Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis.

The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.     

Products of metal exchange reactions of metallothionein

Hepatic metallothionein (MT) isolated from Cd-exposed animals always contains Zn (2-3 mol/mol of protein) in addition to Cd (4-5 mol/mol of protein), and the two metals are distributed in a nonuniform, but reproducible, manner among the seven binding sites of the protein's two metal-thiolate clusters. Different methodologies of preparing rabbit liver Cd, Zn-MT in vitro were investigated to provide insight into why such a distinct mixture of mixed-metal clusters is produced in vivo and by what mechanism they form. 113Cd NMR spectra of the products of stepwise displacement of Zn2+ from Zn7-MT by 113Cd2+ show that Cd binding to the clusters is not cooperative (i.e., clusters containing exclusively Cd are not formed in preference to mixed-metal Cd, Zn clusters), there is no selective occupancy of one cluster before the other, and many clusters are produced with a nonnative metal distribution indicating that this pathway is probably not followed in vivo. In contrast, the surprising discovery was made that the native cluster compositions and their relative concentrations could be reproduced exactly by simply mixing together the appropriate amounts of Cd7-MT and Zn7-MT and allowing intermolecular metal exchange to occur. This heretofore unknown metal interchange reaction occurs readily, and the driving force appears to be the relative thermodynamic instability of three-metal clusters containing Cd. With this new insight into how Cd,Zn-MT is likely to be formed in vivo we are able for the first time to postulate rational explanations for previous observations regarding the response of hepatic Zn and metallothionein levels to Cd administration.     

On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study

113Cd and 31P NMR have been used to investigate the interactions of inhibitors with the metal ion of bovine carboxypeptidase A, using 113Cd as a replacement for the native zinc atom. In the absence of inhibitor and over the pH range 6-9, no 113Cd resonance is visible at room temperature. Upon lowering the temperature to 270 K, however, a broad resonance can be seen at 120 ppm. These results are discussed in terms of possible sources for this resonance modulation. Binding of low molecular weight inhibitors containing potential metal-coordinating moieties results in the appearance of a sharp 113Cd resonance. These inhibitors all bind to the metal ion, a fact which is reflected in the chemical shift of the cadmium resonance and, for L-phenylalanine phosphoramidate phenyl ester, by two-bond 113Cd-31P spin-spin coupling of 30 Hz in the 31P resonance of the bound inhibitor. For inhibitors that coordinate to the metal ion via oxygen, the 113Cd chemical shift is in the range 127-137 ppm, whereas for sulfur coordination there is a downfield shift of approximately 210 ppm. The complexes of 113Cd-substituted carboxypeptidase A with the D and L isomers of thiolactic acid are distinguished by a difference of 11 ppm in the chemical shift of their cadmium resonances. The enzyme complex formed with the macromolecular inhibitor from potatoes, which fills the S1 and S2 subsites, shows one or possibly two closely spaced broad 113Cd resonances. Both the chemical shift and the line width of the 113Cd resonances of the [113Cd]carboxypeptidase-inhibitor complexes give valuable structural and dynamic information about the enzyme active site.     

Phytic acid-zinc ion interactions: a calorimetric and titrimetric study

Using an isoperibolic titration microcalorimeter, the ionization characteristics and associated heat changes of phytic acid (myo-inositol hexaphosphate) and phytic acid in the presence of varying Zn(II) concentrations have been examined over the pH range 2.5–11 at 25°C in 0.2 M KCl. In the absence of Zn(II), ca. 7 of the 12 ionizable protons in phytic acid are titrated in this pH range with ionization heats varying from ca. 2 to −3 kcal-mol^-1. At Zn(II): phytate mol ratios of 4:1 and greater, the dissociation of all protons and complex formation of phytate with Zn(II) occurs below pH 6. From the difference titration curves of phytic acid plus Zn(II) versus Zn(II) alone, ca. 3.5 mol Zn(II) bind per mol phytate. Since Zn(II):phytate complexes are insoluble, the observed heat changes contain contributions not only from heats of precipitation but also from binding, ionization, neutralization, and hydration effects. From the heat change for the titration of (a) phytic acid, pH 2.6–10.4; (b) phytic acid + Zn(II), pH 2.6–6.1; and (c) Zn(II), pH 2.6–6.1 at Zn(II): phytate ratios of 4 to 10, the value of 24.7 ± 0.5 kcal mol⁻¹ phytate has been obtained for the binding of 3.5 mols Zn(II). This figure also includes the heat of precipitation of the complex. In pH-drop experiments, with the initial pH at 8.65, the value of 23.9 kcal mol^-1 was obtained for ΔH°. Hysteresis effects are prevalent in these reaction solutions. Time-dependent changes in pH occur with a change in pH. For the phytate-Zn(II) reactions, the time-course curves are biphasic and fit a rate equation for two simultaneous first order reactions. Hysteresis effects seen in the titration of Zn(II) fit simple first-order kinetics. These effects most probably arise from the ejection of a proton from the aqua ion or aqua ion ligand complex(es).     

