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The ESR spectra of dicupric human serum-transferrin (serum-Tf) were measured from -20 to 37°C in the liquid state (56% glycerol at pH 7.6). Two coordination geometries (types B-1 and B-2) with different ESR parameters were present at the N-site. The contents of the coordination geometry of type B-1 at the N-site increased as the temperature increased. The equilibrium constant between the coordination geometries of types B-1 and B-2 was determined by ESR spectra. The enthalpy value from type B-2 to B-1 was +5.3 kcal/mol, as obtained from a van't Hoff plot. The two conformational energies of the cluster models of the copper-binding site at the N-site of dicupric human serum-Tf, where the Arg124 residue was oriented in two different directions (conformations I and II), were calculated by Density Functional Theory, and the enthalpy value from conformation II to I was +2.1 kcal/mol. The enthalpy value was similar to that (+5.3 kcal/mol) obtained by the coordination geometrical change from type B-2 to B-1 in Cu(II)2 serum-Tf. In conformations I and II, the residue of Arg124 at the N-site is located either far from or near the copper-binding site, respectively, and in both cases the coordination geometry of the cupric ions at the N-site has changed from a flattened tetrahedron to a trigonal bipyramid. This result implies that the ESR spectral change from type B-2 to B-1 is caused by the presence of two different orientations of Arg124 in the change from conformation II to I.

Antioxidant activities of 3-oxygenated and 3,4-dioxygenated carbazole alkaloids and their related carbazoles were comprehensively evaluated. In all assay systems, the 3,8-dihydroxycarbazoles carbazomadurin A (2) and B (3), and their synthetic precursors 2a and 3a exhibited higher antioxidant activities than the 3-monohydroxycarbazoles carazostatin (1), and the synthetic precursors 4a and 4b of carquinostatin A (4). In particular, 2a and 3a exhibited strong scavenging activities due to the reducing ability of formyl group at the C-5 position of carbazoles. The results suggest that these compounds could serve as useful clues for designing and developing novel antioxidants.

The metal dissociation constants of bovine carbonic anhydrase II, bovine carboxypeptidase A, rat aminopeptidase B, and rat dipeptidyl peptidase III were measured using metal buffer solutions. The zinc dissociation constants of bovine carbonic anhydrase II, bovine carboxypeptidase A, rat aminopeptidase B, and rat dipeptidyl peptidase III were 5.8×10(-14), 3.5×10(-12), 3.7×10(-13), and 1.9×10(-13) M, respectively. The ternary complex between metal derivatives of bovine carbonic anhydrase and various chelating agents were characterized using the kinetic method and visible and magnetic circular dichroism spectra. The coordination geometry of the ternary complex was in the equilibrium state between the five and the tetrahedral coordination geometry. The equilibrium state depends on the character of ligands. Dipeptidyl peptidase III which has an abnormal zinc binding motif (HEXXXH) was characterized using the point mutation and computer simulation methods. The abnormal zinc binding motif (HEXXXH) of rat dipeptidyl peptidase III has a large helix part. It is generally known that the cupric derivatives of the zinc peptidase loses enzyme activity, but the cupric derivative of dipeptidyl peptidase III surprisingly has enzyme activity. The measurement of the electron paramagnetic resonance spectra of the cupric rat dipeptidyl peptidase III in the presence of the substrate showed that the coordination geometry is very flexible. The flexibility of the coordination geometry in the cupric rat dipeptidyl peptidase III is important for the expression of enzyme activity. Docking simulation was used to identify the substrate binding site of aminopeptidase B, which is a powerful tool to estimate substrate binding residues in enzymes.

The text mining and full-text searching function were used to analyze the digitalized daily reports of the practical training in pharmacology submitted to the web system in Fukuyama University. Collocations connected to the word "inspection" were searched in the daily reports using the text mining and the full-text search functions of the system. Many collocation groups connected to "inspection" were found in the daily reports and the greatest number of collocations was associated with "preparation of drugs". Practical training in narcotic dispensing has two different aspects: inspection and experience training. The number of people who reported a relation between "inspection" and "narcotics dispensing" in the daily reports was very similar to those who reported a connection between "experience" and "narcotics". Practical training to handle narcotic dispensing is the most fundamental training that the pharmacist must undertake. The progression of team-based medical care has caused medical personnel to recognize the practical training in pharmacology, and the number of the people who reported a relation between "inspection" and "team medical care" in 2011 increased in comparison to 2010. Moreover, the progression of cooperation among hospitals, pharmacies, and local blanches of the Japan Pharmaceutical Association is beneficial to practical training. Practical training in pharmacology is based on the core curriculum, but undergoes periodic modification due to societal circumstances. Therefore, these results suggest that the construction of web system for submitting daily reports is useful for analyzing the daily reports.

Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, “HELLGH,” as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al . (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K m values or in the substrate specificity. However, the k cat values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His 568 residue of rat DPP III is essential for enzyme catalysis. The k cat values of the mutants E507A and E512A were 2.38 and 3.88 s − 1 toward Arg-Arg-NA, and 0.097 and 0.59 s − 1 toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5 × 10 3 to 2 × 10 4 . The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu 507 and Glu 512 form the hydrogen bonds to the nitrogen atoms of His 455 and His 450 . All of these results showed that Glu 507 or Glu 512 stabilizes the coordination bond between the zinc ion and His 455 or His 450 .

