Principles of hydroxamate inhibition of metalloproteases: carboxypeptidase A.

Hydroxamic acids of structure RCON(OH)CH(2)CH(CH(2)C(6)H(5))CO(2)H induce micromolar competitive inhibition of catalysis for the enzyme carboxypeptidase A. Enzyme affinity depends on the nature of the acyl group, for RCO equaling HCO, CH(3)CO, FCH(2)CO, F(2)CHCO, F(3)CCO, CH(3)OCH(2)CO, or CH(3)OCO. In acid dissociation these residues yield hydroxamic acid pK(a) values that vary from 7.6 to 10.3. Profiles of inhibitory pK(i) plotted versus pH indicate characteristically a maximum effectiveness near neutrality. Weaker binding to enzyme is generally displayed in either acidic or alkaline solution, with the position of the alkaline limb of the profiles depending on the pK(a) of the inhibitor. A reverse-protonation pattern of association with the enzyme is indicated, in which the hydroxamate anion of the inhibitor displaces a relatively acidic H(2)O ligand (pK(a) of 6) from the active-site zinc ion of carboxypeptidase A. The metal-coordinating, N-substituted hydroxamic acid functional groups exist in solution as a mixture of syn and anti rotamers, with relative abundances that depend on their pK(a). A pyrrolidinone analogue having a conformationally syn-fixed cyclohydroxamic acid was not an especially potent inhibitor. Structure-activity relationships suggest design criteria for hydroxamic acid inhibitors in order to provide most effective binding with metalloenzymes.

Synergistic inhibition of carboxypeptidase A by zinc ion and imidazole.

Zinc ion in solution yields a 560-fold enhancement in the kinetic inhibition of carboxypeptidase A by the simple heterocycle imidazole, behavior attributed to formation of a ternary complex of the three species. However, the effect is partially negated by formation of less-inhibitory Zn2+(C3H4N2)2-4 coordination complexes, providing for the enzyme an anomalous profile of catalytic rate versus imidazole concentration.

Catalytic activity of carboxypeptidase B and of carboxypeptidase Y with anisylazoformyl substrates.

Anisylazoformyllysine (CH3OC6H4-N = N-CO-Lys-OH) is rapidly hydrolyzed at the acyl-lysine linkage by the zinc-enzyme porcine carboxypeptidase B. The catalytic reaction is readily monitored spectrophotometrically by disappearance of the intense absorption (348.5 nm, epsilon 18400) of the azo chromophore, which chemically fragments after substrate cleavage. Carboxypeptidase Y has no activity toward this type of substrate.

Kinetic characterization of the serralysins: a divergent catalytic mechanism pertaining to astacin-type metalloproteases.

Substrates HO2CCH2CH2CO- and HOCH2CHOHCHOHCO-Phe-Leu-Ala-5-nitro-2-pyridinamide are cleaved efficiently at the acylarenamide linkage, with a convenient spectrophotometric assay, by the Serratia and Pseudomonas approximately 50-kDa extracellular metalloproteases (serralysins). The pH range of catalytic activity extends uniformly from 4 to greater than 10 (k(cat)/Km approximately 10(3) s(-1) M(-1), similar profile for k(cat)). Substrate analogue hydroxamic acid Cbz-Leu-Ala-NHOH competitively inhibits serralysin (Ki 0.04 mM), with a pH dependence indicating that either a displaced metal-bound H2O or a similarly motile enzymic phenol residue (Tyr216) that is crystallographically found ligated to the active-site Zn2+ of the uncomplexed enzyme must have a pKa of approximately 5. A chemical catalytic mechanism of proteolysis consistent with the kinetic data is proposed, in which Tyr216-ArO-, in the course of being released from the active-site metal ion, deprotonates a water molecule attacking the Zn2+-activated substrate linkage, leading to a metal-coordinated tetrahedral oxyanion adduct that subsequently fragments.

Arazoformyl peptide surrogates as spectrophotometric kinetic assay substrates for carboxypeptidase A.

N-(4-Methoxyphenylazoformyl)-L-phenylalanine is efficiently cleaved by the enzyme bovine carboxypeptidase A into fragments anisole, molecular nitrogen, carbonate, and phenylalanine, in the course of which an intense spectral absorption of the substrate (epsilon350 = 19,000 M-1 cm-1) disappears completely. This furnishes a sensitive spectrophotometric detection of the protease. Steady-state catalytic velocity depends upon enzyme and substrate concentrations in the normal manner, and a Michaelis-Menten Km value of 0.11 mM and a kcat value of 44 s-1 were measured at pH 7.5 in saline solution. These parameters have a pH dependence typical for the enzyme. With saturating amounts of substrate, a solution containing 10 nM enzyme produces a spectral absorptivity change at 350 nm of -0.03 a.u./min (1-mm pathlength), suitable for assay purposes. Catalysis may alternatively be monitored at wavelengths as long as 400 nm.

