Circular dichroism-inhibitor titrations of arsanilazotyrosine-248 carboxypeptidase A.

Coupling of carboxypeptidase with diazotized arsanilic acid specifically modifies a single tyrosyl residue. Yet, owing to the fact that the resultant azoTyr-248 can form an intramolecular chelate with zinc, two different circular dichroism probes result: azoTyr-248 itself and the azoTyr-248-Zn chelate. Both are environmentally sensitive and, characteristically, each can signal the same or different perturbations, as is apparent from circular dichroic spectra. This dual probe function greatly magnifies the scope of these chromophores in mapping the topography of the active center with respect to sites of interaction of inhibitors (or substrates). Titration of the azoenzyme with a series of synthetic, competitive inhibitors, e.g., L-benzylsuccinate, L-phenyllactate, and L-Phe, and with the pseudosubstrate, Gly-L-Tyr, in turn generates characteristic circular dichroic spectra. Their analysis yields a single binding constant for each of these agents, one molecule of each binding to the active center. Mixed inhibitions, as seen with beta-phenylpropionate and phenylacetate, resolved previously into competitive and noncompetitive components, are characterized by different spectral effects. Two molecules of these agents bind to the enzyme, consistent with both thermodynamic and enzymatic studies. The interactions leading to competitive and noncompetitive inhibition, respectively, can be recognized and assigned, based on the manner in which the extrema at 340 and 420 nm, reflecting azoTyr-248, and the negative 510-nm circular dichroism band, typical of its chelate with zinc, are affected and on the pH dependence of spectral and kinetic data. Certai4 noncompetitive inhibitors and modifiers induce yet other spectral features. Each probe is very sensitive to changes in its particular active center environment, though both can be relatively insensitive to inhibitors interacting at a distance from the active center.     

Environment and conformation dependent sensitivity of the arsanilazotyrosine-248 carboxypeptidase A chromophore.

Reaction of carboxypeptidase A crystals with diazotized arsanilic acid uniquely modifies Tyr-248 to form a monazo derivative, which-in solution-forms an intramolecular inner-sphere coordination complex in the active site zinc atom. tarsanilazocarboxypeptidase exhibits spectral properties that are closely similar to those of the model complex, tetrazolylazo-N-carbobenzoxytyrosine Zn2+, with a distinctive maximum at 510 nm. In addition, its circular dichroic spectrum reveals a negative extremum at this wavelength, also characteristic of this complex. Both spectra are exquisitely responsive to pth changes and serve to monitor formation and dissociation of the metal-azophenol complex. Two pKapp at 7.7 and 9.5 delineate the pH range over which the probe characteristics most effectively gauge conformational features of the active center of arsanilazcarboxypeptidase. Other environmental parameters, e.g., substrates and inhibitors, as well as crystallization of the enzyme also critically influence the formation and dissociation of the complex; the response of the probe suggests that they induce conformational movement of the azoTyr-248 residue away from the zinc atom. tthe now available chemical, functional, structural data bearing on the spatial relationships of Tyr-248 and Zn, both thought critical to catalysis, are evaluated, based on spectra of arsanilazo- and nitrocarboxypeptidase crystals and solutions as well as on detailed kinetic analyses of the native enzyme in both physical states and based on the X-ray structure analysis of the native enzyme and its Gly-L-Tyr complex. Collectively all of the data show that the conformation of carboxypeptidase in crystals differs from that in solution. Moreover, reexamination of the original X-ray maps reported in 1968 and thought to preclude a Tyr-248-Zn interaction now leads to the conclusion that in up to 25 per cent of the molecules in the crystals ttyr-248 interacts with the active site zinc atom (W.D. Lipscomb (1973), Proc. Nat. Acad. Sci U.S. 70, 3797). Thus, even in the crystals the enzyme exists in at least two different conformations. In one of these Tyr-248 is near while in the other it is far from the zinc atom. The spectral effects of Gly-L-Tyr and beta-phenylpropionate on solutions of arsanilazo- and of nitrocarboxypeptidase demonstrate that during the catalytic process Tyr-248 moves away from the zinc atom. This implies a mechanistic role for Tyr-248 different from that postulated on the basis of X-ray crystallographic analysis. Indeed, the proximity of ttyr-248 to the zinc atom, when altered by substrates and inhibitor, may reflect certain of the properties characteristic of the entatic, active site.     