Effect of zinc ion on the inhibition of carboxypeptidase A by imidazole-bearing substrate analogues

Competitive inhibitors of carboxypeptidase A, 2-(4-imidazoyl)hydrocinnamic acid (1) and its congeners (2-4) that bear an imidazole ring as the zinc-ligating functionality have been evaluated for their CPA inhibitory activity in the presence of zinc ion to find that the zinc ion augments the inhibitory potency by up to 212-fold.     

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.     

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)     

Interprotein metal ion exchange between cadmium-carbonic anhydrase and apo- or zinc-metallothionein

 − 1  s^− 1 at 25 °C and pH 7.4 in Tris.HCl buffer and 0.1 M KCl. At 25 °C, Zn₇-metallothionein also exchanged metal ions with Cd-carbonic anhydrase with a rate constant of 0.33 ± 0.02 M^− 1 s^− 1 to reconstitute enzymatically active protein. Cd-carbonic anhydrase reacted within the time of mixing with the peptide sequence 49–61 of rabbit metallothionein 2 which contains four cysteinyl residues, leading to the exchange of most of the Cd²⁺ into the peptide. At pH 7.4 and 25 °C, Cd²⁺ has higher affinity for apometallothionein than for the apo-peptide. Received: 25 February 1999 / Accepted: 17 September 1999     

Phytic acid-metal ion interactions. II. The effect of pH on Ca(II) binding

The interaction of phytic acid with Ca(II) has been studied by potentiometric titration and by measurement of free Ca(II) concentrations using an ion Selective electrode. With increasing Ca(II) concentration, the titration curve of phytic acid is displaced to regions of lower pH. In the binding of calcium ions to phytic acid, there is no evidence that significant binding occurs below approximately pH 5. Above this pH, the extent of binding is dependent upon both pH and the calcium to phytic acid ratios. Maximum binding obtains at a Ca(II):phytate ratio of 6 with 4.8 mol of Ca(II) bound per mol of phytate above pH ca. 8. Binding constants are apparently very large since binding isotherms at any Ca(II):phytate ratio are a linear function of the total calcium ion concentration. In all cases, binding occurs only when one or more phosphate groups have been converted to the oxo dianion form. The apparent pK' values (curve-fit parameters) that describe the potentiometric titration data are in good agreement with the constants evaluated from the binding of Ca(II) to phytate as a function of pH. Using CPK space-filling models, structures containing six metal ions in coordinate linkage to pairs of oxo dianions have been constructed and discussed within the framework of the axial conformation of phytic acid and the order of proton removal with an increase in pH based upon NMR studies.     

Characterization of enzyme-bound ligand dynamics by solid-state NMR in the presence of ligand exchange: L-phenylalanine on carboxypeptidase A.

Deuterium NMR spectra were obtained for L-phenylalanine-d5, deuterated on the phenyl ring, in cross-linked polycrystalline samples of carboxypeptidase A containing different amounts of water. The deuterium powder pattern line shapes are simulated by extension of the theory to include both a local reorientational motion of the bound L-phenylalanine phenyl ring and exchange of the L-phenylalanine with an intracrystalline isotropic environment. The spectral simulations are consistent with the phenyl ring of the phenylalanine executing pi-flips in the bound environment at rates that vary from 3 x 10(4) Hz at 6% water content to 1 x 10(5) Hz at 21% water content. At all water contents studied, the ligand exchanges with an essentially isotropic environment in the crystal with a rate constant of approximately 2.5 x 10(-3) Hz. Although the dissociation constant for the L-phenylalanine is only 18 mM, the spectral simulations that reproduce the experimental line shape well do not require significant wobble of the phenyl ring rotation axis, which is consistent with the binding interactions identified by x-ray crystallography.