Kinetic and magnetic properties of cobalt(III) ion in the active site of carbonic anhydrase.

Cobalt(III)bovine carbonic anhydrase B was prepared by the oxidation of the cobalt(II) enzyme with hydrogen peroxide and was purified by affinity chromatography. The oxidation reaction is inhibited by specific inhibitors of carbonic anhydrase. The inhibition is explained by the fact that the Co(II)-enzyme . inhibitor complex cannot be directly oxidized by hydrogen peroxide, but has to dissociate to give free Co(II) enzyme which is then oxidized. The Co(III) ion in Co(III) carbonic anhydrase cannot be directly substituted by zinc ions. It can be reduced by either dithionite or BH-4 ions to give, first, their complexes with the Co(II) enzyme, and upon their removal, a fully active Co(II) enzyme. Cyanide and azide bind to cobalt(III) carbonic anhydrase with similar rate constants of 0.060 +/- 0.005 and 0.070 +/- 0.007 M-1 S-1 respectively. These rates are faster than those found for Co(III) inorganic complexes. The Co(III) ion in both Co(III) carbonic anhydrase and Co(III) carboxypeptidase A was found to be diamagnetic, indicating a near octahedral symmetry.     

Exchange inert Co (III)-carboxypeptidase A: a catalytically inactive metallocarboxypeptidase.

Exposure of cobalt (II) carboxypeptidase A_α, [(CPD)Co(II)], to small molar excesses of the oxidizing agent m-chloroperbenzoate rapidly destroys (< 30 sec) both its peptidase and esterase activities in parallel. Concomitantly, the characteristic Co(II) electron paramagentic resonance (EPR) signal is abolished. [(CPD)Co(III)], isolated from the reaction mixture, has the same molecular weight and amino acid composition as [(CPD)Co(II)], contains 0.95 g-atom of Co and 0.01 g-atom of Zn per mole of protein, does not exhibit an EPR spectrum and is catalytically completely inactive towards both peptide and ester substrates. Identical treatment of the native zinc enzyme affects neither its catalytic activity nor its metal content. The reaction of m-chloroperbenzoate with [(CPD)Co(II)] follows saturation kinetics and is prevented by the inhibitor β-phenylpropionate. Furthermore, under the conditions found to oxidize [(CPD)Co(II)] effectively, there is no reaction with Co(II) $\text{E. coli}$ alkaline phosphatase. Thus, m-chloroperbenzoate has the characteristics of an active-site directed oxidizing reagent for [(CPD)Co(II)].     

Effects of mechanism-based reversible inhibitors on the metal environment of cobalt(II)carboxypeptidase A: an electronic spectral study

Electronic absorption, circular dichroic (CD), and magnetic circular dichroic (MCD) spectra have been determined for complexes of cobalt(II)-substituted carboxypeptidase A and five reversible inhibitors. Three of the inhibitors, N-(1-carboxy-5-butyloxycarbonylaminopentyl)-L-phenylalanine, (I); (R,S)-2-benzyl-4-oxobutanoic acid, (III); and 2-benzyl-4-oxo-5,5,5-trifluoropentanoic acid, (IV) are mechanism-based inhibitors. Another, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine, (II), is a tight binding, slowly hydrolyzed substrate. The fifth, phosphoramidon, (V), is a mechanism-based inhibitor of thermolysin, and may also bind to carboxypeptidase in a mechanism-based mode. The absorption and CD spectra of the enzyme-inhibitor complexes all differ from the spectrum of the free enzyme and from each other. The MCD spectra indicate that the tetrahedral coordination geometry of cobalt, which is distorted in the free enzyme, is also distorted in the inhibitor complexes, although to various degrees. The complexes of I and III are spectrally similar despite being structurally dissimilar, and that of IV, whose structure resembles III, is spectrally distinct, indicating that I and III, but not IV, may perturb the metal in nearly the same way. The absorption spectrum of IV is identical to that, at high pH, of Co(II)carboxypeptidase in which Glu-270 has been modified by a carbodiimide reagent, possibly pointing to a common perturbation of this residue. The absorption and CD spectra of II are similar to those of the catalytic intermediate that precedes the rate-limiting step in peptide hydrolysis [D. S. Auld, A. Galdes, K. F. Geoghegan, B. Holmquist, R. Martinelli, and B. L. Vallee, Proc. Natl. Acad. Sci. USA 81, 4675-4681 (1984)]. Since II is a substrate, the steady-state bound species that it generates may therefore be a true productive intermediate rather than a nonproductive mimic of an intermediate. The spectra of the complexes with II and V differ considerably despite structural similarities. The negative CD ellipticity of the free enzyme is reversed in sign in the presence of V, a phenomenon previously observed with complexes of Co(II)carboxypeptidase and dipeptides. This resemblance may result from a similar interaction of cobalt with the phosphoramidate group of phosphoramidon and the N-terminal amine of dipeptides. The spectra of reversible, mechanism-based inhibitors permit general structural predictions about true intermediates but require caution when used for assigning precise conformation and ligands of bound catalytic species.     

