(Received for publication, August 14, 1995)
From the
Carboxyl-terminal amidation, a required post-translational
modification for the bioactivation of many neuropeptides, entails
sequential enzymatic action by peptidylglycine -monooxygenase
(PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5).
The monooxygenase, PAM, first catalyzes conversion of a
glycine-extended pro-peptide to the corresponding
-hydroxyglycine
derivative, and the lyase, PGL, then catalyzes breakdown of this
-hydroxyglycine derivative to the amidated peptide plus
glyoxylate. We have previously established that PAM and PGL exhibit
tandem reaction stereospecificities, with PAM producing, and PGL being
reactive toward, only
-hydroxyglycine derivatives of absolute
configuration (S). We now demonstrate that PAM and PGL exhibit
dramatically different subsite stereospecificities toward the residue
at the penultimate position (the P
residue) in both
substrates and inhibitors. Incubation of Ac-L-Phe-Gly,
Ac-L-Phe-L-Phe-Gly, or (S)-O-Ac-mandelyl-Gly with PAM results in complete
conversion of these substrates to the corresponding
-hydroxylated
products, whereas the corresponding X-D-Phe-Gly compounds
undergo conversions of <1%. The K
of
Ac-D-Phe-Gly is at least 700-fold higher than that of
Ac-L-Phe-Gly, and the same pattern holds for other substrate
stereoisomers. This S
subsite stereospecificity of PAM also
holds for competitive inhibitors; thus, the K
of 45 µM for
Ac-L-Phe-OCH
CO
H increases to 2,247
µM for the -D-Phe- enantiomer. In contrast,
incubation of PGL with Ac-L-Phe-
-hydroxy-Gly,
Ac-D-Phe-
-hydroxy-Gly, (S)-O-Ac-mandelyl-
-hydroxy-Gly, or (R)-O-Ac-mandelyl-
-hydroxy-Gly results in facile
enzymatic conversion of each of these compounds to their corresponding
amide products. The simultaneous expression of high reaction stereospecificity and low S
subsite stereospecificity in the course of PGL catalysis was illustrated
by a series of experiments in which enzymatic conversion of the
diastereomers of Ac-L-Phe-
-hydroxy-Gly and
Ac-D-Phe-
-hydroxy-Gly was monitored directly by HPLC.
Kinetic parameters were determined for both substrates and potent
competitive inhibitors of PGL, and the results confirm that, in sharp
contrast to PAM, the configuration of the chiral moiety at the P
position has virtually no effect on binding or catalysis. These
results illustrate a case where catalytic domains, which must function
sequentially (and with tandem reaction stereochemistry) in a
given metabolic process, nevertheless exhibit sharply contrasting
subsite stereospecificities toward the binding of substrates and
inhibitors.
Tandem enzymatic reactions, which represent sequential steps
along a metabolic pathway, are in many cases catalyzed by
multifunctional proteins comprising two or more distinct catalytic
domains on a single polypeptide chain (for a comprehensive treatment of
early work, see (1) ; see also Refs 2-7). A case in point
is carboxyl-terminal amidation, a required post-translational
modification for the bioactivation of many neuropeptides(8) ,
which entails sequential enzymatic action by peptidylglycine
-monooxygenase (PAM, (
)EC 1.14.17.3) and
peptidylamidoglycolate lyase (PGL, EC
4.3.2.5)(9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .
The monooxygenase, PAM, first catalyzes conversion of a
glycine-extended pro-peptide to the corresponding
-hydroxyglycine
derivative, and the lyase, PGL, then catalyzes breakdown of this
-hydroxyglycine derivative to produce the amidated peptide plus
glyoxylate(9, 10, 19) . The ``amidating
enzyme'' gene in pituitary encodes a multifunctional protein,
which contains the catalytic PAM domain at its NH
terminus
followed by the adjacent catalytic PGL
domain(8, 20, 21) . Subsequent
post-translational endoproteolytic processing of this multifunctional
protein can give rise to various truncated forms of both PAM and PGL (8, 22, 23, 24, 25, 26, 27, 28) .
Elucidation of the stereochemistry of carboxyl-terminal amidation is
a critical issue for detailed mechanistic studies on the enzymology of
this process and for the rational design of pseudosubstrates and
inhibitors targeted at PAM and PGL. In this regard, we recently
demonstrated that PAM and PGL exhibit tandem reaction
stereospecificities in carrying out the two requisite steps in
carboxyl-terminal amidation(29) . Thus, PAM produces only -hydroxyglycine derivatives of absolute configuration (S), and PGL is reactive only toward (S)-
-hydroxyglycines (Fig. S1). While these
results elucidate the reaction stereospecificity of both PAM
and PGL toward the COOH-terminal (S)-
-hydroxyglycine
moiety, the quite distinct question of whether PAM and PGL differ with
respect to subsite stereospecificity remains unresolved.