Arazoformyl dipeptide substrates for thermolysin. Confirmation of a reverse protonation catalytic mechanism.

Cleavage by thermolysin of N-(4-methoxyphenylazoformyl)-L-leucyl-L-leucine plus some congeneric peptides provides a highly sensitive new kinetic assay for proteolytic activity. The pH dependence of Michaelis-Menten parameters Kcat and Km establishes kinetically a reverse protonation catalytic mechanism for this metalloprotease [Mock, W.L., & Aksamawati, M. (1994) Biochem. J. 302, 57-68]. An acidified water molecule (pKa of 5, seen in Km) becomes displaced by substrate carboxamide from the hypercationic Zn2+ of the enzyme, yielding potent Lewis acid activation of the peptide linkage for subsequent hydrolysis. Conversion to product is induced by the side chain of enzymic residue His 231 (pKa of 8, seen in Kcat), which provides general base catalysis for addition of H2O to the zinc-activated scissile carboxamide of the bound substrate. A previously described "superactivation" through chemical modification of the enzyme with acetylphenylalanyl-N-hydroxysuccinimide is nonexistent in the case of the new substrates, which indicates that their binding to thermolysin is largely productive, unlike normal peptides. Correct assignment of kinetically observed pKa values to active site residues, along with recognition of a predominantly nonproductive binding mode for ordinary substrates and thermolysin, forces reinterpretation of previous mechanistic formulations for the enzyme.

Hydrolysis of picolinylprolines by prolidase. A general mechanism for the dual-metal ion containing aminopeptidases.

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 kcat/Km for picolinylproline is sigmoidal, with optimum activity on the acidic limb and a delimiting enzymic pKa 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 pKa 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 Brønsted-type correlation with substrate pKa, 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.

Binding to thermolysin of phenolate-containing inhibitors necessitates a revised mechanism of catalysis.

Competitive inhibition as a function of pH for the metalloendoprotease thermolysin by derivatives of L-alpha-(2-hydroxyphenyl)benzenepropanoyl-L- tryptophanylglycylglycine exhibits a diagnostic bell shape. Binding is maximal between two pKa values: on the acidic limb the apparent Ki value is regulated by an unchanging enzymic ionization (pKa 5.3) which is also seen in the substrate-hydrolysis kinetics (kcat/Km), whereas the alkaline limb for inhibition varies and depends specifically on the pKa of the phenolic group in the inhibitor. Although it should be the phenolate form of the inhibitor that co-ordinates more efficiently to the active-site Zn2+, the apparent Ki shifts from pH-independent at pH values immediately below the inhibitor's pKa to progressively weaker binding at higher pH. This is explained by an anomalous acidity for the exchangeable solvent molecule that is attached to enzymic Zn2+ in the absence of substrate or inhibitor. Since OH- cannot be displaced from the enzyme as readily as H2O, a compensating pKa of 5.3 possessed by Zn(2+)-bound water rationalizes the binding characteristics, yielding the level pH profile exhibited at intermediate pH values. Recognition of the implicit heightened Lewis acidity of the metal ion in thermolysin leads to a revision of the mechanism of catalysis. The substrate amide bond becomes activated for hydrolysis by carbonyl-group co-ordination to the especially acidic Zn2+ ion (completely displacing the H2O/OH- species otherwise bound). The imidazole group of enzymic residue His-231, also discerned in the pH profile for kcat/Km from its pKa of 8, provides general-base assistance for hydration of the activated scissile linkage in the first committed step of catalysis. Additional evidence from inhibition patterns shows how substrate-binding energy may be employed in this scheme to promote hydrolysis of peptides by thermolysin.

Fluxionate Lewis acidity of the Zn2+ ion in carboxypeptidase A.

Competitive inhibition constants Ki for a series of phenol-ring-substituted derivatives of alpha-(2-hydroxyphenyl)benzenepropanoic acid have been ascertained by observing their influence on the catalytic hydrolysis of a peptide substrate by the zinc enzyme carboxypeptidase A. The pH-dependence of Ki shows that binding is maximal between two pKa values: one is that of the phenol group of the inhibitor, and the other uniformly has a value of 6, the pKa of a Zn(2+)-bound water molecule on the enzyme in the absence of substrate or inhibitor. This is the dependence expected if phenolate binds to the Zn2+ displacing its bound H2O/HO-. A log-log plot of the dissociation constants for the productive forms of inhibitor plus enzyme versus the acid dissociation constants of the phenolic residues in the inhibitors yields a straight line with a slope of +0.76. This number indicates that the active-site metal ion has special capacity for dispersing negative charge, such as builds up on the oxygen atom of a carboxamide group undergoing nucleophilic addition.