Thioamide substrate probes of metal-substrate interactions in carboxypeptidase A catalysis

Three thioamide peptides in which the oxygen atom of the scissile peptide bond is replaced by sulfur (denoted by (= S)) were synthesized and found to be good, convenient substrates for carboxypeptidase A. The thioamide bond absorbs strongly in the ultraviolet region, and enzymatic hydrolysis is monitored easily using a continuously recording spectrophotometric assay. The reaction follows Michaelis-Menten kinetics with kcat values of 68, 9.0, and 3.7 sec-1 and Km values of 0.83, 0.81, and 0.53 mM for Z-Glu-Phe(= S)-Phe, Z-Gly-Ala(= S)-Phe, and Z-Phe(= S)-Phe, respectively. Activities of the thioamides and their oxygen amide analogs were determined with a series of metal-substituted carboxypeptidases. The Cd(II), Mn(II), Co(II), and Ni(II) enzymes exhibit 30%-35%, 60%-85%, 150%-190%, and 40%-55% of the Zn(II) enzyme activity with the amide substrates; this compares with 240%-970%, 0%-15%, 340%-840%, and 30%-140% of the Zn(II) activity, respectively, with the thioamides. The activity of the Cu(II) and Hg(II) enzymes is less than 3% toward all substrates. Cadmium, a thiophilic metal, yields an enzyme which is exceedingly active with the thioamides; the kcat/Km values are 2.4-9.7-fold higher than with Zn(II) carboxypeptidase. In contrast, Mn(II), which has a relatively low affinity for sulfur, yields an enzyme with correspondingly low activity toward the thioamides. The results are consistent with a mechanism for peptide bond hydrolysis in which the metal atom interacts with the substrate carbonyl atom during catalysis.     

Enzymatically inactive, exchange-inert Co(III)-carboxypeptidase A: role of inner sphere coordination in peptide and ester catalysis.

Catalytically inactive, exchange-inert Co(III)-carboxypeptidase A has been prepared by reaction of Co(II)-carboxypeptidase A with the active-site-directed oxidizing agent m-chloroperbenzoic acid. Co(III)-carboxypeptidase A, isolated by affinity gel filtration chromatography, has the same amino acid composition and molecular weight as the starting material and contains 0.95 g-atom/mol of cobalt and 0.01 g-atom/mol of zinc. Its electron paramagnetic resonance, circular dichroic, magnetic circular dichroic, and visible absorption spectra are consistent with those of octahedral Co(III) model complexes. Co(III)-caboxypeptidase A is essentially devoid of catalytic activity toward both peptide and ester substrates of the native enzyme, and stopped-flow fluorescence studies with dansylated substrates show that it binds peptides, but not esters. Furthermore, the protein does not react with either type of substrate to yield a single turnover. The implications of these findings to the mechanism of action of carboxypeptidase A are discussed in the light of the "metal-carbonyl" and "metal-hydroxide" hypotheses. Since Co(III)-carboxypeptidase A does not bind esters, inner-sphere coordination to the metal appears to be necessary for ester binding. All attempts to prepare Co(III)-carboxypeptidase A by treatment of Co(II)-carboxypeptidase A with hydrogen peroxide according to previously published procedures (Kang, E.P., Storm, C.B., & Carson, F.W. (1975) J. Am. Chem. Soc. 97, 6723) have been unsuccessful, and the present results do not confirm earlier reports that Co(III)-carboxypeptidase A exhibits esterase activity or that its activity is dependent on the method of preparation of the precursor Co(II)-carboxypeptidase A (Jones, M.M., Hunt, J.B., Storm, C.B., Evans, P.S., Carson, F.W. & Pauli, W.J. (1977) Biochem. Biophys. Res. Commun. 75, 253). These findings call for a reexamination of mechanistic conclusions based on the assumption that Co(III)-carboxypeptidase A is an active esterase.     