Timolol, a beta-blocker, has been shown to be an effective ocular hypotensive agent when used alone or with carbonic anhydrase inhibitor on ocular hypertensive or open angle glaucoma patients. The effect of timolol hemihydrate on the CO(2) hydration activities of human carbonic anhydrase (HCA) I and II and their reaction mechanisms were investigated. Timolol activates the enzyme activities of HCA I and HCA II. In HCA I and II, the enzyme kinetic results clearly showed that timolol increases the value of V(max) but does not influence the value of K(m). The enzyme kinetic method showed that timolol noncompetitively activates HCA I and II activities through the formation of a ternary complex consisting of the enzyme, the substrate, and timolol. These results indicate that timolol binds apart from the narrow cavity of the active site. AutoDocking results showed that timolol binds at the entrance of the active site cavity in a region where the proton shuttle residue, His 64, of HCA I or II, is placed. The enzyme kinetic and AutoDocking results showed that timolol might weakly bind near the proton shuttle residue, His 64, to accelerate the proton transfer rate from His 64 to the buffer components. It is known that efficient activators of carbonic anhydrase possess a bulky aromatic/heterocyclic moiety and a primary/secondary amino group in their molecular structure. Timolol has a heterocyclic moiety and a secondary amino group, which are typical structures in efficient activators of carbonic anhydrase.

Latanoprost, a prostaglandin F2 alpha analogue, has been shown to be an effective ocular hypotensive agent when used alone on ocular hypertensive or open angle glaucoma patients. Carbonic anhydrase (CA) inhibitors are also used to reduce ocular hypertension by decreasing aqueous humor secretion, and are given in combination with prostaglandin F2 alpha analogue. It has been shown that prostaglandin F2 alpha, Minprostin F2 alpha, has been shown to increase the carbonic anhydrase (CA) activity and blood pressure. However, the effects of latanoprost on CA have not been clarified. Therefore, we studied the effects of latanoprost free acid on human carbonic anhydrase (HCA) I and II using the stopped flow method. Latanoprost free acid inhibited the hydration activity of HCA I or II by a noncompetitive mechanism. The inhibition constants (Ki) of latanoprost free acid for HCA I and II were 0.22 and 2.3 mM, respectively. Therefore, latanoprost free acid is a weak inhibitor of HCA I or II. AutoDock simulation of the latanoprost free acid-HCA I or II complex showed that the carboxylic moiety of latanoprost free acid, which is located at the end of the molecule, binds to the zinc ion of the active site by stretching of the chain of latanoprost free acid through the narrow and deep active site cavity of HCA I or II. In the active site cavity of HCA I or II, one side is hydrophilic and the other is hydrophobic. AutoDock simulation results clearly showed that latanoprost free acids lie down on the hydrophobic sides of the active site cavities in HCA I and II. The noncompetitive inhibition mechanism and the binding mode of latanoprost free acid indicate that the behavior of latanoprost free acid is very similar to that of simple anions.

The ESR spectra of dicupric human serum-transferrin (serum-Tf) were measured from -20 to 37°C in the liquid state (56% glycerol at pH 7.6). Two coordination geometries (types B-1 and B-2) with different ESR parameters were present at the N-site. The contents of the coordination geometry of type B-1 at the N-site increased as the temperature increased. The equilibrium constant between the coordination geometries of types B-1 and B-2 was determined by ESR spectra. The enthalpy value from type B-2 to B-1 was +5.3 kcal/mol, as obtained from a van't Hoff plot. The two conformational energies of the cluster models of the copper-binding site at the N-site of dicupric human serum-Tf, where the Arg124 residue was oriented in two different directions (conformations I and II), were calculated by Density Functional Theory, and the enthalpy value from conformation II to I was +2.1 kcal/mol. The enthalpy value was similar to that (+5.3 kcal/mol) obtained by the coordination geometrical change from type B-2 to B-1 in Cu(II)2 serum-Tf. In conformations I and II, the residue of Arg124 at the N-site is located either far from or near the copper-binding site, respectively, and in both cases the coordination geometry of the cupric ions at the N-site has changed from a flattened tetrahedron to a trigonal bipyramid. This result implies that the ESR spectral change from type B-2 to B-1 is caused by the presence of two different orientations of Arg124 in the change from conformation II to I.

Horse heart metmyoglobin (metMb), whose N-terminal Gly1 is linked by an N,N,N′,N″,N″-diethylenetriaminepentaacetatocobaltate(III) ion ([CoIII(dtpa)]) with an amide bond, was prepared and characterized. A one-electron reduced protein, [deoxyMb{CoIII(dtpa)}], was prepared by reduction with a methylviologen-radical cation, which was produced in situ by a photoreduction using a tris(2,2′-bipyridine)ruthenium(II) ion in the presence of disodium dihydrogen ethylenediaminetetraacetate at 25 °C, pH 7.5 (a 0.010 mol dm−3 tris(hydroxymethyl)aminomethane–HCl buffer), and an ionic strength of 0.50 mol dm−3 (NaCl). The reaction of [deoxyMb{CoIII(dtpa)}] to form [metMb{CoII(dtpa)}] obeyed a first-order rate law on the protein concentration. The first-order rate constant was dependent on the concentration of the protein, indicating that both intra- and intermolecular electron-transfer (ET) processes simultaneously occur. The latter is the reaction with excess [metMb{CoIII(dtpa)}] remaining. Both intra- and intermolecula...