Contribution of C-tail residues of potato carboxypeptidase inhibitor to the binding to carboxypeptidase A A mutagenesis analysis.

The role of each residue of the potato carboxypeptidase inhibitor (PCI) C-terminal tail, in the interaction with carboxypeptidase A (CPA), has been studied by the analysis of two main kinds of site-directed mutants: the point substitution of each C-terminal residue by glycine and the sequential deletions of the C-terminal residues. The mutant PCI-CPA interactions have been characterized by the measurement of their inhibition constant, Ki, in several cases, by their kinetic association and dissociation constants determined by presteady-state analysis, and by computational approaches. The role of Pro36 appears to be mainly the restriction of the mobility of the PCI C-tail. In addition, and unexpectedly, both Gly35 and Pro36 have been found to be important for folding of the protein core. Val38 has the greatest enthalpic contribution to the PCI-CPA interaction. Although Tyr37 has a minor contribution to the binding energy of the whole inhibitor, it has been found to be essential for the interaction with the enzyme following the cleavage of the C-terminal Gly39 by CPA. The energetic contribution of the PCI secondary binding site has been evaluated to be about half of the total free energy of dissociation of the PCI-CPA complex.     

Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III).

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.     

The role of Tyr248 probed by mutant bovine carboxypeptidase A: insight into the catalytic mechanism of carboxypeptidase A

We have investigated the function of Tyr248 using bovine wild-type CPA and its Y248F and Y248A mutants to find that the K(M) values were increased by 4.5-11-fold and the k(cat) values were reduced by 4.5-10.7-fold by the replacement of Tyr248 with Phe for the hydrolysis of hippuryl-L-Phe (HPA) and N-[3-(2-furyl)acryloyl]-Phe-Phe (FAPP), respectively. In the case of O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate (ClCPL), an ester substrate, the K(M) value was increased by 2.5-fold, and the k(cat) was reduced by 20-fold. The replacement of Tyr248 with Ala decreased the k(cat) values by about 18- and 237-fold for HPA and ClCPL, respectively, demonstrating that the aromatic ring of Tyr248 plays a critical role in the enzymic reaction. The increases of the K(M) values were only 6- and 5-fold for HPA and ClCPL, respectively. Thus, the present study indicates clearly that Tyr248 plays an important role not only in the binding of substrate but also in the enzymic hydrolysis. The kinetic results may be rationalized by the proposition that the phenolic hydroxyl of Tyr248 forms a hydrogen bond with the zinc-bound water molecule, causing further activation of the water molecule by reducing its pK(a) value. The pH dependency study of k(cat) values and the solvent isotope effects also support the proposition. A unified catalytic mechanism is proposed that can account for the different kinetic behavior observed in the CPA-catalyzed hydrolysis of peptide and ester substrates.     

Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase).

Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.     

The NMR structure and dynamics of the two-domain tick carboxypeptidase inhibitor reveal flexibility in its free form and stiffness upon binding to human carboxypeptidase B

The structure of the tick carboxypeptidase inhibitor (TCI) and its backbone dynamics, free and in complex with human carboxypeptidase B, have been determined by NMR spectroscopy. Although free TCI has the same overall fold as that observed in the crystal structures of its complexes with metallocarboxypeptidase types A and B, there are structural differences at the linker between the two domains. The linker residues have greater flexibility than the globular domains, and the C-terminal residues are highly flexible in free TCI. Upon formation of a complex with carboxypeptidase B, TCI becomes more rigid, especially at the level of the linker and at the C-terminus, which is inserted into the active site groove of the carboxypeptidase. Solvent exchange rates of the backbone amide protons also show a strong reduction of the local TCI dynamics and a stabilization of its structure upon complex formation. The findings are consistent with a recognition mechanism that primarily involves the C-terminal domain, which adjusts its conformation and that of the linker, thus facilitating complex stabilization by further interactions between the N-terminal domain and an exosite of the carboxypeptidase. This adaptability enables TCI to tune its global conformation for proper interaction with distinct types of carboxypeptidases by a mechanism of induced fit. Our results provide new information about the structure-function relationship and stability of a molecule with potential biomedical applications in thrombolytic therapy. Furthermore, the plasticity of TCI makes it an ideal scaffold for developing stronger and/or more specific inhibitors directed toward modulating the activity of metallocarboxypeptidases.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