Role of metal ions in goat carboxypeptidase A-catalysed hydrolysis of acyl peptides

The carboxypeptidase A purified from goat pancreas has been found to have a molecular weight of 34,600 +/- 300. The enzyme is a zinc-protein and the molar ratio of zinc to enzyme protein is 1:1. Removal of zinc yields an inactive apocarboxypeptidase A. The loss of activity of the native enzyme and restoration of the activity of the apoenzyme run parallel with the zinc content of the protein, thus showing the essentiality of zinc for the enzymatic activity. The exact role of zinc in the enzyme catalysed hydrolysis of the acylpeptides has been investigated after preparing metallo proteins by substituting the zinc of carboxypeptidase A with Co2+, Mn2+, Ni2+, Fe2+, Cd2+, Hg2+, and Cu2+ and determining the kinetic parameters of such metalloproteins. These studies indicate that the metal ion is involved in both binding the substrate and polarising the peptide bond.     

(Met)enkephalin and carboxypeptidase processing enzyme are co-released from chromaffin cells by cholinergic stimulation

A carboxypeptidase B-like enzyme is involved in processing of proenkephalin in adrenal medulla. Nicotine stimulated the co-release of this enzyme with (Met)enkephalin pentapeptide from bovine chromaffin cells in primary culture. The ratio of enzyme activity/immunoreactivity was determined for the released carboxypeptidase to provide an index of the level of enzyme activity per unit number of enzyme molecules. The ratio for the Co++-stimulated carboxypeptidase secreted into the cell culture medium upon nicotinic stimulation was 10.1 +/- 1.02 (pmol Met-enkephalin formed per ng carboxypeptidase immunoreactivity), while the Co++-stimulated carboxypeptidase in the soluble and membrane components of purified chromaffin granules had lower ratios of 5.46 +/- 0.70 and 1.07 +/- 0.13, respectively. Hexamethonium, a nicotinic receptor antagonist, blocked the nicotine-induced release of the carboxypeptidase processing enzyme and (Met)enkephalin. These data suggest that a pool of carboxypeptidase enzyme molecules at a high state of activation are present in functionally mature granules whose contents are released by nicotinic receptor stimulation.     

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 the metal ion in the refolding of denatured bovine Co(II)-carbonic anhydrase II

Conditions for reactivation of guanidine-HCl-denatured bovine Co(II)-carbonic anhydrase II are given. The renaturation is accompanied by recovery of the native Co(II)-spectrum of the enzyme. After studying the kinetics of the renaturation process, the metal ion involvement in the refolding pathway can be summarized as follows: (1) Formation of an inactive Co(II)-intermediate with the metal ion firmly bound. No native Co(II)-spectrum is observed in this state, probably due to octahedral coordination of the metal ion in this intermediate. (2) Formation of an inactive Co(II)-intermediate with a native Co(II)-spectrum. The final tetrahedral coordination of the metal ion seems to have been formed in this state. (3) Formation of the active conformation of the enzyme. A functioning active-site is formed after some rearrangements of the polypeptide chain. This isomerisation step does not need to be preceded by formation of the intermediate with a native Co(II)-spectrum. Coordination of Co2+ in a native-like manner is, however, a prerequisite for enzymic activity. It is tentatively suggested that the metal ion is involved in stabilizing a nucleation structure formed at the bottom of the active centre. This probably occurs through binding of Co2+ to some or all of its histidyl ligands in this region after an early structuration of the metal ion binding site. The mechanisms of Co2+ appear to be similar for the refolding enzyme and the native apoenzyme, inferring that the binding site formed as a result of the nucleation process probably has the same structure as in the native conformation.     

A Co(III) derivative of concanavalin A

Co(III) has been stoichiometrically incorporated into jack bean concanavalin A. The Co(III) protein still possesses a binding site for an additional divalent transition metal ion which together with Ca(II) can induce the sugar binding ability. No H₂O₂ oxidation of Co(II) occurs with demetallized concanavalin A activated with Ca(II) and Co(II) unless Co(II) is present in a stoichiometric excess. Evidence is presented to indicate that kinetically stable Co(III) is bound to a completely different location than the thermodynamically stable Co(II) protein site.     

