(Received for publication, March 8, 1995; and in revised form, May 25, 1995)
From the
The velocity of enzymic cleavage of 4-substituted
picolinylprolines by swine kidney prolidase approaches that of
physiological dipeptides, but depends substantially upon the nature of
the pyridine-ring substituent. The pH dependence of k
Prolidase is a widely occurring mammalian dipeptidase that
normally splits aminoacylprolines into the constituent amino acids.
Apparently the enzyme is required because many other catabolic enzymes
are unable to cleave the RCO-proline tertiary carboxamide
linkage(1) , so that C-terminal proline-containing dipeptides
build to toxic concentrations in individuals identified with a
genetically caused deficiency of prolidase(2, 3) .
Previous studies of metalloprotease mechanisms from this laboratory (4, 5, 6, 7) have included an
initiatory examination of catalytic specificity and inhibition for
prolidase(8, 9) . We now extend kinetic analysis to
picolinylprolines, a new class of substrate analogues that affords
unique insight into the catalytic mechanism. Prolidase from a number
of sources has been shown to exist in solution as a dimer of 55-kDa
subunits, each of which consists of an identical single
chain(10, 11, 12, 13, 14, 15) .
A crystallographic structure for prolidase is unavailable, but the
human enzyme has been sequenced(16, 17) . Recently,
apparent sequence homology has been demonstrated with Escherichia
coli methionine aminopeptidase, a metalloenzyme of established
three-dimensional structure, as well as with aminopeptidase P, which
also preferentially hydrolyzes an acylproline
linkage(18, 19, 20) . Because these enzymes
have a similar function and specificity (namely, for cleavage of the
N-terminal residue from a peptide), it is reasonable to infer that they
share a common mechanism, which may be general for aminopeptidases. The related and structurally solved methionine aminopeptidase from E. coli differs from the better-characterized metalloproteases
in that it contains a pair of closely linked metal ions at the active
site (reportedly Co Prolidase has long been recognized as a metallopeptidase. Because
its standard isolation procedure specifies incorporation of aqueous
Mn
Ordinarily, prolidase behaves as an obligatory
aminopeptidase. As earlier shown, the prototypical substrate
glycylproline is cleaved readily, but acetylproline is hydrolyzed
>10
Figure 1:
Structures of substrate analogues for
prolidase. Proline residues are all of L-configuration.
Figure 2:
Plots of k
The same explanation
holds for glycylproline as well. In the bell-shaped pH profile for that
substrate, the alkaline limb inflection (pK The alternative supposition, that the N-cationic form of substrate (e.g. glycyl-ammonium ion in GlyPro) is the catalytically
active species, breaks down in particular for 1-CN, which is one
of the more competent prolidase substrates in spite of yielding a
negligibly small amount of pyridinium ion in the pH region of enzymic
activity (estimated pK Within the strictures provided by the pH
profiles (Fig. 2), there evidently is a profound dependence of
catalytic velocity upon the nature of the 4-substituent within the
picolinyl moiety, with the less basic pyridines yielding the superior
substrates. The overall dependence upon nitrogen basicity seems greater
than can be explained by the relative nucleophile susceptibilities of
the carbonyl groups within the substrates, as was estimated by
hydrazinolysis. Values for limiting k
Figure 3:
Plot
of Zn
Figure 4:
Plot of k
The cognate pH profiles for K
Fig. S1summarizes
the initial stages of the enzymic mechanism that we consider to be
indicated by the data. Step k
Figure S1:
Scheme 1.
Figure 5:
Plot of (k
The foregoing qualitative
explanation for the distribution of the data in Fig. 5is given
quantitative expression in the curve fit shown. Because the velocity of
both steps should depend (in opposite directions) upon substrate
basicity, the Brformalism is introduced for each: k The interpretation of
The
purpose of carrying out the designated fit is so that the derived rate
constants may then be plotted against the pyridine
pK
Figure 6:
Plot of log (k
It is relevant that the anions of glycine and picolinic acid possess
an identical chelative affinity for Zn
Figure S2:
Scheme 2.
That problem is obviated in the second variant
proposed (path b). Here the metallic hydroxide adds across the
scissile linkage, without ever dissociating from the metal ion. The
resulting tetrahedral adduct is superbly set up to expel proline
spontaneously, leaving behind a carboxylate derived from the N-terminal
substrate moiety, which emerges from the catalytic process immediately
bridging both metal ions. In this scheme two Lewis acids are brought
into action concurrently to facilitate cleavage of the amide linkage,
and the Lewis acidity-robbing influence of the substrate's
coordinating nitrogen upon the first metal ion is compensated by the
presence of the second. The hypothesized addition of H-O(metal)
across the activated substrate carboxamide in this latter mechanism
might benefit from an auxiliary base to function as acceptor for the
tacitly required proton transfer, but such a group could be unnecessary
should this process, unaided, normally be faster by itself than are
earlier steps in the sequence (k More probable
is that both metal ions have similar intrinsic Lewis acidity but that
the ionizations of their ligated water molecules are coupled (mutually
perturbed) due to proximity. In this interpretation the
pK As regards subsequent steps
of the proposed mechanism (Fig. S2, path b), the
bridging tetrahedral intermediate incorporating two acidic metal ions,
both functioning so as to accept the transient oxyanionic charge which
builds at the reaction center in the course of reaction, appears
capable of accommodating the electronic and geometric transition state
demands especially well. This mechanism perhaps acquires greater
acceptability if one examines it in the reverse direction, i.e. for amide bond synthesis. Since in principle prolidase must
catalyze its reaction in either direction, the chemical plausibility of
amine addition to such a doubly activated carboxylate group as produced
in this scheme supports transition state stabilization in both
directions.
In a model-building exercise, the tetrahedral adduct
from Fig. S2(path b) has been fitted into this
hypothetical active site for prolidase, by allowing the usual doubly
H-bonded motif of ion-pairing between the cited arginine guanidinium
ion (position 233) and the carboxylate of picolinylproline, plus
chelation of the N terminus of the substrate with one metal ion
(designated M2), and with simultaneous bridging of the other oxyanion
of the tetrahedral adduct to the second metal ion (M1) in the active
site. The resulting structure, which has required no molecular
distortions to assemble, is stereo depicted in Fig. 7. No
unreasonable intermolecular contacts result. The pyridine nitrogen of
substrate appears to compete with Asp
Figure 7:
Model for picolinylproline tetrahedral
reaction intermediate (bold), docked into the active site of
prolidase, as recovered from the structure of the sequentially
homologous metalloprotein methionine aminopeptidase. Residues are
numbered as for the latter enzyme. Substrate carboxylate is shown
salt-linked to Arg
It is concluded that the mechanism of Fig. S2(path b) is compatible with the available
structural information for the enzyme. Fig. 7might also suit
the alternative formulation (path a), if it is taken as an
approximation of the transition state for transfer of
HO
/K
for
picolinylproline is sigmoidal, with optimum activity on the acidic limb
and a delimiting enzymic pK
of 6.6,
unlike glycylproline (bell-shaped pH profile, maximum at pH 7.7).