The electron-transfer reactions from Fe(II)(CN)6 4− to the cupric ion in human- or bovine-copper, zinc-superoxide dismutase were followed at various pHs from pH 5.0 to 8.0 by the micro-stopped-flow method. From pH 5.0 to 8.0, Fe(II)(CN)6 4− first forms an adduct through the charge interaction with Arg 143 or 141 of the active cavity; electron-transfer then occurs from Fe(II)(CN)6 4− to the cupric ion of the enzyme in human- or bovine-copper, zinc-superoxide dismutase. The formation-constant values of the adducts in human- and bovine-copper, zinc-superoxide dismutase are almost constant between pH 7.0 and 8.0, but increase with decreasing pH at pH values lower than 7.0. In contrast, the electron-transfer rate constants from Fe(II)(CN)6 4− to the cupric ion of the enzyme after adduct-formation in human- and bovine-copper, zinc-superoxide dismutase are nearly constant from pH 5.0 to 8.0 and give almost the same values in bovine- or human-copper, zinc-superoxide dismutase. The pH dependencies of adduct-formation-constants in human- and bovine superoxide dismutase indicate that the protonation of some residue with pKa value less than 5.0 accelerates the formation of adducts formed through the charge interaction between Arg 143 (human) or Arg 141 (bovine) and Fe(II)(CN)6 4−. X-ray diffraction analysis data show that the arrangement of Arg 143 and Glu 133 around the copper ion in the active cavity of human-copper, zinc-superoxide dismutase is very similar to that of bovine copper, zinc-superoxide dismutase. The low pKa value of the residue involved in adduct-formation suggests that the protonation group should be the carboxylate. Protonation of the carboxylate group of Glu 133 (Glu 131) results in neutralization of the minus charge of the carboxylate group and a decrease in the electronic repulsion for Fe(II)(CN)6 4−, so that the formation-constant of the adduct should increase with decreases in pH at lower pH values.

The electron transfer reactions from Fe II (CN) 4− 6 and W IV (CN) 4− 8 to the cupric ions in human copper, zinc superoxide dismutase were followed by the micro-stopped-flow method. The kinetic rate data clearly indicate that Fe II (CN) 4− 6 or W IV (CN) 4− 8 first forms an adduct with the enzyme through the interaction with Arg143 of the active cavity and then an electron from Fe II (CN) 4− 6 or W IV (CN) 4− 8 of the adduct transfers to the cupric ion in the enzyme. The dissociation constants of the adducts of Fe II (CN) 4− 6 and W IV (CN) 4− 8 were 4.0(±0.3) × 10 −3 and 2.2(±0.3) × 10 −3 M, respectively. In spite of the difference between the standard redox potentials of Fe III (CN) 3− 6 /Fe II (CN) 4− 6 (468 mV) and W V (CN) 3− 8 /W IV (CN) 4− 8 (556 mV), the electron transfer rate constant (0.148(±0.005) s −1 ) of Fe II (CN) 4− 6 at 25°C is very similar to that of W IV (CN) 4− 8 (0.072(±0.011) s −1 ). The entropy values of the adduct formations and the activation energies of the electron transfer rates were determined by the temperature dependence of the dissociation constants of the adducts and the electron transfer rates. The enthalpy values of the formation of adducts are almost zero, so that the driving forces to form the adducts are mainly derived from the entropy. The activation energy of the electron transfer rate of Fe II (CN) 4− 6 is very similar to that of W IV (CN) 4− 8 . The formation of the adduct between Fe II (CN) 4− 6 and the enzyme was inhibited by the presence of various anions (ClO − 4 , SO 2− 4 , SCN − , and N − 3 ). The bulky anions SO 2− 4 and ClO − 4 behave as competitive inhibitors for Fe II (CN) 4− 6 ; these anions should interact mainly with Arg143, as it has a positive charge at the entrance of the active cavity. The competitive inhibition constants of ClO − 4 , SO 2− 4 , and SCN − were 0.010, 0.012, and 0.008 M. The azide ion, which is smaller than SO 2− 4 or ClO − 4 , shows mixed inhibition, because N − 3 can interact with Arg143 (competitive inhibition) and also directly binds to the cupric ion in h-SOD (noncompetitive inhibition). The competitive and noncompetitive inhibition constants of N − 3 were 0.004 and 0.016 M, respectively.

Bilirubin oxidase obtained from Trachyderma tsunodae K-2593 is a multi-copper enzyme which has type 1:type 2:type 3 copper atoms in the ratio 1:1:2. Anion binding properties of bilirubin oxidase were investigated to determine the structural and functional properties of bilirubin oxidase. The anions N3−, SCN−, F−, Cl−, and Br− were non-competitive inhibitors against the substrate, bilirubin ditaurate, and the inhibition constants of the anions were in the following order: N3−

We have purified dipeptidyl peptidase III (EC 3.4.14.4) from human placenta. It had a pH optimum of 8.8 and readily hydrolysed Arg-Arg-β-naphthylamide. Monoamino acid-, Gly-Phe-, Gly-Pro- and Bz-Arg-β-naphthylamides were not hydrolysed at all. The enzyme was inhibited by p-chloromercuriphenylsulphonic acid, metal chelators and 3,4-dichloroisocoumarin and contained 1 mol of zinc per mol of enzyme. The zinc dissociation constant was 250 fM at pH 7.4 as determined by the zinc binding study. We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNA species, a cDNA clone with 2633 bp encoding the rat enzyme. The longest open reading frame encodes a 827-residue protein with a theoretical molecular mass of 92790 Da. Escherichia coli SOLR cells were infected with the pBluescript phagemid containing the cloned cDNA and established the overexpression of a protein that hydrolysed Arg-Arg-β-naphthylamide. The recombinant protein was purified and the amino acid sequence of the protein was confirmed. We presumed that the putative zinc-binding domain involved in catalysis was present in the recombinant enzyme. It was a novel zinc-binding motif in that one amino acid residue was inserted into the conserved HEXXH motif characteristic of the metalloproteinases.