Figure S1: Scheme 1
We
report here a series of experiments, which demonstrate that PAM and PGL
indeed exhibit dramatically different subsite stereospecificities
toward the residue at the penultimate position (the P
residue) (
)of their respective substrates. In addition, we
introduce new competitive inhibitors for PAM and PGL, and we
demonstrate that the distinctive difference in S
subsite
stereospecificities for substrates is also
reflected in the binding of competitive inhibitors targeted at either
enzyme, respectively. Finally, we illustrate the simultaneous
expression of high reaction stereospecificity and low S
subsite stereospecificity in the course of PGL catalysis by a
series of experiments in which enzymatic conversion of all four
diastereomers of Ac-Phe-
-hydroxy-Gly was monitored directly by
HPLC. Thus, these results provide a clear example of a case where
catalytic domains, which must function sequentially (and with tandem reaction stereochemistry) in a given metabolic process,
nevertheless exhibit contrasting subsite stereospecificities toward the
binding of both substrates and inhibitors.
Synthetic Ac-L-Phe-L-Phe-Gly and Ac-L-Phe-D-Phe-Gly have specific rotation values of -32.9° and 44.8°, respectively.
The crude Ac-D-Phe-Gly-OEt (0.5 g, 1.7 mmol)
was dissolved in a mixture of methanol and water (20 ml, 1:1), and 3.8
ml of 0.5 M NaOH (1.9 mmol) was then added. The mixture was
stirred at room temperature, and the hydrolysis of
Ac-D-Phe-Gly-OEt was monitored on a C8 PR-HPLC column with an
isocratic solution of HO/CH
CN (80/20, 0.2%
phosphoric acid). Nearly complete hydrolysis was achieved in about 2 h,
after which the pH of the solution was adjusted to neutrality with
dilute HCl. The mixture was extracted with methylene chloride (2
30 ml) to remove any unreacted Ac-D-Phe-Gly-OEt. The
pH of the solution pH was then adjusted to about 3.0 and extracted with
ethyl acetate (2
30 ml). The ethyl acetate layer was dried over
MgSO
and evaporated to dryness to give
Ac-D-Phe-Gly (0.3 g).
H NMR
(Me
SO-d
, 300 MHz)
: 8.20 (d, 1H,
CO
H), 7.15-7.30 (m, 5H, Ph), 4.50 (m, 1H,
NHCHCONH), 3.85 (d, 2H,
NHCH
CO
H), 3.05 (dd, 1H,
PhCHHCH), 2.73 (dd, 1H, PhCHHCH), 1.75 (s, 3H,
CH
CO). Mass spectroscopy (electron impact): m/z 264 (M
, molecular ion).
Synthetic Ac-D-Phe-Gly has a specific rotation value of -11.8° in methanol.
Synthetic Ac-L-Phe-Gly has a specific rotation value of 11.9° in methanol.
(R)-2-Phenylbutyramide (0.28 g, 1.70 mmol) was then reacted
with glyoxylic acid in acetone, as described in the
literature(34) , to give (R)-2-phenylbutyl--hydroxy-Gly (0.21 g).
[
] = -29.48° in methanol;
H
NMR (Me
SO-d
, 300 MHz)
: 8.82 (d,
1H, OH),7.29 (m, 5H, Ph), 6.32 (bs, 1H, NH), 5.37 (t, 1H,
CH
CHCO), 3.43 (m, 1H, NHCHOH),
1.59-1.95 (m, 2H, CH
CH
CH), 0.79
(q, 3H, CH
); mass spectroscopy (FAB-MS): m/z 238
(M + 1).
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
To a solution of
Ac-glycine (1.0 g, 8.54 mmol) in 30 ml of acetonitrile were added
2-bromophenyl acetate (2.2 g, 8.56 mmol) and 1,8-diazabicyclo
[5.4.0] undec-7-ene (1.3 ml, 8.69 mmol)(36) . The
reaction mixture was stirred at room temperature overnight. Water was
then added (50 ml) and the mixture extracted with ethyl acetate (2
50 ml). The combined organic extracts were washed with water
(50 ml), dried over NaSO
, and concentrated under reduced
pressure. Recrystallization from EtOAc/hexane gave pure Ac-Gly phenacyl
acetate (1.6 g). Melting point: 119-121 °C;
H NMR
(CDCl
, 300 MHz)
: 7.88 (d, 2H), 7.61 (t, 1H), 7.48 (t,
2H), 5.42 (s, 2H, CO
CH
COPh), 4.88 (s,
2H, CONHCH
CO
), 4.17 (d, 2H,
CO
CH
CO
), 2.03 (s, 3H,
CH
CO).
