©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hydrolysis of Picolinylprolines by Prolidase
A GENERAL MECHANISM FOR THE DUAL-METAL ION CONTAINING AMINOPEPTIDASES (*)

(Received for publication, March 8, 1995; and in revised form, May 25, 1995)

William L. Mock (§) Yaya Liu

From the Department of Chemistry, University of Illinois, Chicago, Illinois 60607-7061

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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/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 pKof 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.


INTRODUCTION

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 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) .

Prolidase has long been recognized as a metallopeptidase. Because its standard isolation procedure specifies incorporation of aqueous Mn (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.


MATERIALS AND METHODS

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(r) 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(r) of 55,000 according to SDS-polyacrylamide gel electrophoresis, and with M(r) 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 (^1H 400 MHz, C 100 MHz), IR (^1)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(3), mp >125 °C (dec), dicyclohexylamine salt mp 158-160 °C; 4-methoxypicolinylproline, from 4-methoxypicolinic acid (37) ; 1-OCH(3), mp >125 °C (dec), dicyclohexylamine salt mp 158-160 °C; 4-(dimethylamino)picolinylproline, from 4-(dimethylamino)picolinic acid(38) , 1-N(CH(3))(2), 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(3))OCH(3), 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(3)CH(2)OTs and MeNH(2), 160 °C, 3 days) and 4-chloropicolinic acid (H(2)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(3))CH(2)CF(3), dicyclohexylamine salt mp 155-156 °C. Picolinic acid esters were obtained by a Fischer esterification procedure (EtOH, HCl, 2 h reflux, subsequent H(2)O-K(2)CO(3) 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 geq8.1 (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(0) = DeltaA/{1 + (K/[M])bullet(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.


RESULTS

Ordinarily, prolidase behaves as an obligatory aminopeptidase. As earlier shown, the prototypical substrate glycylproline is cleaved readily, but acetylproline is hydrolyzed >10^4-fold more slowly, even though the latter NH(2)-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.


Figure 1: Structures of substrate analogues for prolidase. Proline residues are all of L-configuration.



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(2)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 N(2)H(4), 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 pKvalues 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 pKvalues 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(m)

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(2)O from a metal ion, they cannot compete as coordinating agents with HO.


Figure 2: Plots of k /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(3))CH(2)CF(3) 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(3))OCH(3)). 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)).



The same explanation holds for glycylproline as well. In the bell-shaped pH profile for that substrate, the alkaline limb inflection (pK approx 8.8) must be attributed to the glycyl NH(2) 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(3))OCH(3) and 1-N(CH(3))CH(2)CF(3). For these picolinyl substrates the pyridine-ring pKvalue 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(3))(2) 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.

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 approx -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/Kperturbations 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) .

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/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 and Intrinsic Metal Ion Affinity

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 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 alpha) 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.


Figure 3: Plot of Zn 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 alpha (``Discussion'').



Enzyme Activity, [S] K(m)

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(3))OCH(3), 1-N(CH(3))OCH(2)CF(3), 1-N(CH(3))(2)) 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 EbulletS complex, but a fuller accounting is deferred under ``Discussion.''


Figure 4: Plot of k 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.



The cognate pH profiles for K 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).


DISCUSSION

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. (^2)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(2)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.

Fig. S1summarizes the initial stages of the enzymic mechanism that we consider to be indicated by the data. Step k(1)/k(1) represents productive substrate binding in which H(2)O becomes displaced from an active site metal ion (LM). 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(2)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(3))OCH(3), and for normal substrates such as glycylproline(8) . Step k(2) 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).


Figure S1: Scheme 1.



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(2) 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(1) 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(2)O. Because steps k(1) and k(2) 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.


Figure 5: Plot of (k/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.''



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 = 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(1)k(2)/(k(1) + k(2)), it follows that (k/K) = k(1)`(K)k(2)`(K)/{k(1)`(K) + k(2)`(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(1)` = 0.19 (± 0.01) and k(2)` = 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 alpha1 = +0.11 (±0.03), and that for the second alpha2 = -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(1) step, which is thought to be replacement of an active site H(2)O ligand on metal ion by substrate, the value of alpha1 (+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, alpha approx +0.3). Should alpha1 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(2)O displacement from the metal ion, but not with a dissociative process in which H(2)O departs in a slow step, followed by a diffusion-limited substrate insertion. The latter process should have a negligible value for alpha1. On the other hand, a purely associative process, where H(2)O leaves slowly subsequent to the incoming pyridine having become fully bonded to the metal, might be expected to have a higher value (geqalpha), especially if adherence of the nucleofuge H(2)O to the metal ion were loosened proportionate to pyridine basicity.