The dependence on pH of the absorption and circular dichroic spectra of iron(III), cobalt(III) and copper(II) transferrins has been (re)investigated. In the alkaline region, the CD profiles of iron(III) and cobalt(III) transferrin are essentially pH independent up to pH 11; only for very high pH values (pH > 11) is breakdown of the cobalt(III) and iron(III) transferrin derivatives observed, without evidence of conformational rearrangements. By contrast, the CD profiles of copper transferrin show drastic changes in shape around pH 10; these spectral changes, which are fitted to a pKa of ~10.4, are interpreted in terms of a substantial rearrangement of the local environment of the copper ions at high pH. Although the CD spectra of copper transferrin at alkaline pH strictly resemble those observed upon addition of modifier anions, the mechanism of site destabilization in the two cases is different; at variance with the case of modifier anions, our results suggest that the high pH form of copper transferrin still contains the synergistic anion. A13C NMR experiment has confirmed this view. In the acidic region, iron(III) and cobalt(III) transferrins are stable down to pH ~6. For lower pH values progressive metal detachment is observed without evidence of conformational changes; around pH 4.5 most bound metals are released. In the case of the less stable copper-transferrin, metal removal from the specific binding sites is already complete around pH 6.0; in concomitance with release from the primary sites, binding of copper ions to secondary sites is observed. Additional information has been gained from CD experiments in the far UV. The pH dependent properties of iron(III), cobalt(III) and copper(II) transferrin are discussed in the frame of the present knowledge of transferrin chemistry, particular emphasis being attributed to the comparison between tripositive and bipositive metal derivatives.

Dipeptidyl peptidase III (DPP III), the zinc peptidase, has a unique helix portion in the metal-binding motif (HELLGH). The enzyme activity of the cupric derivative of rat DPP III (Cu(II)-rat DPP III) for Lys-Ala-β-NA is about 30% of that of the wild-type enzyme. On the other hand, the enzyme activity of Cu(II)-rat del-DPP III, in which Leu453 is deleted from the metal-binding motif, possesses only 1–2% of the enzyme activity of rat del-DPP III. The EPR spectra of Cu(II)-rat DPP III in the presence of various concentrations of the substrate, Lys-Ala-β-NA, changed dramatically, showing formation of the enzyme–metal–substrate complex. The EPR spectra of Cu(II)-rat del-DPP III did not change in the presence of excess Lys-Ala-β-NA. The deletion of Leu453 from the HELLGH motif of rat DPP III leads to a complete loss of flexibility in the ligand geometry around the cupric ions. Under the formation of the enzyme–metal–substrate complex, Glu451 of Cu(II)-rat DPP III is sufficiently able to approach the water molecule via a very different orientation from that of the resting state; however, Glu451 of Cu(II)-rat del-DPP III is not able to access the water molecule.

The change in fluorescence spectra of crystalline pullulanase from Klebsiella pneumoniae caused by the addition of alpha-, beta-, and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin was investigated at 25 degrees C and pH 5.6. The fluorescence intensity at around 325 nm (excitation at 280 nm) was increased by the addition of all the cyclodextrins studied. The dissociation constant, Kd, of the enzyme-cyclodextrin complex was evaluated by fluorometric titration for each cyclodextrin, and was consistent with the inhibitor constant, Ki, obtained previously [Iwamoto et al. (1993) J. Biochem. 113, 93-96]. The Kd values of beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin were approximately two orders of magnitude smaller than those of alpha- and gamma-cyclodextrins. Fluorescence titration of a cyclodextrin in the presence of another cyclodextrin revealed competition among alpha-, beta-, and gamma-cyclodextrins for binding with the enzyme, which indicates that the binding region of beta-cyclodextrin overlaps those of alpha- and gamma-cyclodextrins. On the other hand, with excitation at 295 nm, a fluorescence spectral change similar to that excited at 280 nm was observed for alpha- and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin, whereas beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin did not show any such change. These results suggest that the binding site or the binding mode of beta-cyclodextrin is slightly different from those of alpha- and gamma-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Almost all naturally occurring metalloproteases are monozinc enzymes. The zinc in any number of zinc metalloproteases has been substituted by some other divalent cation. Almost all Co(II)- or Mn(II)-substituted enzymes maintain the catalytic activity of their zinc counterparts. However, in the case of Cu(II) substitution of zinc proteases, a great number of enzymes are not active, for example, thermolysin, carboxypeptidase A, endopeptidase fromLactococcus lactis, or aminopeptidase B, while some do have catalytic activity, for example, astacin (37%) and DPP III (100%). Based on structural studies of various metal-substituted enzymes, for example, thermolysin, astacin, aminopeptidase B, dipeptidyl peptidase (DPP) III, and del-DPP III, the metal coordination geometries of both active and inactive Cu(II)-substituted enzymes are shown to be the same as those of the wild-type Zn(II) enzymes. Therefore, the enzyme activity of a copper-ion-substituted zinc metalloprotease may depend on the flexibility of catalytic domain.

The electron-transfer rate between oxidized copper, zinc superoxide dismutase and Fe(CN) 6 4- has been measured for several mutants of human SOD and compared to the rate obtained with the native enzyme. Among the results, the following are meaningful: (i) substitution of the positive Arg at position 143 with a neutral group decreases the electron transfer rate, (ii) mutation of Thr-137 has little influence, and (iii) substitution of Glu-133 with the neutral Gln increases the rate.