Chemical modification locates guanidinyl and carboxylate groups within the active site of prolidase.

Reagents phenylglyoxal or 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate inactivate the enzyme prolidase, with protection conferred by the competitive inhibitor N-acetylproline. The presence of arginine and carboxylate (aspartic/glutamic acid) residues at the active site of this metallodipeptidase may be inferred.

Mechanistically significant diastereoselection in the sulfoximine inhibition of carboxypeptidase A.

The diastereomers of L-alpha-[ [S-(2-phenylethyl)sulfonimidoyl]methyl]benzenepropanoic acid bind differentially to carboxypeptidase A. These putative transition state-analogue inhibitors show unique and interpretationally significant pH dependences for Ki, as well as for the visible absorption spectra of their E.I complexes in the case of the cobalt-substituted enzyme. From the geometry of the enzymically preferred isomer, it may be concluded that the mechanism of peptide scission by the enzyme entails addition of a nucleophile to the si face of the bound-substrate prochiral carboxamide linkage. New interpretational constraints on the mode of action of the enzyme are thereby imposed.

Specificity and pH dependence for acylproline cleavage by prolidase.

Catalytic pH dependence for the hydrolytic activity of the enzyme prolidase with a series of dipeptide substrates is found to be generally bell-shaped (kcat/Km) or simple sigmoidal (kcat). An enzymic residue with a pKa value of 6.6 is found to be critically involved in the catalytic mechanism, as is the substrate amino group. Significant catalysis at a pH of 6.6 is also observed for prolidase with (alkylthio)acetylprolines and with haloacetylprolines. A reverse-protonation state mechanism for substrate binding and activation is postulated, involving a chelative interaction of the aminoacylamide portion of substrate with a strongly Lewis-acidic active site metal ion.

Mechanism and inhibition of prolidase.

The pH dependence of Ki for inhibition of prolidase by acetylproline, proline, and trans-1,2-cyclopentanedicarboxylate follows a different pattern in each case, although deprotonation of an enzymic functional group with a pKa value of 6.6 perturbs ligand binding in every instance. Results are most easily explained with prolidase active as a metalloenzyme dimer exhibiting selective cooperative interactions.

pK values for active site residues of carboxypeptidase A.

The phenolic group of active site residue Tyr-248 in carboxypeptidase A has a pKa value of 10.06, as determined from the pH dependence of its rate of nitration by tetranitromethane. The decrease in enzyme activity (kcat/Km) in alkaline solution, characterized by a pKa value of approximately 9.0 (for cobalt carboxypeptidase A), is associated with the protonation state of an imidazole ligand of the active-site metal ion, as indicated by a selective pH dependence of the 1H NMR spectrum of the enzyme. Inhibition of the cobalt-substituted enzyme by 2-(1-carboxy-2-phenylethyl)phenol and its 4,6-dichloro- and 4-phenylazo-derivatives confirms that the decrease in enzyme activity (kcat/Km) in acidic solution, characterized by a pKa value of 5.8, is due to the protonation state of a water molecule bound to the active-site metal ion in the absence of substrate. Changes in the coordination number of the active-site metal ion are seen in its visible absorption spectrum as a consequence of binding of the phenolic inhibitors. Conventional concepts regarding the mechanisms of the enzyme are brought into question.

A probe of the active site acidity of carboxypeptidase A.

The substrate analogue 2-(1-carboxy-2-phenylethyl)-4-phenylazophenol is a potent competitive inhibitor of carboxypeptidase A. Upon ligation to the active site, the azophenol moiety undergoes a shift of pKa from a value of 8.76 to a value of 4.9; this provides an index of the Lewis acidity of the active site zinc ion. Examination of the pH dependence of Ki for the inhibitor shows maximum effectiveness in neutral solution (limiting Ki = 7.6 X 10(-7) M), with an increase in Ki in acid (pK1 = 6.16) and in alkaline solution (pK2 = 9.71, pK3 = 8.76). It is concluded that a proton-accepting enzymic functional group with the lower pKa (6.2) controls inhibitor binding, that ionization of this group is also manifested in the hydrolysis of peptide substrates (kcat/Km), and that the identity of this group is the water molecule that binds to the active site metal ion in the uncomplexed enzyme (H2OZn2+L3). Reverse protonation state inhibition is demonstrated, and conventional concepts regarding the mechanism of peptide hydrolysis by the enzyme are brought into question.