The interpretation of alpha2 is less obvious, although its magnitude is more securely established by the data. The value of alpha2 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/Kvalues, they should also reflect pre-equilibrium binding of substrates in those cases for which k(2) is rate-limiting (on the high-pK limb of Fig. 5). Since in those cases k/K k(2)k(1)/k, the Brcoefficient alpha2 additionally incorporates a factor derived from the ratio of k and k(1). We are unable to measure that pseudo-K value directly (Kvalues 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 alpha obtained from those data is intrinsically positive, it should mask the true extent of the dependence of k(2) 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 alpha2. For an estimate, one may further subtract the picolinic acid model equilibrium binding coefficient (alpha) from alpha2, 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) approx -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 pKvalues). 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 ‖alpha‖ 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 ‖alpha‖ = 0.76(5) . In both instances the active site metal ion appears hyperacidic relative to the wholly solvated parent species (H(2)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(3))OCH(3), 1-N(CH(3))CH(2)CF(3), 1-N(CH(3))(2)) 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(1) 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 pKaccounting 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(1)k(2)/(k(1) + k(2)). The alkaline limb dependence was accommodated by postulating that k(1) has in each case the same pK(1) of 6.6 as was seen in the k/K profiles (in the absence of evidence for a perturbation within EbulletS)(47) . The acidic limb, attributed to a step k(2) (probably, but not necessarily, an identical step as the previously considered k(2) 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(2) value of 5.5 was specified. The appropriate equation then can be shown to be k = k(1)`k(2)``/{k(1)`(1 + [H]/K) + k(2)``(1 + Ka(1)/[H])}, which is the expression fitted to the data in Fig. 4. By this expediency an approximate value for k(2)``, 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.

The purpose of carrying out the designated fit is so that the derived rate constants may then be plotted against the pyridine pK 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 alpha2` = -0.41 (±0.07). There appears to be agreement between this value for alpha2` and the value of -0.37 previously determined for alpha2 (also for a step k(2)), 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(2) 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(2) 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. (^3)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 EbulletS complex might by steric interactions perturb the relative rates of the steps designated by k(1) and k(2) 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(1)/k(1)and k(2) as formulated in Fig. S1. For such reasons, the alpha2` coefficient from log (k) values has limited interpretability, and overall concurrence with Fig. 5is adequate.


Figure 6: Plot of log (k) (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(2)). 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(2)O:M dissociation exclusively) might loom as rate limiting there.

It is relevant that the anions of glycine and picolinic acid possess an identical chelative affinity for Zn as judged by metal-ion dissociation constant in homogeneous aqueous solution (K approx 10M 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 EbulletS complex, as is reflected in alpha2. Consequently, 1-N(CH(3))(2) 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(2)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.


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(1)) 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.

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 of 5.4-6.4, seen in k when only one H(2)O remains in the chelated EbulletS 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 leq5, respectively, for the two H(2)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(2)O would still remain after deprotonation in that instance. The suggested weakening of acidity for the remaining metal-bound H(2)O, consequent to productive substrate binding involving its neighboring metal ion, results in a pK elevation from a value of leq5 into the pH range accessible to kinetic investigation. (^4)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(2) 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(2)O:M ionization than with that of an ancillary side chain functionality.

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.

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 beta-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 beta-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) (^5)(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 beta-sheet trough comprising the active site, most likely provides the cationic element responsible for binding the carboxylate group of substrates. The RCO(2) 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 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 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(2)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.


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 (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.



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. 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 EbulletS 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.


FOOTNOTES

*
This work was supported by the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

^1
The abbreviations used are: IR, infrared; MES, 2-(N-morpholino) ethanesulfonic acid; PIPES, piperazine-N,N`-bis[2-ethanesulfonic acid]; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid.