Deprotection of the terminal carboxylic acid was
achieved by stirring the ester with zinc dust in glacial acetic acid
(20 ml) at room temperature, for 3 h(37) . The zinc dust was
filtered off. The filtrate was concentrated under reduced pressure and
dried under vacuum overnight to remove all traces of acetic acid. The
residue was then dissolved in water (50 ml) and washed twice with ethyl
acetate (50 ml). The aqueous layer was then concentrated, dried under
vacuum over PO
, and the residue recrystallized
from ethanol/hexane, giving N-Ac-Gly acetic acid ester (0.56
g). Melting point: 144-146 °C;
H NMR
(Me
SO-d
, 300 MHz)
: 4.57 (s, 2H,
NHCH
CO
), 3.89 (d, 2H,
CO
CH
CO
H), 1.85 (s, 3H,
CH
CO); mass spectroscopy (electron impact): m/z 175 (M
, molecular
ion).
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Figure 1:
The time courses of PAM-catalyzed
conversion of Ac-L-Phe-Gly and Ac-D-Phe-Gly.
Ac-L-Phe-Gly (100 µM) () and
Ac-D-Phe-Gly (100 µM) (
) were incubated
with the same amount of PAM, and the time-dependent conversions were
measured using C8 RP-HPLC. The mobile phase is 40% CH
CN,
60% H
O, 0.2% phosphoric acid. The standard curve of
Ac-L-Phe-
-hydroxy-Gly was used to calculate both the
concentration of Ac-L-Phe-
-hydroxy-Gly derived from
Ac-L-Phe-Gly, and the concentration of the new product derived
from Ac-D-Phe-Gly.
In contrast, as shown in Fig. 1, when
Ac-D-Phe-Gly is incubated with a large quantity of PAM,
product formation is barely detectable. Examination of HPLC traces at
high sensitivity revealed appearance of a very small amount, i.e. at most, 0.5% conversion, of product with retention time
corresponding to that of authentic Ac-L-Phe--hydroxy-Gly
within the first few minutes of incubation, with no further conversion
being detectable even after 20 h. Similarly, incubation of PAM with
either Ac-L-Phe-D-Phe-Gly or (R)-O-Ac-mandelyl-Gly results in conversions of less
than 0.2% and 0.9%, respectively. Control experiments confirmed that
PAM remains fully active after incubation with these substrates.
In
order to demonstrate directly that this S subsite
stereospecificity affects substrate binding, apparent K
values were determined for both
Ac-L-Phe-Gly and its non-reactive -D-Phe- enantiomer
using our standard assay substrate,
TNP-D-Tyr-L-Val-Gly. As is evident from the data in Table 1, there is a dramatic 700-fold difference between the
apparent K
values for the two Ac-Phe-Gly
enantiomers. Moreover, the true K
of
Ac-L-Phe-Gly is actually even lower than the value of 2
µM listed in the table, since some reaction of
Ac-L-Phe-Gly itself occurs during the initial rate
measurements. Although reaction of Ac-Phe-Gly is transparent to our
assay, it causes the measured K
to be higher than
the true value. The K
of 580 µM for
Ac-L-Phe-D-Phe-Gly is 480-fold higher than the K
for its L-Phe-L-Phe-diastereomer, and the same pattern is
true for (R)- and (S)-O-Ac-mandelyl-Gly.
Thus, it is clearly evident from these results that substrate
reactivity toward PAM is virtually abolished by the presence of a D-amino acid residue, or of the stereotopically equivalent (R)-mandelyl residue, at the P
position.
Figure 2:
The time courses of PGL-catalyzed
conversion of Ac-D-Phe--hydroxy-Gly and (R)-O-Ac-mandelyl-
-hydroxy-Gly to their amide
products. Ac-D-Phe-
-hydroxy-Gly (200 µM)
(
) and (R) O-Ac-mandelyl-
-hydroxy-Gly (200
µM) (
) were each incubated with 0.2 µg/ml
purified PGL at 37 °C, and the formation of the amide products were
measured using C8 RP-HPLC.