Productive chelation to an active site metal ion by the N terminus of
substrates is indicated, with a water molecule ligated to that
hyper(Lewis)acidic center prior to substrate binding supplying the
pK
of 6.6. The rate-governing catalytic
step differs according to the 4-substituent on the picolinyl residue;
productive binding is slow in the case of electron-withdrawing groups,
but subsequent nucleophilic addition to the metal ion-activated
scissile linkage becomes controlling with more basic pyridine rings.
Rate constants yield a Br-type correlation with substrate
pK
, providing a gauge of active-site
Lewis acidity. A mechanism is suggested involving the cooperative
participation of two especially acidic metal ions positioned adjacently
within the active site (situated as in an homologous and structurally
characterized aminopeptidase), with both serving to stabilize a
bridging carboxamide-hydrate intermediate.
for optimum activity), rather
than a single coordinatively bound cationic center. This binary-metal
characteristic, employing Zn
, is shared by leucine
aminopeptidase (which has an unrelated chain-fold conformation,
however)(21, 22, 23) . An aminopeptidase from Aeromonas proteolytica has also been solved, and it conforms
to the pattern of dual metal ions at the active site(24) .
Therefore, the presently uncertain chemical mechanism of these
aminopeptidases must involve unique features relative to such
lone-metal enzymes as carboxypeptidase A or thermolysin(25) .
(at much higher than physiological
concentrations), that element has been thought to be involved in the
enzyme mechanism. However, our investigations have suggested that this
particular metal ion is not of great importance, and while it seems to
have a stabilizing role for some enzyme preparations, there is no
direct evidence that manganese rather than the more common
Zn
, for example, is an obligatory active site
component. (We find no advantage to the standard post-isolation
Mn
incubation in our current enzyme procurement, and
do not use it). Because various divalent transition metal ions
possessing similar chemical properties are functionally interchangeable
in other
metalloproteases(26, 27, 28, 29, 30) ,
atomic identity may be regarded as a less essential point compared to
the broader question of mechanistic role that is here addressed by
structure-activity relationships among substrates.
Enzyme
Prolidase (EC 3.4.13.9) was isolated from
fresh swine kidneys by a modification of the procedure of Manao et
al.(10) . The steps involve (i) formation of an acetone
powder from the filtrate of homogenized kidneys, (ii) ammonium sulfate
and acetone fractionation followed by lyophilization, and (iii) final
chromatographic purification. The first two steps were carried out as
described previously, except that ethanolic phenylmethanesulfonyl
fluoride (150 mg/liter) was incorporated in the homogenization mixture
as a serine protease inhibitor. Without this precaution the final
55-kDa enzyme obtained was found by SDS-polyacrylamide gel
electrophoresis to be contaminated with proteins of M of approximately 20,000 and 35,000. A commercially obtained
specimen of prolidase was found to consist mostly of the latter
mixture. The apparent chain nicking does not have observable kinetic
consequences except at the low-pH end of the catalytic efficacy range,
where the evidently fragmented enzyme loses activity that the protected
material does not. Also, the intact enzyme was found not to benefit
kinetically or in terms of stability from the aqueous Mn
incubation procedure that hitherto has been stipulated for
prolidase, and consequently that ``activation'' was not
applied by us. Step iii, chromatographic purification, was in our case
carried out by high performance liquid chromatography with a 15-cm
polymeric hydroxyethyl methacrylate, macroporous, amine-type
ion-exchange column (Alltech, packing: HEMA-IEC BIO 1000 DEAE 10U),
employing an eluent of 0.05 M Tris, 0.4 N NaCl, pH of
7.9, and with monitoring at 280 nm. After pressure-induced dialytic
concentration of the dipeptidolytically active fraction, this gave high
activity prolidase as a single band with M
of
55,000 according to SDS-polyacrylamide gel electrophoresis, and with M
of approximately 110,000 (dimer) when the
electrophoresis was carried out without protein denaturation. Crude
(unchromatographed) enzyme gave with some substrates K
values approximately twice that
obtained with purified enzyme, suggesting adventitious inhibition, but
with otherwise similar kinetic behavior. The crude preparation was used
for estimating rates with some very slowly cleaved substrates, where
adequate amounts of purified prolidase could not be secured for the
purpose.
Substrates
Picolinylprolines were generally
prepared by coupling the appropriately substituted picolinic acids to L-proline methyl ester with 1,3-dicyclohexylcarbodiimide, with
subsequent saponification. Structurally confirmatory NMR (H
400 MHz,
C 100 MHz), IR (
)spectra, and
elemental analyses (C, H, N) were obtained for the following:
picolinylproline, 1-H, mp 169-170
°C(31) ; nicotinylproline, 2, mp
176-177 °C(32) ; isonicotinylproline, 3, mp 230-231 °C(33) ;
4-cyanopicolinylproline, from ethyl 4-cyanopicolinate (34) via the acyl hydrazide and azide, 1-CN, mp
156-157 °C; 4-chloropicolinylproline, from
4-chloropicolinic acid (35) , 1-Cl, mp 119.5-121
°C; 4-methylpicolinylproline, from 4-methylpicolinic
acid(36) , 1-CH
, mp >125 °C (dec),
dicyclohexylamine salt mp 158-160 °C;
4-methoxypicolinylproline, from 4-methoxypicolinic acid (37) ; 1-OCH
, mp >125 °C (dec),
dicyclohexylamine salt mp 158-160 °C;
4-(dimethylamino)picolinylproline, from
4-(dimethylamino)picolinic acid(38) , 1-N(CH
)
, mp >125 °C (dec),
dicyclohexylamine salt mp 154-155 °C;
4-(N,O-dimethylhydroxylamino)picolinylproline, via 4-chloropicolinic acid and N,O-dimethylhydroxylamine (MeOH, 25 °C, 3 days)
giving 4-(N,O-dimethylhydroxylamino)picolinic acid,
mp 139-141 °C, subsequently yielding 1-N(CH
)OCH
, mp >100 °C (dec),
dicyclohexylamine salt mp 133-134 °C;
4-(N,N-(trifluoroethyl)methylamino)picolinylproline, via N,N-(trifluoroethyl)methylamine p-toluenesulfonate (mp 113-115 °C, from
CF
CH
OTs and MeNH
, 160 °C, 3
days) and 4-chloropicolinic acid (H
O, KOH, 160 °C, 24
h) was obtained
4-(N,N-(trifluoroethyl)methylamino)picolinic acid (as p-toluenesulfonate) mp 130-135 °C, and thence 1-N(CH
)CH
CF
,
dicyclohexylamine salt mp 155-156 °C. Picolinic acid esters were obtained by a Fischer esterification procedure (EtOH,
HCl, 2 h reflux, subsequent H
O-K
CO
neutralization).