^2
The potent metalloprotease inhibitors bestatin and amastatin, conjectured transition state analogues, apparently bind to bovine lens leucine aminopeptidase through chelative coordination of their vicinal amino alcohol moiety to one of the active site metal ions (43, 44).

^3
The acidic limb curvature in the k 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).

^4
An oxide bridge between the metal ions at pH > 6.6, as reported for one aminopeptidase (24), would be functionally equivalent to two ligated hydroxides.

^5
As regards the actual prolidase protein sequence, the metal-binding residues would be D276, D287, H366, E412, and E452. The methionine aminopeptidase numbering has been adopted to facilitate structural comparison.


REFERENCES

  1. Bizzozero, S. A., and Zweifel, B. O.(1975)FEBS Lett.59,105-108 [CrossRef][Medline] [Order article via Infotrieve]
  2. Yaron, A., and Naider, F. (1993)Crit. Rev. Biochem. Mol. Biol.28,31-81 [Abstract]
  3. Myara, I., Cosson, C., Moatti, N., and Lemonnier, A.(1994)Int. J. Biochem. 26,207-214 [CrossRef][Medline] [Order article via Infotrieve]
  4. Mock, W. L., and Zhang, J. Z.(1991)J. Biol. Chem.266,6393-6400 [Abstract/Free Full Text]
  5. Mock, W. L., Freeman, D. J., and Aksamawati, M.(1993)Biochem. J. 289,185-193 [Medline] [Order article via Infotrieve]
  6. Mock, W. L., and Aksamawati, M.(1994)Biochem. J.302,57-68 [Medline] [Order article via Infotrieve]
  7. Mock, W. L., and Xu, X. (1994)Bioorg. Chem.22,373-386 [CrossRef]
  8. Mock, W. L., Green, P. C., and Boyer, K. D.(1990)J. Biol. Chem. 265,19600-19605 [Abstract/Free Full Text]
  9. Mock, W. L., and Green, P. C.(1990)J. Biol. Chem.265,19606-19610 [Abstract/Free Full Text]
  10. Manao, G., Nassi, P., Capuggi, G., Camici, G., and Ramponi, G.(1972) Physiol. Chem. Phys.4,75-87 [Medline] [Order article via Infotrieve]
  11. Sjstrm, H., and Norn, O.(1974) Biochim. Biophys. Acta359,177-185 [Medline] [Order article via Infotrieve]
  12. Endo, F., Matsuda, I., Ogata, A., and Tanaka, S.(1982)Pediatric Res. 16,227-231 [Abstract]
  13. Yoshimoto, T., Matsubara, F., Kawano, E., and Tsuru, D.(1983)J. Biochem. (Tokyo) 94,1889-1896 [Abstract]
  14. Browne, P., and O'Cuinn, G.(1983)J. Biol. Chem.258,6147-6154 [Abstract/Free Full Text]
  15. Richter, A. M., Lancaster, G. L., Choy, F. Y. M., and Hechtman, P.(1989) Biochem. Cell Biol.67,34-41 [Medline] [Order article via Infotrieve]
  16. Endo, F., Tanoue, A., Nakai, H., Hata, A., Indo, Y., Titani, K., and Matsuda, I.(1989) J. Biol. Chem.264,4476-4481 [Abstract/Free Full Text]
  17. Tanoue, A., Endo, F., and Matsuda, I.(1990)J. Biol. Chem.265,11306-11311 [Abstract/Free Full Text]
  18. Roderick, S. L., and Matthews, B. W.(1993)Biochemistry32,3907-3912 [Medline] [Order article via Infotrieve]
  19. Bazan, J. F., Weaver, L. H., Roderick, S. L., Huber, R., and Matthews, B. W.(1994) Proc. Natl. Acad. Sci. U. S. A.91,2473-2477 [Abstract]
  20. Denslow, N. D., Ryan, J. W., and Nguyen, H. P.(1994)Biochem. Biophys. Res. Commun.205,1790-1795 [CrossRef][Medline] [Order article via Infotrieve]