The interaction between pullulanase from Klebsiella pneumoniae and alpha-, beta-, and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin was examined by means of inhibition studies of the enzyme activity, UV difference spectroscopy, and flow calorimetry. All the above cyclodextrins were found to be competitive inhibitors, but beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin showed strong inhibition, the inhibitor constants being two orders of magnitude less than those of alpha- and gamma-cyclodextrins. The difference spectra of beta-cyclodextrin were slightly but significantly different from those of the other cyclodextrins, showing blue shift of a few nanometers. Moreover, only beta-cyclodextrin has a positive entropy change upon binding with the enzyme; all the other cyclodextrins have negative values. These results show that the binding mode of beta-cyclodextrin is subtly different from those of alpha- and gamma-cyclodextrins.

Several Pt(IV) and Pt(II) complexes containing 1R, 2R-cyclohexanediamine (1R, 2R-dach) as a carrier ligand were synthesized. The cytotoxicities and the uptake of the platinum complexes by leukemia L1210 cells were compared in order to study the correlation between their structures and cytotoxicities.[Pt(II)Cl2(1R, 2R-dach)], [(Pt(II)(oxalato)(1R, 2R, -dach)], and [Pt(II)(malonato)(1R, 2R-dach)], which have excellent anticancer properties, exhibited very high cytotoxicities and were easily taken up by leukemia L1210 cells. [Pt(IV)Cl4(1R, 2R-dach)], trans(Cl)-[Pt(IV)Cl2(oxalato)(1R, 2R-dach)], and trans(Cl)-[Pt(IV)Cl2(malonato)(1R, 2R-dach)] also had high cytotoxicities. After a short incubation time, the uptake of [Pt(II)Cl2(1R, 2R-dach)], [Pt(II)(oxalato)(1R, 2R-dach)], and [Pt(II)(malonato)(1R, 2R-dach)] by leukemia L1210 cells were respectively very similar to those of [Pt(IV)Cl4(1R, 2R-dach)], trans(Cl)-[Pt(IV)Cl2(oxalato)(1R, 2R-dach)], and trans(Cl)-[Pt(IV)Cl2(malonato)(1R, 2R-dach)].In addition, trans(OH)-[Pt(IV)(OH)2Y2(1R, 2R-dach)] (Y2 : oxalato or malonato) did not exhibit cytotoxicity towards leukemia L1210 cells, whereas trans(Cl)-[Pt(IV)Cl2Y2(1R, 2R-dach)] (Y2 : oxalato or malonato) were highly cytotoxic. The accumulation of trans(OH)-[Pt(IV)(OH)2Y2(1R, 2R-dach)] in leukemia L1210 cells was much lower than that of trans(Cl)-[Pt(IV)Cl2Y2(1R, 2R-dach)]. Platinum(IV) complexes, in which leaving groups are replaced by hydroxide groups, have decreased cytotoxic activity, because the hydroxide groups of the platinum(IV) complex reduce the uptake of platinum by the cells. trans(OH), cis(Cl)-[Pt(IV)(OH)2Cl2(1R, 2R-dach)], which has hydroxide and chloride groups, was easily incorporated into the cells and exhibited the high cytotoxic activity. This behavior indicates that the chloride group apparently overcomes the ameliorating effect of the hydroxide group.

Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, "HELLGH," as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al. (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K(m) values or in the substrate specificity. However, the k(cat) values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His⁵⁶⁸ residue of rat DPP III is essential for enzyme catalysis. The k(cat) values of the mutants E507A and E512A were 2.38 and 3.88 s⁻¹ toward Arg-Arg-NA, and 0.097 and 0.59 s⁻¹ toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5 x 10³ to 2 x 10⁴. The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu⁵⁰⁷ and Glu⁵¹² form the hydrogen bonds to the nitrogen atoms of His⁴⁵⁵ and His⁴⁵⁰. All of these results showed that Glu⁵⁰⁷ or Glu⁵¹² stabilizes the coordination bond between the zinc ion and His⁴⁵⁵ or His⁴⁵⁰.

Square-planar complexes with the formula [PtCl2(S-Hp-Mepn)], where S-Hp-Mepn is (S)-1-(2-amino-2-methylpropyl)amino-2-propanol, CH3CH(OH)CH2NHCH2C(CH3)2NH2, were synthesized and separated into three types of crystals: small imbricate crystals (1), needles (2), and plates (3). 1H NMR spectroscopy has revealed that 1 and 2 are diastereoisomers each other but that 3 consists of both diastereoisomers in 1 : 1 composition. X-Ray crystallography has shown that the diastereoisomer 2 has the absolute configuration of R about the nitrogen atom of secondary amine and 3 is a quasi-racemic compound of S(N) and R(N) isomers: 2; orthorhombic, space group P212121 with a = 10.447(1), b = 13.017(2), c = 8.764(2) A, R = 0.043, Rw = 0.052: 3; monoclinic, space group C 2 with a = 21.530(1), b = 11.019(2), c = 13.491(5) A, β = 125.73(1)°, R = 0.042, Rw = 0.054. But the conformations of R(N)-isomers in 2 and 3 were different. The analysis of the CD and 1H NMR spectra of 1 and 2 shows that 1 has the absolute configuration of S ...