Kinetic Analysis
Enzymic hydrolyses were followed
at 25.0 (± 0.1) °C by continuous spectrophotometric
analysis, following the decrease in light absorption at a suitable
wavelength in the range 230-240 nm (region of significant
pyridine-ring absorption) subsequent to addition of 1-20 µl
of purified enzyme to 2.5 ml of buffered substrate solution. Relative
rates (k/K
) are in
every case expressed as a fraction of the limiting maximum activity for
glycylproline substrate similarly determined. Estimated enzyme
concentrations are typically in the micro- to nanomolar range. Buffers
employed were MES, pH of 5.3-6.9, PIPES, pH of 7.1-7.7,
CHES, pH of
8.1 (all shown not to perturb kinetics). The method of
initial rates was employed with a computer-assisted line fitting
procedure; kinetic parameters were secured from a direct fit of v/[E] to the Michaelis-Menten
equation using substrate concentrations spanning at least two points
with values below K
and two above.
Catalysis was shown to be first-order in enzyme at the concentrations
employed. In all kinetic runs v/[E] was
adjusted so as to avoid complications from slow cis-trans isomerization in the substrate(39, 40) .
Hydrazinolysis kinetics for ethyl picolinates were carried out
similarly, except that rate constants were obtained by exponential
curve fitting to the complete pseudo-first-order kinetic runs. Enzymic
cleavage products were detected by thin layer chromatographic analysis,
with comparison to authentic materials.
Controls
Chelative metal ion dissociation
constants for 1 and for the corresponding picolinic acids were
secured by spectrophotometric titration in aqueous solution. At a fixed
pH (varying from 2 to 6.5, depending on substrate), a 0.1 mM solution of the picolinate was allowed to coordinate with varying
concentrations of metal ion, Mn or
Zn
. The family of resulting spectra commonly
exhibited perturbations having an isosbestic point, and plotting of
neighboring absorption readings versus log
[M
] yielded sigmoidal curves from which the
extent of chelation could be quantified. Dissociation constants, K
, were obtained by fitting absorptions
to the expression A - A
=
A/{1 + (K
/[M
])
(1
+
[H
]/K
)},
where K
is an acid dissociation constant
for competing nitrogen protonation previously determined. All pH values
in this article are calibrated pH meter readings uncorrected for ionic
strength effects. Tolerances listed are standard errors from
least-squares analysis.
-fold more slowly, even though the latter
NH
-abridged analogue binds comparably as well when employed
as an inhibitor of the enzyme (with allowance for pH
effects)(8, 9) . N-Acylglycylprolines also
are not substrates(41) . Contemplation of the enzyme mechanism
prompted us to attempt amide-bond cleavage by prolidase of a dipeptide
surrogate, picolinylproline. It proved to be an excellent substrate,
with spectral properties providing a convenient continuous
spectrophotometric kinetic analysis. That has led to a systematic
investigation of the potential prolidase substrates depicted in Fig. 1.
Specificity
Although picolinylproline (1-H) is
cleaved comparably rapidly as the normal substrate glycylproline,
isomers nicotinylproline (2) and isonicotinylproline (3)
were not detectably hydrolyzed by swine-kidney prolidase. This result,
in conjunction with the inertness of acetylproline and other
specificity studies previously reported(8) , led to a
conjecture that productive binding of substrates to prolidase requires
a chelative interaction between an active site metal ion and the N
terminus of the substrate. Specifically, coordination is postulated to
involve the oxygen atom of the scissile carboxamide together with an
unprotonated amino group (of the glycyl residue) or the heteroaromatic
nitrogen (of the picolinyl residue), with both carbonyl and nitrogen
functionality simultaneously interacting electrodatively with an active
site Lewis acid. The pH dependence of enzymic catalysis in the case of
physiological dipeptide substrates has been shown to be compatible with
such an interpretation(8) . Because that interaction should
respond in an informative manner to variation in basicity within the
pyridine nitrogen in 1, the series of 4-substituted
picolinylprolines shown in Fig. 1was prepared for submission to
prolidase.Substrate Basicity
The proton affinity of a
pyridine nitrogen depends strongly upon the electron-donating or
electron-withdrawing properties of ring substituents that are situated para to it. This is demonstrated by the pK values from ring protonation of the various members of the
substrate series having structure 1, which as listed in Table 1cover a range of >8 pK units. These are microscopic pK
values,
specifically for protonation on nitrogen, inasmuch as they have been
obtained by
C NMR spectroscopy. The method involves
preparation of an aqueous solution (containing
5% D
O)
of the picolinylproline derivative, and recording of the chemical shift
of the methine carbon ortho to the aromatic ring nitrogen for
various pH values. When
C is plotted against pH, a
sigmoidal curve results, from which the pK
value can be ascertained analytically. Although the
substrate carboxyl group ionizes concurrently in some cases (and may
also be assigned a pK
value from its
chemical shift dependence), that causes no interference with
measurement of pyridine basicity by this technique.
Substrate Nucleophilic Susceptibility
Because a
substantial dependence upon the nature of the ring substituent is noted
in the enzymic rate of amide bond scission for various representatives
of 1, it became desirable to examine independently the intrinsic
acyl-group reactivities of such pyridinecarboxylic acid derivatives.
The innate influence of the substituent upon addition to the picolinyl
C=O group was weighed by measuring the relative rates of
nonenzymic hydrazinolysis of ethyl esters of the picolinic acids
utilized in this investigation. Under the conditions employed, the
acylhydrazide-forming reaction of esters ordinarily is second-order in
NH
, and the rate-limiting step is thought to be
attack of nucleophile on the carbonyl group, with general base
assistance by a second hydrazine molecule(42) . Second-order
dependence on hydrazine was confirmed for the ethyl picolinates. As
anticipated because the reacting carbonyl group is cross-conjugated
with respect to the substituent borne by the pyridine ring, the latter
was found to have only a small effect upon the velocity of the acyl
substitution reaction in the case of the esters. Respective
pseudo-first-order rate constants for a hydrazine concentration of 1 M are listed in Table 1. When log k
is plotted versus pK
values for
ring-nitrogen protonation of the corresponding picolinic acid anions
(with pK
values measured as for the
substrates), a linear dependence is seen, with a Brcoefficient slope of -0.06 (±0.03). Exactly the same
coefficient is obtained when the hydrazinolysis rate constants for the
esters are plotted logarithmically against the
pK
values for 1. From the low
absolute magnitude of this parameter (reflecting the similarity of
rates), there appears to be relatively little direct interaction
between the ring substituent and the reaction center in the course of
acyl transfer.
Enzyme Activity, [S] K
The
kinetics of picolinylproline hydrolysis by prolidase show a different
pH profile than observed for the normal substrate glycylproline.
Illustrative data are presented in Fig. 2for the specificity
constant k/K
(the
second-order rate constant for enzymic amide hydrolysis at low
substrate concentration). This kinetic parameter is particularly
valuable in identifying functionality involved in the enzyme mechanism,
for in most instances pH-induced perturbations of the so-measured
catalytic activity yield true pK
values
of the groups responsible, either for binding of substrate or for
participation within the first committed step of the catalytic cycle.