Phytic acid-enhanced metal ion exchange reactions: the effect on carboxypeptidase A

On the basis of the known interaction of phytic acid to form soluble or insoluble complexes with cations, the effect of this naturally occurring polydentate ligand on carboxypeptidase A, a zinc-containing metalloenzyme, and its Co(II)-substituted derivative, has been studied. Under conditions of rigorous exclusion of adventitious metal ions, phytate showed no inhibitory effect. However, the addition of Cu(II) ions to form soluble phytate-Cu(II) complexes at pH 7.2 and 25 degrees C caused more than a 95% decrease in activity. The Cd(II) ion was nearly as effective but other ions showed only a small or no effect. In the absence of phytate, incubation of the enzyme with Cu(II) or Cd(II) at the same concentration produced only about a 25% reduction in activity. The decrease in activity followed first-order kinetics, and the rate constant was the same (1.2 x 10(-4) sec-1) as seen upon incubation with EDTA. However, in contrast to that observed upon incubation of the enzyme with phytate and Cu(II), exposure to EDTA produced a complete loss in activity which could be regained by addition of Zn(II) to the assay solution. In the former case, not only was there residual activity left after incubation at pH 7.2 for 24 hrs at 25 degrees C, but the initial activity could not be regained under similar assay treatment. An increase in either the Cu(II) or phytate concentration while the other was kept constant, yielded saturation curves with maximal effect at 3 x 10(-5) M for Cu(II) and at 5 x 10(-5) M for phytate (enzyme at ca. 10(-6) M). At these ratios, all of the cupric ions are completely bound to phytate as determined by ion-selective potentiometry. A preparative scale reaction of phytate and Cu(II) with carboxypeptidase A (kcat 8460 min-1; K'M 0.23 mM with CBZ-glycyl-glycyl-L-phenylalanine as substrate at pH 7.5, 25 degrees C) gave a product isolated in 95% yield but with lower activity (kcat 198 min-1; K'M 0.25 mM). A Cu(II)-carboxypeptidase preparation had similar kinetic parameters (kcat 207 min-1; K'M 0.34 mM). This near identity of constants suggested that a metal exchange reaction had occurred, i.e., incubation of Zn(II)-carboxypeptidase with a phytate-Cu(II) complex resulted in not only the removal of the zinc ion from the active site but also the sequential and rapid incorporation of a cupric ion into the apoenzyme so formed.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

The kininases of Bothrops jararaca plasma

Evidence is presented to suggest that kininase activity of Bothrops jararaca plasma is due to the presence of at least three distinct enzymes: a carboxypeptidase B type enzyme, similar to that found in human plasma in that its activity is enhanced by Co2+ (1 X 10(-4) M); a carboxypeptidase B type enzyme whose activity is unaffected by Co2+, and an enzyme which cleaves bradykinin to liberate Phe-Arg as the major peptide fragment formed. The latter enzyme is responsible for the major kininase activity of this snake plasma and is identified as a dipeptide hydrolase.     

Proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Purification and characterization of the carboxypeptidase.

We described earlier the facilitated purifications of the trypsin and aminopeptidase components present in Pronase (Vosbeck, K. D., Chow, K. -F., and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 6029-6034). A partially resolved protein mixture left over after one of the steps in that procedure was passed through a Sephadex G-75 column. By this means, a component with carboxypeptidase activity was separated from associated serine endopeptidases. Further purification of this exopeptidase to apparent homogeneity was acheived by refiltration through the same Sephadex column and by CM-cellulose chromatography. A single protein band was observed after acrylamide gel electrophoresis; analysis by sedimentation equilibrium using the meniscus depletion method gave a molecular weight of 30,300. This enzyme demonstrates activity against Nalpha-benzyloxycarbonylglycyl-L-leucine and hippuryl-D,L-phenyllactate; no activity was found against Nalpha-acetyl-L-tyrosine ethyl ester, Nalpha-benzoyl-D,L-arginine-p-nitroanilide, or L-leuckne-p-nitroanilide. The maximum activity lies between pH values of 7 and 8; the enzyme is stable between pH values of 6 and 10. At room temperature 1,10-phenanthroline inactivates the enzyme completely whereas EDTA has no effect. Of the many cations tested, only Co2+, Ni2+, or Zn2+ restores activity to the 1,10-phenanthroline-treated enzyme; Co2+ provided 3 times the native activity. The metal in the native protein was found to be zinc. These findings are similar to those recorded with bovine pancreatic carboxypeptidase A, and suggest the possibility that the present enzyme may ge genetically related to the mammalian protein, as in previously noted examples of homology of three Pronase endopeptidases to pancreatic serine enzymes.     