Square-planar complexes with the formula [PtCl2(R-Hb-Mepn)], where R-Hb-Mepn is (R)-2-[(2-methyl-2-aminopropyl)amino]-1-butanol CH3CH2CH(CH2OH)NHCH2C(CH3)2NH2, were synthesized and separated into two diastereoisomers. On the basis of CD spectroscopic and X-ray crystallographic analyses, (+)300- and (−)300-isomers were determined to have the R-nitrogen center with λ chelate ring and the S-nitrogen center with δ chelate ring, respectively. R(N)-[PtCl2(R-Hb-Mepn)] crystallizes in the monoclinic space group P21 with a = 8.809 (1), b = 8.9960 (4), c = 8.536 (1) A, β = 108.787 (5)°, Z = 2, R = 0.042, and RW = 0.062. S(N)-[PtCl2(R-Hb-Mepn)] crystallizes in the orthorhombic space group P212121 with a = 9.287 (1), b = 14.831 (1), c = 9.141 (1) A, Z = 4, R = 0.029, and RW = 0.034. 1H NMR spectra indicate that R-Hb-Mepn chelate rings take fixed conformation in acidic D2O solution. At high temeprature (90–95 °C), the conversion of the R(N)-isomer to the S(N)-isomer occurred easily. The R(N)-[PtCl2(R-Hb-Mepn)] and S(N...

A recombinant rat aminopeptidase-B (Ap-B) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli BL21 harboring a plasmid pGEX-Ap-B and was purified by glutathione-Sepharose 4B and Q-Sepharose columns. The metal-substituted derivatives of Ap-B, Co(II)- and Cu(II)-Ap-B contain almost 1 mole of cobalt(II) and copper(II) ions per enzyme molecule, respectively. The specific activity of Co(II)-Ap-B is very similar to that of recombinant Ap-B but that of Cu(II)-Ap-B is very low. The dissociation constants of the zinc ions of recombinant Ap-B and of the cobalt ions of Co(II)-Ap-B calculated from the relationships between the free metal ions and the residual enzyme activities are 3.7(+/-1.0)x10(-13) and 4.7(+/-1.0)x10(-12) M, respectively. The EPR parameters (gperpendicular), g// and A//) of Cu(II)-Ap-B were 2.06, 2.27, and 156x10(-4) cm-1. The A// value and the g// of Cu(II)-Ap-B are very similar to those of Cu(II)-thermolysin or Cu(II)-dipeptidyl peptidase III, in which the coordination geometry is a distorted tetrahedral.

Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.

Two monoclonal antibodies raised against Pt(II)(1R, 2R-cyclohexanediamine)-DNA were prepared, and the specificity of the monoclonal antibodies against Pt(II)(cyclohexanediamine)-DNA derivatives was determined by competitive enzyme-linked immunosorbent assay. The binding affinity of the monoclonal antibodies is apparently influenced by the cyclohexanediamine moiety of Pt(II)(cyclohexanediamine)-DNA adducts, but the monoclonal antibodies can not bind to the low molecular analogue, Pt(II)(1R, 2R-cyclohexanediamine)-d(GpG), which is the intrastrand binding model compound of Pt(II)(1R, 2R-cyclohexanediamine)-DNA. Therefore, the monoclonal antibodies recognize the macromolecular parts, including DNA duplex, in addition to the cyclohexane moieties of the platinum complexes on Pt(II)(cyclohexanediamine)-DNA adducts.

The zinc-binding motif (HELLGH) of dipeptidyl peptidase III (DPP III) is different from the common zinc-binding motif (HExxH) of metallopeptidases. To clarify the importance of the zinc-binding motif part of DPP III for enzymatic activity, we measured the recovery of the enzyme activity of apo-Leu453-deleted dipeptidyl peptidase III (apo-Leu453-del-DPP III), which has a motif (HELGH) like that of the common peptidase (HExxH), in the presence of various metal ions. The enzyme activity of apo-Leu453-deleted DPP III could not be recovered by the addition of cupric ions, while apo-DPP III could be easily reactivated by the addition of cupric ions. The visible and electron paramagnetic resonance spectra of the isolated Cu(II)-Leu453-del DPP III clearly show that the cupric ions of Cu(II)-Leu453-del-DPP III bound to the motif part (HELGH) but did not exhibit any enzyme activity. The motif part of DPP III directly influences the expression of the enzyme activity in the copper derivative of DPP III. The competitive inhibitor that is not at all digested by DPP III, Hisprophen (His-Pro-Phe-His-Leu-d-Leu-Val-Tyr), has been determined. The inhibition constant (Ki) of Hisprophen for DPP III or Cu(II)-DPP III was 4.1 × 10−5 or 3.8 × 10−5 M, respectively. In the presence of the competitive peptide inhibitor, Hisprophen, the EPR spectra of Cu(II)-DPP III were completely different from that of Cu(II)-DPP III itself. This result clearly indicates that the metal ions of DPP III are located in the active site and directly interact with the substrate.

The ascorbate oxidase obtained from a microorganism, Acremonium sp. HI-25 (molecular weight, 80 kDa; monomeric protein), was studied with respect to atomic absorption, EPR, absorption spectra, circular dichroism (CD) spectra, and steady-state kinetics. The enzyme was found to be a multicopper protein, containing four copper atoms of three kinds, types 1, 2, and 3 copper, in the ratio of 1:1:2. The EPR parameters of the type 1 and 2 copper atoms in the ascorbate oxidase are very similar to those in the case of the ascorbate oxidase obtained from cucumber, which is a dimeric protein. The apparent Km and kcat values for ascorbic acid of the ascorbate oxidase from Acremonium sp. HI-25 are almost the same as those of the monomeric unit of the ascorbate oxidase from cucumber.

Cyclic octapeptides, cyclo[L-Tyr(Bzl)-L-Pro]4 (2) and cyclo(L-Tyr-L-Pro)4 (3), were prepared and their conformations investigated. A C2-symmetric conformation containing two cis peptide bonds was found in 2 in CDCl3 and 3 in CD3OD. Cyclo(L-Phe-L-Pro)4 (1) formed a 1 : 1 complex with L-phenylalanine methylester hydrochloride (L-PheOMe·HCl). The 13C-NMR spectra of the complexes of 1 and 2 with 1 eq of D- and L-PheOMe·HCl (D/L ratio=1/2) in CDCl3 displayed separate resonances for several carbon atoms of D-PheOMe·HCl and L-PheOMe·HCl owing to the formation of diastereomeric pairs of the complexes.

Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(453)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-13), 5.8 (+/-0.7) x 10(-12), and 3.2 (+/-0.9) x 10(-10) M, respectively. The recoveries of the enzyme activities by the addition of various metal ions to apo-DPP III were also measured, and Co(2+), Ni(2+), and Cu(2+) ions completely recovered the enzyme activities as did Zn(2+). The dissociation constants of Co(2+), Ni(2+), and Cu(2+) ions for apo-DPP III at pH 7.4 were 8.2 (+/-0.9) x 10(-13), 2.7 (+/-0.3) x 10(-12), and 1.1 (+/-0.1) x 10(-14) M, respectively. The shape of the absorption spectrum of Co(2+)-DPP III was very similar to that of Co(2+)-carboxypeptidase A or Co(2+)-thermolysin, in which the Co(2+) is bound to two histidyl nitrogens, a water molecule, and a glutamate residue. The absorption spectrum of Cu(2+)-DPP III is also very similar to that of Cu(2+)-thermolysin. The EPR spectrum and the EPR parameters of Cu(2+)-DPP III were very similar to those of Cu(2+)-thermolysin but slightly different from those of Cu(2+)-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu(2+)-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu(2+)-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.

The absolute configuration of the anti-tumor complex trans(OH)-Pt(OH)2(malonato)(1R, 2R-cyclohexanediamine) was determined by X-ray anomalous scattering technique. The final unit cell was monoclinic, space group P21 with a = 9.142A, b = 7.788A, c = 11.946A, β = 96.48°, Z = 2. The crystal structure was determined by direct method and difference Fourier synthesis, and refined to R = 0.025 and Rw = 0.033 based on 2768 independent reflections. The platinum atom has roughly octahedral coordination. The cyclohexane ring has the expected chair configuration, with two amino groups in equatorial positions while the malonato ligand, in contrast, shows a boat conformation for six membered Pt O-C-C-C-O ring.

The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. None of the expressed mutated proteins exhibited DPP III activity. The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry. The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10%. These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity.

Aminopeptidase B (Ap B; EC 3.4.11.6) is a zinc-binding protein that contains the consensus sequence HEXXHX18E (324-347), conserved among the M1 family of metallopeptidases. To determine if these putative zinc-binding residues (His324, His328 and Glu347) and the active-site Glu325 are essential for the enzyme activity, we replaced the histidines with tyrosines and the glutamic acid residues with alanines using site-directed mutagenesis. The cDNAs were expressed in Escherichia coli, and the resulting recombinant proteins, named H324Y, E325A, H328Y and E347A, were purified to apparent homogeneity. None of the expressed mutated proteins showed aminopeptidase activity. The E325A enzyme contained 1 mol of zinc per mol of protein, and the other three mutants, H324Y, H328Y and E347A, did not contain significant amounts of zinc, as determined by atomic absorption spectrometry. From sequence-homology searches, Ap B is known to be closely related to leukotriene (LT)-A4 hydrolase (EC 3.3.2.6). We examined human placental Ap B and recombinant rat Ap B, both of which had been purified previously [Fukasawa, Fukasawa, Kanai, Fujii and Harada (1996) J. Biol. Chem. 271, 30731-30735], to determine whether or not they had epoxide hydrolase activities. However, neither enzyme hydrolysed LTA4 into LTB4. We then replaced some amino acids in the domain of the rat enzyme similar to the LTA4-binding site of LTA4 hydrolase. However, these mutants, Y408F, N409S and NE409-410SS also did not possess any epoxide hydrolase activity. We concluded that Ap B is an M1-family zinc metallopeptidase without epoxide hydrolase activity.

Copper binding selectivity of the N- and C-sites in serum (human)- and ovo-transferrin was investigated through copper binding constants, copper dissociation rate constants, and EPR spectra. At pH 7.4, stepwise copper binding constants of serum (human)-transferrin were K1 = 1.8 (±0.6) × 1012M−1and K2 = 1.2 (±0.5) × 1011M−1, and those of ovo-transferrin were K1 = 1.9 (±0.5) × 1011M−1 and K2 = 2.1 (±0.4) × 1011M−1. Absorbance changes resulting from copper binding to the C- or N-site at various ratios of Cu2+/apo-transferrin were separated by a kinetic method. It was clearly indicated that, in serum (human)-transferrin, the copper binding affinity for the C-site was much larger than that for the N-site, whereas in ovo-transferrin, the C- and N-sites have almost the same affinity for copper ions. In the presence of anions (0.1 M KCl or 0.1 M NaClO4), the stepwise copper binding constants of serum (human)-transferrin were almost 10-times smaller than those in the absence of the anions. The selectivity in binding the copper ions to both sites of serum (human)-transferrin in the presence of 0.1 M NaClO4 is much smaller than that in the presence of 0.1 M KCl or in the absence of anions. The copper dissociation rate constants from the N- and C-sites of dicupric serum-Tf were slightly changed by the addition of the anions (0.1 M KCl and 0.1 M NaClO4). EPR spectra of the copper ions of the N-site in dicupric serum-transferrin are dramatically changed respectively by the addition of 0.1 M KCl, 0.1 M NaCl, and 0.1 M NaClO4. This suggests that the change in the coordination geometry of the copper ions occurs at the N-site.