The dependence for glycylproline is bell-shaped, with peak activity
near neutral pH and with delimiting pK
values of 6.6 and 8.8 (Fig. 2, inset).
However, that for 1-H is merely sigmoidal with the maximum in
acidic solution and with the single governing pK
having a value of
6.6, matching the acidic-limb
pK
for glycylproline, but with an
opposite inflection. Similar behavior was observed previously and has
been explained(8) . The key realization is that an essential
enzymic functionality with a pK
value of
6.6 exists within the active site of prolidase and that it controls
binding of substrates absolutely, according to the pH profiles.
Obviously from the data that group must adopt its conjugate acid form
for productive association and catalytic conversion of chelating
substrates such as picolinylproline. Because the control exerted is
absolute (the enzymic rate drops to zero at high pH), the most
plausible interpretation would seem to be that a water molecule that
binds to an active site metal ion in the absence of substrate has
become acidified to this pK
value by the
enzyme(5, 8) . While pyridine ligands are capable of
displacing H
O from a metal ion, they cannot compete as
coordinating agents with HO
.
/K
(activity relative to
glycylproline) versus pH for 4-substituted picolinylprolines.
Symbol code (pyridine substituents) is indicated on diagram; data for
1-Me, and 1-N(CH
)CH
CF
are not
shown, but are similar to that for other picolinylprolines of
comparable pK
. Limiting values for k
/K
on acidic limb
are recorded in Table 1, relative to the value for glycylproline
at its pH optimum, after correction for substrate protonation as
necessary (i.e.dashed line extrapolation in the case
of 1-N(CH
)OCH
). The inset contains a
similar pH profile for glycylproline (data from (8) ). The
equations for the fitted lines are (k
/K
)
= (k
/K
)
/(1
+ K
/[H
]) for the
sigmoidal curves, and (k
/K
)
= (k
/K
)
/{(1
+ K
/[H
])(1
+
[H
]/K
`)}
for the bell-shaped curves (K
for enzyme
and K
` for substrate nitrogen, but with
assignments reversed for glycylproline ( Table 1and
text)).
8.8) must be attributed to the glycyl NH
group within the substrate. This has been experimentally
confirmed previously by an engendering of small offsets in the
substrate's titrimetric pK
value,
as purposely introduced by N-substituents on the glycyl residue, with
those pK
perturbations detected also in
the catalytic kinetics(8) . While at first glance it might
appear from the pH profile that the substrate ammonium ion is the
species bound in the case of glycylproline, that is an illusion.
Binding of the conjugate base form of the substrate (unprotonated
amine) to the conjugate acid form of the enzyme (as for
picolinylproline) produces exactly the same pH dependence as if the
protonation states were reversed. The validity of this unitary
explanation for all kinds of substrates for prolidase is confirmed by 1-N(CH
)OCH
and 1-N(CH
)CH
CF
. For these
picolinyl substrates the pyridine-ring pK
value falls within the window of pH values that can be examined
kinetically. They show the bell-shaped pH profile of dipeptide
substrates, with delimiting pK
values
congruent with that of enzyme (alkaline limb pK
of
7) and of the picolinyl ring (acidic limb
pK
of 5.6-5.8). Substrate 1-N(CH
)
was hydrolyzed too slowly by
prolidase for measurement of an adequate pH profile, but qualitative
observations suggest that it conforms to the pattern of the other
picolinylprolines, and a corresponding relative rate constant could be
estimated from the Michaelis profile at a single intermediate pH.
-1.6).
Furthermore, any conjectured preferential enzymic binding of pyridinium
ions from 1 seems incompatible with the substantial rate
dependence upon hetero-ring basicity seen in Fig. 2, inasmuch as
all such substrates in their protonated form should be equivalently
cationic as regards ion pairing within the active site. Our conclusion
that observed k
/K
perturbations as a function of pH are connected with productive
substrate ligation to a metal ion, rather than being the manifestation
of a subsequent kinetic step in the catalytic mechanism, is
corroborated by prolidase inhibition studies, wherein a cognate pattern
has been seen for enzymic equilibrium association of catalytically
inert substrate analogues that are also metal ion
interacting(9) .
/K
rate constants
on the acidic limb (corrected for substrate protonation as necessary)
are listed in Table 1, together with the enzymic
pK
value (
6.6) obtained also by
curve fitting in each case. The pattern of kinetic behavior (dependence
upon pH and substituent) provides the basis for formulation of a
detailed catalytic mechanism, as is subsequently considered under
``Discussion.''
Picolinate pK
Because of the preceding evidence indicating that
chelation involving the picolinyl residue is a obligatory feature of
productive substrate binding, it became necessary to acquire
independent substantiation as to how that might depend upon pyridine
basicity. Dissociation constants (practical values) for the
complexation of aqueous Zn and Intrinsic Metal Ion
Affinity
with various 4-substituted
picolinic acids were obtained by spectrophotometric analysis. Some
results are summarized in Fig. 3, wherein a satisfactory linear
free energy relationship may be noted between nitrogen basicity
(pK
value for uncomplexed ligand) and
zinc ion affinity (also expressed logarithmically,
pK
). The slope of the fitted line is 0.31
(±0.02), registering a modest-but-positive correlation
(designated
) between proton affinity and that for
the divalent transition metal ion. A similar dissection was carried out
for chelation of the same picolinates with aqueous
Mn
, as well as for the picolinylproline substrates 1 with Zn
. In these cases coordination is
several orders of magnitude weaker overall, but an essentially
identical correlation coefficient was found. The connection between
chelative metal ion affinity for picolinates and enzyme catalytic
activity will also figure in the subsequent Discussion.
affinity (negative logarithm of dissociation
constant) versus pK
for
4-substituted picolinic acids. Values for pK
(±0.07) were obtained from spectrophotometric
perturbations in the presence of variable amounts of excess zinc ion in
moderately acidic solution, with correction for competition by protons,
but uncorrected for activity coefficient of Zn
. Slope
of least-squares regression line corresponds to
(``Discussion'').
Enzyme Activity, [S] K
The
pH profiles for k, the first-order rate constant
for the enzymic reaction with saturating amounts of substrate, are
presented in Fig. 4. The overall pattern is complex, but at
least for some substrates (filled symbols: 1-H, 1-N(CH
)OCH
, 1-N(CH
)OCH
CF
, 1-N(CH
)
) the pH dependence may be fitted
with a simple sigmoidal function, yielding a limiting value for k
on the alkaline limb, and an acidic-limb
pK
value of 5.4-6.4 in each case.