Synthetic preparations
of Ac-L-Phe--hydroxy-Gly and (S)-O-Ac-mandelyl-
-hydroxy-Gly actually consist
of equimolar mixtures of their respective (S,S) and (S,R) diastereomers; similarly, synthetic
Ac-D-Phe-
-hydroxy-Gly and (R)-O-Ac-mandelyl-
-hydroxy-Gly actually consist
of equimolar mixtures of the respective (R,S) and (R,R) diastereomers. Previously, we demonstrated that
PGL is stereospecific toward the terminal
-hydroxyglycine
moiety(29) , i.e. PGL reacts only with
-hydroxyglycine moieties of the (S) configuration.
Accordingly, if PGL exhibits the same S
subsite
stereospecificity as PAM, we would expect 50% conversion only for
Ac-L-Phe-
-hydroxy-Gly and (S)-O-Ac-mandelyl-
-hydroxy-Gly, while
Ac-D-Phe-
-hydroxy-Gly and (R)-O-Ac-mandelyl-
-hydroxy-Gly should have been unreactive toward PGL. Therefore, our finding that 50%
conversion actually occurs with all these substrates (Table 3) is
a strong indication that, unlike PAM, PGL reacts with substrates
possessing residues of either the (S) or (R)
configuration at the P
position.
In order to demonstrate
directly this simultaneous expression of high reaction stereospecificity and low Ssubsite stereospecificity in the course of PGL catalysis, the series of
HPLC experiments illustrated in Fig. 3was carried out. Panel A shows the HPLC elution profile of
Ac-L-Phe-
-hydroxy-Gly produced from Ac-L-Phe-Gly
by PAM. Since PAM catalysis produces only
-hydroxyglycines of the (S) configuration, this peak represents the retention time of
the (S,S) diastereomer of
Ac-L-Phe-
-hydroxy-Gly. Panels B and C show the elution profiles of synthetic
Ac-L-Phe-
-hydroxy-Gly and
Ac-D-Phe-
-hydroxy-Gly, respectively. It is evident from panels B and C that the two diastereomers of
synthetic Ac-L-Phe-
-hydroxy-Gly (with the configurations (S,R) and (S,S)) and the two
diastereomers of synthetic Ac-D-Phe-
-hydroxy-Gly (with
the configurations (R,S) and (R,R))
are well separated on a C8 RP-HPLC column using a mobile phase of 2%
CH
CN, 98% H
O, 0.2% H
PO
. Panels D and E show the elution profiles obtained
after reaction of Ac-L-Phe-
-hydroxy-Gly and
Ac-D-Phe-
-hydroxy-Gly, respectively, with PGL. It is
quite clear from panel D that reaction of PGL with synthetic
Ac-L-Phe-
-hydroxy-Gly results in conversion of only one
substrate diastereomer to Ac-L-Phe-NH
. Since the
reactive diastereomer has retention time identical to that of
Ac-L-Phe-
-hydroxy-Gly (with (S,S)
configuration) produced by PAM (Fig. 3, panel A), it is
clear that this reactive diastereomer has the (S,S)
configuration. Correspondingly, as shown in panel E, reaction
of PGL with synthetic Ac-D-Phe-
-hydroxy-Gly also results
in conversion of only one diastereomer of this substrate to
Ac-D-Phe-NH
. From a comparison with the previous
panels, it is quite clear that the reactive diastereomer of this
substrate has the (R,S) configuration. Taken
together, these experiments unequivocally confirm the sharp contrast
between the reaction and the subsite stereospecificities of PGL; while PGL is reactive only toward an
-hydroxyglycine moiety of the (S)
configuration, this lyase reacts readily with substrate diastereomers
of either the (S) or (R) configuration at the
P
position.
Figure 3:
Simultaneous expression of high reaction
stereospecificity and low S subsite stereospecificity in
PGL catalysis. The two diasteromers of synthetic
Ac-L-Phe-
-hydroxy-Gly and the two diasteromers of
synthetic Ac-D-Phe-
-hydroxy-Gly were separated on a C8
RP-HPLC with a mobile phase of 2% CH
CN, 98% H
O,
0.2% H
PO
. Panel A, HPLC chromatogram
of Ac-L-Phe-
-hydroxy-Gly produced by PAM. Since PAM only
produces the peptidyl-
-hydroxyGlycine derivatives with the S configuration at the
-hydroxyglycine moiety, the peak at 46.5
min has the configuration (S,S). The small peak at 51
min is Ac-L-Phe-NH
. Panel B, HPLC
chromatogram of synthetic Ac-L-Phe-
-hydroxy-Gly. By
comparison with panel A, it is evident that the two
diastereomers with retention times of 42 and 46.5 min, respectively,
have the configurations (S,R) and (S,S). Panel C, HPLC chromatogram of
synthetic Ac-D-Phe-
-hydroxy-Gly. By comparison with panel A, it is evident that the two diastereomers with
retention times of 42 and 46.5 min, respectively, have the
configurations (R,S) and (R,R). The
small peak at 51 min is Ac-D-Phe-NH
. Panel
D, HPLC chromatogram of the sample obtained after PGL-catalyzed
conversion of Ac-L-Phe-
-hydroxy-Gly to
Ac-L-Phe-NH
. This panel demonstrates that only the
diastereomer of the (S,S) configuration was converted
to Ac-L-Phe-NH
. Panel E, HPLC
chromatogram of the sample obtained after PGL-catalyzed conversion of
Ac-D-Phe-
-hydroxy-Gly to
Ac-D-Phe-NH
. This panel demonstrates that only the
diastereomer of the (R,S) configuration was converted
to Ac-D-Phe-NH
.