Ascorbate oxidase from the fungus Acremonium sp. HI-25 is a copper-containing glycoprotein that catalyzes the oxidation of ascorbic acid to dehydroascorbic acid. Monosaccharide composition analysis showed that the enzyme contains exclusively N-linked oligosaccharide chains. Following liberation by hydrazinolysis/re-N-acetylation, and fractionation by HPLC on anion exchange, Amide-80 and/or octadecyl silica columns after derivatization with P-aminobenzoic ethyl ester, the structures of the twelve major neutral oligosaccharides were identified by FAB-MS, 400 MHz 1H-NMR, methylation analysis, mild acid hydrolysis, and/or sequential exoglycosidase digestions. Acremonium sp. ascorbate oxidase was found to consist of high-mannose type oligosaccharides (76.3%) having 4 to 9 mannose residues and a series of novel D-galactofuranose-containing high-mannose type oligosaccharides (18.6%) with the following structure.

Zinc deficient bovine superoxide dismutase (Cu2E2SOD (E = empty)) was prepared and purified by high performance liquid chromatography (HPLC). Each peak was characterized as to protein, copper content and specific activity. The Cu2E2SOD peak fractionated by HPLC has a low specific activity at pH 7.8 (about 10% of the native enzyme (Cu2Zn2SOD)). With the addition of zinc ions, the specific activity of Cu2E2SOD was quantitatively restored to that of the native enzyme. This behavior implies that the zinc ion is very important for the appearance of enzyme activity.

Antibodies reactive to (1R,2R)-cyclohexanediamineplatinum(II)-DNA ((1R,2R)-cyclohexanediamine: 1R,2R-dach) adducts were elicited by immunization of rabbit with calf thymus DNA modified by Pt(1R,2R-dach)Cl2 at a ratio of bound platinum per nucleotide ((D/N)b) of 0.0335. In an enzyme-linked immunosorbent assay (ELISA), the binding of specific antibodies to Pt(1R,2R-dach)-DNA adduct (60 microliters of 1.235 x 10(-7) M Pt in each wells) on the assay plate was competitively inhibited by Pt(1R,2R-dach)-DNA adduct ((D/N)b = 0.0653) in the solution. Almost equal inhibition was observed with Pt(1S,2S-dach)-DNA ((D/N)b = 0.0412), an optical isomer of 1R,2R-dach. Pt(1R,2S-dach)-DNA ((D/N)b = 0.0371) and Pt(1R,3S-dach)-DNA ((D/N)b = 0.0281) in which the cyclohexane ring is stereochemically perpendicular to the platinum chelate plane, also inhibited antibody binding, but these adducts gave only incomplete inhibition at higher Pt-DNA adduct concentrations. Although Pt(1R,2R-dach)-d(GpG) and Pt(1R,2R-dach)(NH3)2 inhibited antibody binding, the affinity of the antibody for Pt(1R,2R-dach)(NH3)2 was lower than with Pt(1R,2R-dach)-DNA, and the inhibition behavior of Pt(1R,2R-dach)-d(GpG) was biphasic, i.e., at the lower concentration the inhibition curve was consistent with that of Pt(1R,2R-dach)-DNA, but at the higher concentration it shifted to that of Pt(1R,2R-dach)(NH3)2. The affinity of the antibody for cis-DDP was markedly lower than with Pt(1R,2R-dach)(NH3)2. These facts suggest that the antibodies may bind to the substituents (the platinum and its surroundings) of the various Pt complexes rather than the DNA structure altered by platinum binding.

(1992). Bilirubin Oxidase from Trachyderma tsunodae K-2593, a Multi-copper Enzyme. Bioscience, Biotechnology, and Biochemistry: Vol. 56, No. 8, pp. 1349-1350.

In order to determine a micro amount of cis- and trans-platin (cis- and trans-diamminedichloroplatinum(II)) separately, we have utilized the trans effect of SCN-.cis-Platin reacted with potassium thiocyanate to give anionic [Pt(SCN)4]2- which was extracted into nitroethane as an ion pair with zephiramine. On the other hand, trans-platin gave neutral trans-[Pt(SCN)2(NH3)2] under the same conditions, which was not extracted into nitroethane. Therefore, it is possible to determine cis- and trans-platin, separately, by the solvent extraction method. In the determination of the mixture of cis- and trans-platin (4.00×10-6 and 2.67×10-6M, respectively), the recovery of each platinum complex was 94±4 and 101±10% for cis- and trans-platin, respectively.

When the two copper ions at the active sites of bovine erythrocyte superoxide dismutase were reduced by Fe(CN)64−, a single stage kinetic process was observed. This result indicates that the two copper sites in bovine erythrocyte superoxide dismutase are equivalent for reduction by Fe(CN)64−.

The behavior of complexing agents for the copper removal reaction was studied by the equilibrium dialysis method. In the copper removal reaction, complexing agents are divided into two types: those that are reducing agents and those that are not. Sodium cyanide and sodium thiosulfate are of the first type, and 8-hydroxyquinoline-5-sulfonic acid, 2,2′-bipyridyl, and picolinic acid are of the second type. From equilibrium dialysis with the first type of complexing agent, the apparent binding constant (pH 6.0) between cuprous ions and apotyrosinase was calculated to be 10 15 m −1 . Similarly, the apparent binding constant (pH 6.0) between cupric ions and apo-tyrosinase was about 10 13 m −1 , which was calculated from equilibrium dialysis with the second type of complexing agent. The apparent binding constant between cuprous ions and apo-tyrosinase was larger than that between cupric ions and apo-tyrosinase.