The more active substrates seemingly show a bell-shaped profile (with
some ambiguity, e.g.1-Cl). They display a comparable
acid dependence at low pH, which consequently appears to be common to
all picolinylprolines, but they exhibit an additional extinction of
catalytic activity on the alkaline limb, corresponding to that noted in k
/K
. Curves have
been drawn consonant with that interpretation, and appropriate (k
)
values (directly measured, or
corrected by extrapolation for decreases on the alkaline limb) are
recorded in Table 1, along with fitted pK
values for the cleanly sigmoidal cases. The most plausible
explanation for the transformation of line shape within the observed
pattern of activity is a substituent-engendered change in rate-limiting
step within the E
S complex, but a fuller accounting is
deferred under ``Discussion.''
versus pH
for substrates 1. Symbol code is indicated on diagram; data for 1-OMe
is not shown, but is similar to that for 1-Me. The inset contains pH profiles with an expanded scale for poorer substrates.
Limiting values for k
on alkaline limb are
recorded in Table 1, extrapolating where necessary, and scaled
according to the specificity constant
K
. The equation for the sigmoidal fit is (k
)
= (k
)
/(1 +
[H
]/K
), and
for the bell-shaped curves an equivalent double sigmoid as described in
the text was used.
are not shown. Data within such plots
exhibit considerable variability as a function of substituent and pH
(with the electron-rich pyridine species apparently binding more
tightly), but in actuality K
values are
merely a ratio of the primary kinetic parameters k
and k
/K
, so
that no independent mechanistic evidence is contained therein. For
comparison, the K
value for
picolinylproline (1-H) at pH 5.4 is 0.3 mM; that for
glycylproline at pH 7.6 is 0.7 mM(8) (experimental K
value in each case for the pH
corresponding to optimum specificity constant).
Substrate Binding and Activation
The preeminent
conclusion to be drawn from this and previous kinetic examinations of
prolidase (8, 9) is that productive binding to the
enzyme involves a chelative interaction between the substrate N
terminus and an active site metal ion. ()In addition to
conferring specificity, that interaction presumably activates the
scissile amide linkage toward nucleophilic addition due to Lewis acid
character on the part of the implicated metal ion, a faculty that would
serve directly to polarize the carbonyl group within the substrate. One
measure of that metal ion's electron deficiency is the
distinctive pK
(value of 6.6) of the
water molecule which occupies the metallic ligation site in the absence
of substrate. While such proton acidity might initially seem
surprising, it is in line with that recently shown for ligated
H
O in carboxypeptidase A and in
thermolysin(5, 6) . The apparent pK
offset from higher values commonly observed for model
hydrated divalent transition metal ions in aqueous solution is
explained by evolutionary selection favoring optimum enzyme activity,
which has promoted the greatest Lewis acidity effectively attainable
within the substrate-activating metal ion. Chemically, a metalloprotein
can realize such acidity by restrictions on the number of electrodative
ligands holding M
to the active site, and perhaps by
the juxtaposition of two such dicationic centers, as occurs within
certain bimetallo-aminopeptidases to which prolidase seems to be
related. The original objective of this study was to assay the
importance of active site Lewis acidity with respect to catalytic rate
by probing the properties of the substrate-engaged metal ion with the
aid of the series of dipeptide analogues of structure 1, of
varying pyridine basicity. We believe that this goal has been achieved,
and as a bonus a detailed mechanistic insight into the complex kinetic
behavior of the substrates with prolidase has emerged. An explanation
of the relative rates of cleavage for picolinylprolines of disparate
basicity, in terms of sequential steps within the peptide hydrolysis
mechanism, needs to be considered first.
/k
represents productive substrate binding in which H
O
becomes displaced from an active site metal ion
(L
M
). The actual water
substitution reaction may involve a prior recognitional association
between enzyme and substrate (i.e. an encounter complex).
Deprotonation of the indicated enzymic H
O molecule
(pK
of 6.6) accounts for the sigmoidal pH
dependence in k
/K
exhibited in Fig. 2, inasmuch as HO
cannot be displaced. Competitive protonation of the
substrate's chelating nitrogen (when it is sufficiently basic to
appear in the pH profile) likewise prevents coordination, explaining
the bell-shaped contour shown in that figure for 1-N(CH
)OCH
, and for normal substrates
such as glycylproline(8) . Step k
represents an ensuing addition to the activated substrate by a
nucleophile (:Nu), the chemical nature of which will be considered
subsequently. The tetrahedral adduct is almost certainly a transient
species, and it collapses so as to release proline (concurrently with,
or subsequent to N-protonation, which facilitates its departure).
Quantitative Evaluation
Structure-activity
correlations support and flesh out this interpretation. In Fig. 5is shown a replot of (k/K
)
values, the maximum specific activities secured from Fig. 2as listed in Table 1, versus the
pK
values for N-protonation of the
various pyridine-containing substrates. Catalytic activity peaks for
derivatized picolinylamides occupying the middle of the plot and tails
off on both limbs, very much so for the more basic members of the
series. The optimal substrate analogue by the usual criterion of
specificity constant is chloropicolinylproline (1-Cl). We
suggest that the most plausible explanation for this pattern of
behavior is a change in rate-limiting step for catalysis, occurring
across the series of substrates. Those substrates with a highly basic
pyridine residue are hydrolyzed very poorly by prolidase, in spite of
the fact that they bind to the enzyme as well or better than the
substrates with lesser proton affinity. The only plausible explanation
is that coordination of a strongly electron-donating pyridine to the
active site metal ion diminishes its available Lewis acidity toward the
scissile carbonyl group, producing less activation for nucleophilic
addition (stepk
in Fig. S1).
Activity also tapers off on the other limb of the plot in Fig. 5, especially for the relatively nonbasic cyano derivative.
That becomes explicable when one realizes that step k
in Fig. S1should be retarded by an absence of basicity
within the substrate. For this step reactivity should respond in the
reverse sense to electron availability on the pyridine nitrogen, for
here it is functioning as a nucleophile in competition with
H
O. Because steps k
and k
make opposing demands upon the substrate
nitrogen, a changeover in rate-limiting step should occur at some point
on the profile, and appears to do so.
/K
)
versus the pK
values for substrates
1. Lower trace is for rate constants themselves (left-hand
axis); upper trace is the same data presented in log
format (right-hand axis). Dashed lines are asymptotes
for a curve fit given under
``Discussion.''
= k`(K
)
. This
assumes that velocities for each step obey a linear free energy
relationship with respect to the proton affinity of the pyridine
nitrogen in the substrates. Since the overall rate constant for
consecutive reactions is always of the form k
k
/(k
+ k
), it follows that (k
/K
)
= k
`(K
)
k
`(K
)
/{k
`(K
)
+ k
`(K
)
}.
Directly fitting the data to this expression by the method of
least-squares gives the curve shown in Fig. 5, with parameters
(relative rate factors) k
` = 0.19 (±
0.01) and k
` = 2.3 (± 1.1). These
numbers have no particular significance; they simply represent
incidental intercepts for pK
= 0.