Kinetic parameters for the two pairs of
diastereomeric PGL substrates are listed in Table 4. It is
apparent that the V values are virtually
unaffected by whether the residue at the P
position has the (S) or (R) configuration, whereas small effects are
apparent on the K
values. Both pairs of compounds
are comparable in reactivity to N-benzoyl-
-hydroxy-Gly.
Both (R)-2-phenylbutyl--hydroxy-Gly and (S)-2-phenylbutyl-
-hydroxy-Gly were found to indeed be
PGL inhibitors. We also observed that they both undergo very slow
enzymatic conversion to the corresponding phenylbutyramides (as
confirmed by HPLC analysis). The substrate activity (V/K) of these compounds is
10,000 times slower
than that of our normal PGL assay substrate,
TNP-D-Tyr-L-Val-
-hydroxy-Gly; therefore,
inhibition kinetics are readily performed in the usual manner. Both
compounds are highly potent competitive inhibitors, and the K
values obtained are 280 µM and 310
µM for (R)-2-phenylbutyl-
-OH-Gly and (S)-2-phenylbutyl-
-OH-Gly, respectively (Table 5).
It is thus clear that, in sharp contrast to PAM, the configuration of
the chiral moiety at the P
position has no effect on the
potency of these PGL inhibitors.
Table 5also lists the
kinetic constants measured for the very slow PGL-catalyzed reaction of
these two compounds. As expected, V and K
are identical for (R)- and (S)-2-phenylbutyl-
-OH-Gly. Moreover, the measured K
values are identical to the K
values obtained in the inhibition experiments against
TNP-D-Tyr-L-Val-
-hydroxy-Gly, thus confirming
that the K
values represent true binding
constants.
Taken together, the results reported here establish that
despite their tandem reaction stereospecificities with respect to the
COOH-terminal (S)--hydroxyglycine moiety, PAM and PGL
exhibit sharply contrasting S
subsite stereospecificities
in the binding of both substrates and inhibitors. Physiologically,
these two enzymatic steps must occur sequentially in order to convert a
given glycine-extended pro-peptide to the corresponding mature amidated
peptide(9, 11) . Thus, from a metabolic viewpoint, the
stereospecificities of these catalytic domains would have been expected
to correlate. From a mechanistic perspective, there is, of course, a
vast difference between the catalytic site of a metallo-monooxygenase
on the one hand, which entails participation of both active-site copper
and an electron donor in catalysis, and that of a lyase on the other.
Indeed, the contrasting stereospecificities we report here are likely
reflections of corresponding differences in the active site
topographies of PAM and PGL.
To our knowledge, this is the first demonstration of a case where catalytic domains that must function sequentially nevertheless exhibit contrasting binding stereospecificities. Most likely, the possibility that stereospecificities of such domains might differ has not been addressed since, of course, metabolites flow through the catalytic domains in sequence; a given domain would never ``see'' a metabolite of altered chirality such that it would have been precluded from binding at the preceding domain. Thus, inspection of the stereochemical configurations of metabolic intermediates does not reveal possible differences in the subsite stereospecificities of the domains; stereochemical studies using substrate analogs and inhibitors of deliberately altered chirality are required for this purpose. For example, in the case of chorismate mutase-prephenate dehydrogenase, a multifunctional protein catalyzing sequential reactions that has been the subject of much recent interest(41, 42, 43) , the relative stereochemistry at the ring position para to the site of enzymatic reaction clearly affects reactivity of substrate analogs in the dehydrogenase reaction. However, a comparison of the effect of commensurate changes in subsite stereochemistry on binding and turnover for the individual catalytic domains has not been carried out, and, unfortunately, synthetic approaches to the synthesis of individual substrate analog stereoisomers that might be used in such investigations have been unsuccessful(41, 42, 43) .