  21. Burley, S. K., David, P. R., Taylor, A., and Lipscomb, W. N.(1990)Proc. Natl. Acad. Sci. U. S. A.87,6878-6882 [Abstract]
  22. Burley, S. K., David, P. R., and Lipscomb, W. N.(1991)Proc. Natl. Acad. Sci. U. S. A.88,6916-6920 [Abstract]
  23. Burley, S. K., David, P. R., Sweet, R. M., Taylor, A., and Lipscomb, W. N.(1992) J. Mol. Biol.224,113-140 [Medline] [Order article via Infotrieve]
  24. Chevrier, B., Schalk, C., D'Orchymont, H., Rondeau, J.-M., Moras, D., and Tarnus, C. (1994)Structure2,283-291 [Medline] [Order article via Infotrieve]
  25. Taylor, A.(1993) FASEB J. 7,290-298 [Abstract/Free Full Text]
  26. Allen, M. P., Yamada, A. H., and Carpenter, F. H.(1983)Biochemistry 22,3778-3783 [Medline] [Order article via Infotrieve]
  27. Thompson, G. A., and Carpenter, F. H.(1976)J. Biol. Chem.251,53-60 [Abstract]
  28. Thompson, G. A., and Carpenter, F. H.(1976)J. Biol. Chem.251,1618-1624 [Abstract]
  29. King, S. W., and Fife, T. H.(1983)Biochemistry22,3603-3610 [Medline] [Order article via Infotrieve]
  30. Auld, D. S., and Vallee, B. L.(1970)Biochemistry9,602-609 [Medline] [Order article via Infotrieve]
  31. Fabre, J. L., Farge, D., and Lav, C. J. et D.(1985) Tetrahedron Lett.26,5447-5450 [CrossRef]
  32. Kuznetsova, E. A., Voronina, T. A., Khromova, I. V., Garibova, T. L., Tosina, E. K., Stolyarova, L. G., Troitskaya, V. S., and Smirnov, L. D.(1989) Khim. Farm. Zh.23, 1425-1431; (1990) Chem. Abstr.112,172135v
  33. Samvelyan, V. M., Kazaryan, S. A., Matevosyan, L. A., Dzhanpoladyan, E. G., Khoetsyan, E. L., and Mndzhoyan, O. L.(1992)Eksp. Klin. Farmakol. 55, 11-13;(1993) Chem. Abstr.118,204725e
  34. Heinisch, G., and Ltsch, G.(1985)Angew. Chem. Int. Ed. Engl.24,692-693
  35. Mosher, H. S., and Look, M.(1955)J. Org. Chem.20,283-286
  36. Clemo, G. R., and Gourlay, W. M. (1938) J. Chem. Soc. 478-479
  37. Endo, M., and Nakashima, T.(1960)Yakugaku Zasshi80, 875-879;(1960) Chem. Abstr.54,24705b
  38. Deady, L. W., Shanks, R. A., Campbell, A. D., and Chooi, S. Y.(1971) Aust. J. Chem.24,385-392
  39. King, G. F., Middlehurst, C. R., and Kuchel, P. W.(1986) Biochemistry25,1054-1062 [Medline] [Order article via Infotrieve]
  40. Lin, L.-N., and Brandts, J. F.(1979)Biochemistry18,43-47 [Medline] [Order article via Infotrieve]
  41. Davis, N. C., and Smith, E. L.(1957)J. Biol. Chem.224,261-275 [Free Full Text]
  42. Blackburn, G. M., and Jencks, W. P.(1968)J. Am. Chem. Soc.90,2638-2645
  43. Kim, H., and Lipscomb, W. N.(1993)Proc. Natl. Acad. Sci. U. S. A. 90,5006-5010 [Abstract]
  44. Kim, H., and Lipscomb, W. N.(1993)Biochemistry32,8465-8478 [Medline] [Order article via Infotrieve]
  45. Postmus, C., Jr., Magnusson, L. B., and Craig, C. A.(1966)Inorg. Chem. 5,1154-1157
  46. Leussing, D. L., and Bai, K. S.(1968)Anal. Chem.40,575-581
  47. Mock, W. L.(1992) Bioorg. Chem.20,377-381
  48. Mock, W. L., and Zhuang, H.(1991)Biochem. Biophys. Res. Commun. 180,401-406 [Medline] [Order article via Infotrieve]

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