More relevantly, the Brcoefficient for the first step
comes out to be
1 = +0.11 (±0.03), and that for
the second
2 = -0.37 (±0.06). The latter
values correspond to the slopes of the asymptotic straight lines in the
log-log plot at the top of Fig. 5, in which the kinetic order
with respect to substrate pK
for each of
the sequential steps may be perceived clearly. While some
qualifications should be attached to these numbers (there is
considerable covariance in such a multiparameter fit, and the
Brrelationship may not hold rigorously over such a spread
of pK
values), the resulting coefficients
seem reasonable mechanistically. For the k
step,
which is thought to be replacement of an active site H
O
ligand on metal ion by substrate, the value of
1 (+0.11) has
the same sign but is roughly one-third of that noted for equilibrium
binding of metal ions to picolinic acids in aqueous solution (Fig. 3,
+0.3). Should
1
correspond to a kinetic process in which the substrate has not fully
bonded to the metal ion, the apparent expression of such a fraction of
the pyridine nitrogen's basicity within the transition state for
ligand substitution becomes plausible. That would be consistent with a
concerted mechanism of H
O displacement from the metal ion,
but not with a dissociative process in which H
O departs in
a slow step, followed by a diffusion-limited substrate insertion. The
latter process should have a negligible value for
1. On the other
hand, a purely associative process, where H
O leaves slowly
subsequent to the incoming pyridine having become fully bonded to the
metal, might be expected to have a higher value
(
), especially if adherence of the nucleofuge
H
O to the metal ion were loosened proportionate to pyridine
basicity.
2 is less obvious, although its
magnitude is more securely established by the data. The value of
2
is negative (-0.37), reflecting a quenching of enzyme activity
for the electron-rich pyridines. However, the slope of this asymptotic
regression line is considerably greater in absolute terms than the
coefficient of -0.06 noted for hydazinolysis of picolinate
esters. This establishes that the substituent effect is not a direct
perturbation of the reaction center, but instead probably occurs by
means of a coordinated metal ion which relays the effect from the
pyridine nitrogen through to the substrate carbonyl group. A further
complication comes from the observed values for the rate constant not
being for the second reaction step alone. Because the measured
velocities are k
/K
values, they should also reflect pre-equilibrium binding of
substrates in those cases for which k
is
rate-limiting (on the high-pK
limb of Fig. 5). Since in those cases k
/K
k
k
/k
,
the Brcoefficient
2 additionally incorporates a
factor derived from the ratio of k
and k
. We are unable to measure that
pseudo-K
value directly (K
values will not suffice, due to the
possible change in rate-limiting step), but the coefficient for
equilibrium binding of metal ions to picolinic acids or amides (Fig. 3) may stand in for the purpose. Because the
obtained from those data is intrinsically positive, it should
mask the true extent of the dependence of k
upon
substrate basicity, i.e. the coefficient of -0.37 is
only a measure of how much more important active site Lewis acidity is
for carbonyl addition than for chelative binding. In order to calculate
the actual kinetic Lewis base susceptibility of the enzymic metal ion,
corrections must be applied to
2. For an estimate, one may further
subtract the picolinic acid model equilibrium binding coefficient
(
) from
2, but then one should also incorporate
the small direct contribution from the substituent to reaction rate as
secured from hydrazinolysis of esters: -0.37 - (0.3 -
0.06)
-0.6. The absolute value of the latter number
approximates a fractional measure for the :Nu addition step of the
degree of ``proton-like'' character of the
substrate-recognizing metal ion in the active site of prolidase (since
it comes from a reflexive correlation with pK
values). That coefficient matters mechanistically, for while the
availability of the highly efficacious specific acid H
to a catalytic mechanism is controlled by pH and is slight at
aqueous neutrality, enzyme-substrate binding energy may be used to
impress a nearly equipotent metal ion stoichiometrically onto the
substrate, so that the closer the value of ‖
‖ should
tend to unity, the greater the benefit of M
. In the
case of carboxypeptidase A, the ``fluxionate Lewis acidity''
of the active site zinc ion (which amounts to the same concept) has
been evaluated similarly from K
values,
in an inhibition study correlating enzymic affinity for a series of
metal-liganding phenolate inhibitors with the latter's
pK
values: in that case
‖
‖ = 0.76(5) . In both instances the
active site metal ion appears hyperacidic relative to the wholly
solvated parent species
(H
O)
Zn
or to other
similar divalent metal ions in aqueous
solution(45, 46) . That is a mechanistically
significant observation, connecting straightforwardly with an
apparently enhanced proton acidity for the water molecule that binds to
the Lewis acid center in the case of the uncomplexed enzyme (enzymic
pK
of 6.6 for prolidase, 6.2 for
carboxypeptidase A)(5) . These aspects of metalloprotease
catalysis, which are crucial to an appreciation of mechanism, have been
explored elsewhere(4, 5, 6, 7) .
Rate-limiting Step
The foregoing interpretation
would be too speculative were it not supported by the pH dependence of k that is displayed by the various
picolinylprolines. As shown in Fig. 4, this kinetic parameter
generally decreases in acidic solution, in contradistinction to the
behavior of k
/K
which reaches a maximum there (Fig. 2). With some of
the more basic pyridines (1-H, 1-N(CH
)OCH
, 1-N(CH
)CH
CF
, 1-N(CH
)
) the saturation rate constant k
levels off at a finite limiting value on the
alkaline limb. However, the less basic substrates exhibit an additional
fall-off on the alkaline limb (especially 1-CN). The combination
is indicative of a change in rate-limiting step and suggests that for
the latter substrates the k
step as seen in the k
/K
profiles is
intruding into the k
profile at high pH, in the
manner just suggested by analysis of Fig. 5. The curve fits
displayed in Fig. 4were constructed accordingly. First, the
experimentally sigmoidal curves were fitted to give an alkaline limb (k
)
value for each applicable
substrate (recorded in Table 1) plus a pK
accounting for the decrease on the acidic limb of the profile
(range of values 5.4-6.4, apparently varying proportionately with
substrate basicity). By designation of corresponding acidic and
alkaline limb pK
values, the bell-shaped
curves could be fitted too. As before, the net observed velocity is
assumed to be for the slower of two consecutive reactions: k
= k
k
/(k
+ k
). The alkaline limb dependence was
accommodated by postulating that k
has in each
case the same pK
of 6.6 as
was seen in the k
/K
profiles (in the absence of evidence for a perturbation
within E
S)(47) . The acidic limb, attributed to
a step k
(probably, but not necessarily, an
identical step as the previously considered k
in Fig. S1), was presumed to have always the same downward
inflection seen in the sigmoidal k
profiles of
the other substrates, for which a
pK
value of 5.5 was
specified. The appropriate equation then can be shown to be k
= k
`k
``/{k
`(1
+ [H
]/K
)
+ k
``(1 + K
a
/[H
])},
which is the expression fitted to the data in Fig. 4. By this
expediency an approximate value for k
``, an
extrapolated limiting value on the alkaline limb for turnover of
chelatively bound substrate, may be obtained even for those substrates
that exhibit bell-shaped profiles. These are the estimates listed in
the final column of Table 1under (k
)
, along with the more firmly
established rate constants and pK
values
that are also recorded there for the cleanly sigmoidal curves.