A potent mercapto bi-product analogue inhibitor for human carboxypeptidase N

The bi-product analogue inhibitor, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, has been synthesized in high yield and exhibits a K_i of 2.0 nM with human plasma carboxypeptidase N. The ease of synthesis and subsequent availability make it an ideal compound to study potentiation of bradykinin and other vasoactive peptides.     

Differences between the conformations of nitrotyrosyl-248 carboxypeptidase A in the crystalline state and in solution

In solution, nitrocarboxypeptidase A, modified at tyrosyl-248, exhibits a nitrotyrosyl pK apparent of 6.3. In the crystalline state, the pK apparent is about 8.2. This change in ionization is consistent with the hypothesis that crystallization of the enzyme causes a displacement of tyrosine-248 away from the active site zinc ion.     

Secondary binding site of the potato carboxypeptidase inhibitor. Contribution to its structure, folding, and biological properties

The contribution of each residue of the potato carboxypeptidase inhibitor (PCI) secondary binding site to the overall properties of this protein has been examined using alanine-scanning mutagenesis. Structural and enzymatic studies, performed on a series of PCI mutants, demonstrate that the proper positioning of the primary site for efficient binding and inhibition of carboxypeptidase A is significantly dependent on such a secondary contact region. The aromatic residues in this region play a key role in the stabilization of the PCI-enzyme complex, whereas polar residues contribute little to this task. A comparative study of the oxidative folding of these PCI mutants has been carried out using the disulfide quenching approach. The data, together with the structural characterization of some of these mutants, clearly indicate that noncovalent forces drive the refolding of this small disulfide-rich protein at the reshuffling stage, the rate-limiting step of the process. Moreover, it reveals that by introducing new noncovalent intramolecular contacts in PCI, we may create more stable variants, which also show improved folding efficiency. Taken together, the collected results clarify the folding determinants of the primary and secondary binding sites of PCI and their contribution to the inhibition of the carboxypeptidase, providing clues about PCI evolution and knowledge for its biotechnological redesign.     

The unfolding pathway of leech carboxypeptidase inhibitor

The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable.     

Characterization of an inhibitory metal binding site in carboxypeptidase A

The specificity of metal ion inhibition of bovine carboxypeptidase A ([(CPD)Zn]) catalysis is examined under stopped-flow conditions with use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent KI values of 2.4 x 10(-5), 4.8 x 10(-5), and 1.1 x 10(-2) M in 0.5 M NaCl at pH 7.5 and 25 degrees C. The kcat/Km value, 7.3 x 10(6) M-1 s-1, is affected less than 10% at 1 x 10(-4) M Mn(II) or Cu(II) and at 1 x 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([(CPD)Co]).(ABSTRACT TRUNCATED AT 250 WORDS)     

2-Benzyl-3,4-iminobutanoic acid as inhibitor of carboxypeptidase A.

2-Benzyl-3,4-iminobutanoic acid (3) was evaluated as a novel class of inhibitor for carboxypeptidase A (CPA). All four stereoisomers of 3 are found to have competitive inhibitory activity for CPA, although their inhibitory potencies differ widely with (2R,3R)-3 being most potent. The molecular modeling study for CPA(2R,3R)-3 complex suggested that the lone pair electrons on the nitrogen of the aziridine ring in the inhibitor forms a coordinative bond with the active site zinc ion and the proton on the nitrogen is engaged in hydrogen bonding with one of the carboxylate oxygens of Glu-270.