The effect of binding of cobalt(III)--nucleotide complexes on the kinetic properties of adenosine triphosphatase activity in coupling factor 1 from chloroplasts.

Co(III)-ATP and Co(III)-ADP as well as the parent complexes containing phenanthroline as an additional ligand were found to inhibit ATPase activity competitively in coupling factor 1 from chloroplasts. The K_i values were at the micromolar range and were found to decrease with time of preincubation of the enzyme with the Co(III) complexes. Co(III)-phenanthroline-ATP was found to bind to the enzyme at two sites with dissociation constants of 1 and 3 μm. The labeling as well as the inhibition was completely reversed by dithiothrietol. In addition, the complexes caused a time-dependent release of enzyme-bound Mn(II) ions, thus labeling the metal binding site. The results were interpreted with regard to the mechanism of ATPase activity in coupling factor 1.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Activation of rabbit kidney fructose diphosphatase by Mg-EDTA, Mn-EDTA and Co-EDTA complexes

Purified rabbit kidney fructose diphosphatase requires both a free cation and a metal-chelate when assayed at pH 8 or below. In the presence Mg²⁺ or Mn²⁺, effective metal chelates were Mn(II)-EDTA, Mg(II)-EDTA, and Co(III)-EDTA. With Mg²⁺ as the cation the affinity of the enzyme for Mn(II)-EDTA or Mg(II)-EDTA was approximately the same, and 300-fold greater than that for Co(III)-EDTA.Activation of the enzyme by the very stable Co(III)-EDTA complex, as well as failure of an ionophore antibiotic to replace EDTA as activator, exclude the possibility that the effects of EDTA are due to removal of metal inhibitors.Inhibition of fructose diphosphatase by Ca²⁺ was competitive with Mg²⁺, and noncompetitive with Mg(II)-EDTA, or Co(III)-EDTA. Conversely inhibition by Zn(II)-EDTA was competitive with Mg(II)-EDTA and noncompetitive with free Mg²⁺. The data suggest that the free metals bind to one site on the enzyme while the metal-EDTA chelates bind to a second site.     

Enkephalin-degrading dipeptidyl carboxypeptidase in guinea pig serum: its properties and action on bioactive peptides

A dipeptidyl carboxypeptidase, which cleaved the Gly3-Phe4 bond of enkephalins, was purified from guinea pig serum 420-fold. The optimum pH of the enzyme was in the neutral range (pH 7.25), and the molecular weight was estimated to be approx. 280,000. The enzyme hydrolyzed Met- and Leu-enkephalin with Km values of 0.30 and 0.50 mM, respectively. The enzyme was inhibited by metal chelators and p-chloro-mercuribenzoate. Captopril showed high inhibitory potency, while phosphoramidon and Phe-Ala showed no effect on the enzyme activity. Therefore, the obtained enzyme can be classified as an angiotensin-converting enzyme (EC 3.4.15.1). Among the bioactive peptides examined, bradykinin and angiotensin I were hydrolyzed by the enzyme. Angiotensin III showed a stronger inhibitory effect than that of angiotensin II. Substance P, gastrin I, and secretin were also inhibitory toward the enzyme activity. On high-performance liquid chromatography analysis, Met-enkephalin-Arg6-Phe7 and Leu-enkephalin-Arg6 were cleaved sequentially at the second peptide bond of the C terminus. Thus, the dipeptidyl carboxypeptidase in guinea pig serum may play a role not only in the angiotensin-bradykinin system but also in the metabolism of circulating enkephalins and other bioactive peptides.     

Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli

The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 +/- 0.2 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 50% of the catalytic activity. Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 +/- 0.5 mM for the second metal binding site. Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry. Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of [CoCo(MetAP)] also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry. EPR studies on [CoCo(MetAP)] also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion ( approximately 5%) of the sample exhibited detectable spin-spin interactions at pH values > 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. (1)H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H(2)O and D(2)O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171. Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E. coli are discussed.     

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues

The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 Å of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.