values of the substrates, as done
previously for k
/K
(Fig. 5). The results are summarized in Fig. 6, for those substrates that are considered to permit a
trustworthy extrapolation onto the alkaline limb (excluding 1-CN
and 1-Cl). As before, a reasonable Brrelationship
is noted in the log plot for (k
)
,
with coefficient
2` = -0.41 (±0.07). There
appears to be agreement between this value for
2` and the value of
-0.37 previously determined for
2 (also for a step k
), from the analysis of Fig. 5, although
the theoretically anticipated value of approximately -0.6 is not
realized for this very approximate Brcoefficient. It does
appear that a nonlinear curve fit could yield that value as an
asymptote for the high pK
substrates. In
that connection the reservation should be mentioned that step k
need not be the same for both the k
/K
and the k
profiles for all substrates, even though the
simplest interpretation might have it so. The parameter from the k
/K
profile is for
the first committed step of the catalytic mechanism, i.e. the
first step for which net flux in the reverse direction is negligible in
the steady state. However, a reaction intermediate might accumulate
subsequently within the mechanism, so that its conversion (different
step k
corresponding to the k
profile) could then become rate limiting overall. A candidate
slow step might be product dissociation of chelated picolinic acid from
the enzyme, occurring after departure of proline, so that the
antecedent step is effectively irreversible. That process could
sensibly also show a negative Brcoefficient, although it
is not immediately obvious why that step should have the observed pH
dependence for k
. (
)The scatter (and
slope) in Fig. 6may also reflect other ambiguities associated
with interpreting k
values. For example, the
pyridine substituents of 1 within an E
S complex
might by steric interactions perturb the relative rates of the steps
designated by k
and k
in
additional ways that are unrelated to their influence on nitrogen
basicity. However, those same interactions might also tend to cancel in k
/K
due to
opposing effects on k
/k
and k
as formulated in Fig. S1.
For such reasons, the
2` coefficient from log (k
)
values has limited
interpretability, and overall concurrence with Fig. 5is
adequate.
)
(alkaline limb of pH profile) versus the pK
values for
substrates 1.
Applicability to Dipeptides
The corresponding kversus pH profiles with prolidase for
physiological-type substrates related to glycylproline have previously
been examined(8) . The results were complex but now appear to
be explicable by the two-step mechanism just proposed. For example,
glycylthiazolidinecarboxylate (a catalytically very active thia
analogue of glycylproline, penultimately obtained from cysteine plus
formaldehyde) shows only an acidic limb fall-off in k
, as for the more basic members of the series 1. This suggests that it is experiencing the same rate-limiting
step (k
). However, the enzymically less
susceptible dipeptide valylproline shows exactly the opposite pH
profile, with a maximum for k
in acidic solution
and with an extinction of catalytic activity on the alkaline limb.
Because the chelating amino group is more sterically congested in the
case of a valine N-terminal residue, it seems plausible that a
substrate binding step (reflecting H
O:M
dissociation exclusively) might loom as rate limiting there.
as judged by
metal-ion dissociation constant in homogeneous aqueous solution (K
10
M
for each), despite a >4-unit difference between them in
nitrogen-atom pK
value. The greater
polarizability of pyridine nitrogen within metal ion complexes accounts
for this chelative alikeness. That explains why relatively non-basic
picolinylprolines are nonetheless competent bidentate-ligating
substrates for prolidase. However, in electrodative interaction with
the active site M
, such polarizability apparently
leads to a greater ``killing effect'' upon enzymic Lewis
acidity within the E
S complex, as is reflected in
2. Consequently, 1-N(CH
)
is a poor
substrate kinetically, even though it has a basicity similar to that of
the less polarizable glycylproline. Overall, the evidence implies that
the same steps as described in Fig. S1also occur and are
competitive with one another in the case of normal dipeptide substrates
undergoing catalysis with prolidase, and that a common mechanism
prevails for all hydrolytically susceptible acylproline derivatives.
Amide Scission Mechanism
Although the initial
stages of the prolidase catalytic mechanism appear to be defined by the
kinetic patterns previously described, the nature of the nucleophile
required for cleaving the amide linkage in Fig. S1is not
identified by that data. As cited in the Introduction to this article,
there appear to be a pair of linked metal ions suitably disposed for
simultaneous interaction with a substrate in the active site of the
structurally characterized aminopeptidases that are related to
prolidase by amino acid sequence (19) or by specificity. Fig. S2provides two variants of a metallic hydroxide-induced
amide cleavage process that we should like to consider. For simplicity,
in this latter scheme the acidic limb pK seen in the k
profile is assigned to
a water molecule on the second metal ion in the active site, which
assumes an acidity similar to that of the first
H
O:M
, once hydroxide has been excluded
from the latter site by a neutral substrate ligand in the initial step
of the mechanism. Alternatively, the second metal-bound water could be
much less acidic, and the pK
could be
that of a general base residue (possibly a carboxylate) responsible for
carrying out a deprotonation in the course of reaction. In either case
the conjugate acid species would be catalytically inactive, explaining
the pH profile for k
. In the first variant (path a) the second metal ion delivers its (incipient)
hydroxide to the scissile amide linkage, yielding a tetrahedral adduct
which subsequently dissociates to yield the products of cleavage.
Something similar to this scheme has been claimed in model systems, and
the process is akin to that formerly thought to be operational in
thermolysin(6) . However, it does not appear that this
HO
transfer would be sufficiently favorable
energetically, without introduction of a supernumerary ligand to
satisfy electron deficiency on the cationic metal center which
hydroxide vacates.
) or some
subsequent step (e.g. product release). If there were such a
general base, and if it were the residue giving rise to the
pK
of 5.4-6.4 in the k
contour, then the second metal-bound water
shown ionizing in Fig. S2would have to be even more acidic than
that, so as not to show up in the kinetic pH profiles.
of 5.4-6.4, seen in k
when only one H
O remains in the
chelated E
S complex, corresponds to a mean value between
what is observed for the second and first ionizations of the
uncomplexed enzyme, in which both lyate species are present
(pK
values of 6.6 and
5,
respectively, for the two H
O:M
). In
support of the latter version, it is chemically implausible that
productive substrate binding should be completely suppressed in
alkaline solution as appears to be the case (Fig. 2), should the
pK
of 6.6 (from the k
/K
profiles)
merely be the first ionization of the coupled system of two metal-bound
waters. A non-ionized and hence displaceable H
O would still
remain after deprotonation in that instance. The suggested weakening of
acidity for the remaining metal-bound H
O, consequent to
productive substrate binding involving its neighboring metal ion,
results in a pK
elevation from a value of
5 into the pH range accessible to kinetic investigation. (
)That explains why the acidic limb ionization seen in k
does not show up as a similar pH dependence in
the profile for k
/K
, even though
the same k
step may be manifested in each.
