TRH-DE. We also thank Noel Breslin for
excellent technical assistance.
| |
FOOTNOTES |
*
This research was supported by Wellcome Trust Grant
046172/Z/95/Z.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. Tel.: 353-1-608-1608;
Fax: 353-1-677-2400; e-mail: kellyja@tcd.ie.
Published, JBC Papers in Press, March 28, 2000, DOI 10.1074/jbc.M910386199
| |
ABBREVIATIONS |
The abbreviations used are:
TRH-DE, thyrotropin-releasing hormone-degrading ectoenzyme;
TRH, thyrotropin-releasing hormone;
Glp-His-ProNH2,
L-pyroglutamyl-L-histidyl-L-prolineamide;
Glp, pyroglutamic acid;
CPHNA, N-[1-carboxy-2-phenylethyl]N-imidazole benzyl
histidyl--naphthylamide;
TRHAMC, pyroglutamyl-histidyl-prolylamido-4-methyl coumarin;
AMC, 7-amino-4-methyl coumarin;
Fmoc, N-(9-fluorenyl)methyloxycarbonyl;
tBu, t-butyl ether;
Trt, triphenylmethyl;
Pmc, 2,2,5,7,8-pentamethylchromane-6-sulfonyl;
Boc, N--t-butyloxycarbonyl;
OtBu, t-butylester;
HBTU, 2-(1H-benztriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate;
HOBt, N-hydroxybenzotriazole;
DIPEA, N,N-diisopropylethylamine;
HPLC, high
pressure liquid chromatography;
DPP-IV, dipeptidyl peptidase IV;
Thi, thienylalanine. All amino acids are in the L configuration
unless otherwise stated.
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ABSTRACT
INTRODUCTION