Furthermore, there is a correlation between substrate basicity and the
value of that kinetically detected pK
(as
perceived within k
in Fig. 4, acidic
limb). The more strongly basic pyridines appear to cause the
pK
to have a slightly higher value (Table 1). That is explicable by enzymically enforced
juxtaposition of the two metal ions; increased electron donation to one
by a substrate-pyridine moiety can induce a coordination shift of
enzymic bridging ligands (likely to be carboxylates according to
crystallography (see later)), so as to relay a decrease in Lewis
acidity onto the second metal ion. Consequently, the observation that
substrate basicity becomes reflected in attenuated form in the k
profile is more in line with detection of
enzymic H
O:M
ionization than with that of
an ancillary side chain functionality.
Enzymic Structural Model
For a test of this
mechanistic hypothesis, recourse may be made to protein
crystallographic evidence. Although a structure for prolidase is
unavailable, as previously noted the methionine aminopeptidase from E. coli has been solved (18) , and sequence data for
the respective enzymes strongly suggests homology with prolidase.
Although reservations might be harbored about comparison of bacterial
and mammalian enzymes, E. coli concomitantly produces an
homologous prolidase of its own(19) , providing additional
credibility for the following suppositions. We have constructed a model
for the active site of prolidase, consisting of appropriate portions of
the methionine aminopeptidase structure surrounding the two metal ions,
with amino acid residue replacements according to the sequence which is
available for human (and bacterial) prolidase. The active site region
is comprised entirely of core -sheet structure, which is not
likely to be breached between the two enzymes. The enzymatic
M
-capturing side chains are completely identical for
the two proteins. This dual-metal ion binding locus consists of one
side chain functional group from each of five adjacent strands of
-sheet that in turn shape a concave surface subsuming the active
site. The metal-coordinating residues are Asp
,
Asp
, His
, Glu
, and
Glu
(numbered according to the methionine aminopeptidase
sequence) (
)(19) . With regard to other side chain
alterations that are noted within the vicinity of the catalytic metal
ions upon comparison of the enzymes, the observed residue replacements
in prolidase represent excision of functionality that potentially could
have been chemically participatory, or the changes are otherwise
conservative (N95L, T99G, H106A, S110T, G170P, I172L, F177L, and
H178G), with one exception (Q233R). The latter arginine introduction,
next to a metal-coordinating glutamate at position 235 in the bottom of
the
-sheet trough comprising the active site, most likely provides
the cationic element responsible for binding the carboxylate group of
substrates. The RCO
anionic entity in the
C terminus proline residue of substrates is a specificity feature with
regard to the enzyme prolidase (a dipeptidase), but not for methionine
aminopeptidase, which accepts extended chains (and so has Gln at
position 233). An essential arginine at the active site of prolidase
has previously been identified by chemical modification of its
guanidinium ion with phenylglyoxal, which entirely inactivates the
enzyme (but with complete protection conferred by the presence of a
competitive inhibitor)(48) . No other arginine residue (or
equivalently cationic lysine) appears to be available within the
vicinity of the enzymic metal ions, according to our prolidase
reconstruction.
in attachment to
M2, but that is explicable. Because the chelative substrate ligation to
that cationic center is bidentate, at least partial displacement of one
of the enzymic ligands is needed if expansion of the coordination
sphere of the metal ion is to be avoided. In the antecedent structure
of the enzyme (uncomplexed with a substrate), Asp
and
Glu
are seen to be shared by M1 and M2; the ligation of
one or both of these carboxylates may become shifted predominately to
M1 upon chelative assimilation of substrate onto M2 (consequently
affecting the pK
of
H
O:M1
as previously suggested).
Compensatory adjustments might be anticipated in the other carboxylate
side chains that participate in holding the metal ions in place, but in
the present model all have been left in their native positions as found
crystallographically.
(dashed lines). Metastable
hydrated carbonyl of scissile carboxamide ligates one oxygen (anion) to
each of the metal ions, M1 and M2, which are separated by 0.29 nm
within the active site. Substrate pyridyl residue also coordinates to
M2.
. Regarding the possibility that the
pK
of 5.4-6.4 seen in the k
profiles is due to an enzymic carboxylate
serving as a general base, rather than to a metal-bound water, the
enzyme structure provides no support. The only glutamate or aspartate
residues found in the active site appear to be engaged in ligation to
the metal ions, which intervention should lower their
pK
values rather than raise them
abnormally. The only histidine is similarly prevented from
participation as a base. There is a possibility that Glu
could become disengaged from M1 as a consequence of substrate
binding (attendant to the chelation-induced shifting of Asp
or Glu
ligation mainly to M1). The Glu
carboxylate would then be in a position to aid addition of
H-O(M1) to the substrate carbonyl by proton acceptance. Such an
obligatory cascade of enzymic side chain adjustments could explain why
non-chelating peptide surrogates such as acetylproline are not
substrates. However, even in that eventuality the kinetically detected
pK
of 5.4-6.4 in k
need not correspond to ionization of a carboxyl group; freed
Glu
could have a normal pK
value and still fulfill the general-base role. Chemical
modification evidence (inactivation with a water-soluble carbodiimide)
has suggested involvement of an enzymic carboxylate in the
mechanism(48) . However, incomplete protection was conferred by
competitive inhibitors, creating doubt as to whether an active site
residue is responsible. Alternatively, the carboxylate group that is
present within the substrate, and which becomes ionically linked to the
arginine occupying position 233 within the E
S complex,
appears suitably poised next to the nucleophile and could also function
in the role of proton acceptor, as seems to be the situation in the
mechanism for carboxypeptidase A(4, 7) . The latter
hypothesis offers an explanation as to why nucleophilic attack on the
substrate carbonyl group appears from Fig. 7to take place cis to the proline carboxylate, rather than from the
sterically less-encumbered opposite face of the scissile carboxamide
linkage.
Conclusion
In summary, the most plausible
catalytic amide cleavage mechanism for prolidase, and by inference for
other dual metal-containing aminopeptidases, is chelative activation of
the substrate by one of a pair of active site metal ions, with
nucleophilic addition to the scissile carbonyl group by the neighboring
(or second) metal hydroxide. The key property of the enzyme as revealed
by kinetics is an anomalously low pK value for at least one (and probably both) of the water
molecules that bind to the enzymic metal ions in the absence of
substrate. While on the one hand such enzymic hydroxide coordination
makes substrate insertion into the M
ligand sphere
difficult, the compensating factor is a greater activation of substrate
once bound, due to the implicitly enhanced Lewis acidity of the
coordinating metal ion, plus the possible engendering of a properly
promoted nucleophile from the second metallic Lewis acid present in the
enzyme active site. Because the challenge for understanding enzyme
mechanisms chemically is one of explaining transition state
stabilization, the postulated role of two especially Lewis acidic metal
ions engaged in simultaneous ligation to the anionic tetrahedral adduct
intermediate renders the reaction path in the case of prolidase more
comprehensible than for many other, more thoroughly studied proteases.
profiles might also be explained as a
``phantom'' pK resulting from a changeover between
pH-dependent and pH-independent rate-limiting steps, but this would
necessitate an implausible requirement that both such steps vary
similarly in velocity for all substrates within the series 1 (spanning
1000-fold change of rates, or greater, if dipeptide
substrates are included).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.