Yasuko Kawamura-Konishi

Purification, Characterization, and Molecular Cloning of Tyrosinase from Pholiota nameko

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.

Peroxidase activity of an antibody–ferric porphyrin complex

Abstract The catalytic antibody 2B4 which catalyzes insertion of a cupric ion into porphyrin also combines with ferric porphyrin to form an antibody–ferric porphyrin complex. The antibody has distinct amino acid sequences in complementarity-determining regions compared to other anti-porphyrin antibodies reported. The 2B4-ferric porphyrin complex oxidized o -dianisidine and 2,2′-azino- bis (3-ethylbenz-thiazoline-6-sulfonic acid) utilizing hydrogen peroxide more efficiently than ferric porphyrin, but did not oxidize pyrogallol nor hydroquinone. The peroxidase reaction of the complex was examined kinetically for o -dianisidine, and compared with that of ferric porphyrin. With increasing concentrations of o -dianisidine, the reaction rate obtained for ferric porphyrin increased gradually, in contrast, that for the complex increased steeply and then saturated. These results indicated that the interaction of the complex with o -dianisidine was much higher than that of ferric porphyrin. At a constant concentration of o -dianisidine, the reaction rates obtained for the complex and for ferric porphyrin both showed saturation behavior against hydrogen peroxide concentration. The K m value for hydrogen peroxide of the complex was similar to that of ferric porphyrin but much larger than that of natural peroxidase, suggesting that the antibody did not have a residue facilitating the binding of hydrogen peroxide as in natural peroxidase. In the reaction of the complex with hydrogen peroxide, active intermediates were not observed. Based on the results, a scheme for the peroxidase reaction by the complex was proposed. It was considered that the enhancement of the peroxidase activity by the antibody was mainly attributed to an increase in the interaction with o -dianisidine, and that the substrate specificity of the complex resulted from the difference in the interaction.

Kinetics of the reconstitution of hemoglobin from semihemoglobins α and β with heme

Kinetics of the reconstitution of hemoglobin from semihemoglobins α and β with hemin dicyanide have been investigated using three kinds of stopped-flow technique (Soret absorption, fluorescence quenching of tryptophan, and Soret CD). The semihemoglobins α and β are occupied by heme in the α and β chains, respectively, the other chain being heme-free. Based on the kinetic results, the following scheme for the reconstitution is proposed; First, hemin dicyanide enters the pocket-like site of the apo chains. Second, in semihemoglobin α, the CN-ligand in the fifth coordination position of iron is replaced by the imidazole ring of the proximal His immediately after the heme insertion. In contrast, semihemoglobin β changes its conformation after the heme insertion, and this is followed by the ligand replacement. Finally, the partial structure changes induced by the ligand replacement propagate onto the whole molecule and the final conformation is attained. The results indicate that semihemoglobin α retains a more rigid and organized structure, and more closely approaches its final structure than does semihemoglobin β.

Interaction between αl and β1 subunits of human hemoglobin

We prepared normal and modified α and β globin chains in which C-terminal residues were enzymatically removed. The CD spectra of the deoxy form of these chains and the reconstituted modified Hbs were measured in the Soret region. The CD spectra of the modified Hbs were markedly different from the arithmetic means of respective spectra of their constituent chains. This difference was ascribed to the interaction between αl and β1 subunits to make the α1βl dimer. The peak wavelength of the difference CD spectra could be classified into two groups, one was 433±1 nm and the other 437±1 nm. A comparison of this classification with the previously identified quaternary structures revealed that the R and T structures showed a maximum of the difference CD spectra at 437±1 nm and 433±1 nm, respectively. These results indicated that the R and T structures differed in the interaction between α 1 and β1 subunits.

The reaction of Aspergillus niger catalase with methyl hydroperoxide

The formation of Compound I from Aspergillus niger catalase and methyl hydroperoxide (CH3OOH) has been investigated kinetically by means of rapid-scanning stopped-flow techniques. The spectral changes during the reaction showed distinct isobestic points. The second-order rate constant and the activation energy for the formation of Compound I were 6.4 x 10(3) M-1s-1 and 10.4 kcal.mol-1, respectively. After formation of Compound I, the absorbance at the Soret peak returned slowly to the level of ferric enzyme with a first-order rate constant of 1.7 x 10(-3) s-1. Spectrophotometric titration of the enzyme with CH3OOH indicates that 4 mol of peroxide react with 1 mol of enzyme to form 1 mol of Compound I. The amount of Compound I formed was proportional to the specific activity of the catalase. The irreversible inhibition of catalase by 3-amino-1,2,4-triazole (AT) was observed in the presence of CH3OOH or H2O2. The second-order rate constant of the catalase-AT formation in CH3OOH was 3.0 M-1 min-1 at 37 degrees C and pH 6.8 and the pKa value was estimated to be 6.10 from the pH profile of the rate constant of the AT-inhibition. These results indicate that A. niger catalase forms Compound I with the same properties as other catalases and peroxidases, but the velocity of the Compound I formation is lower than that of the others.

Kinetic studies on the reaction mechanism of sarcosine oxidase

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC 1.5.3.1) isolated from Corynebacterium sp. U-96 contains both covalently bound FAD and noncovalently bound FAD. The noncovalent FAD reacts with sarcosine, the covalent FAD with molecular oxygen (Jorns, M.S. (1985) Biochemistry 24, 3189-3194). To clarify the reaction mechanism of the enzyme, kinetic investigations were performed by the stopped-flow method as well as by analysis of the overall reaction. The absorption spectrum of the enzyme in the steady state was very similar to that of the oxidized enzyme, and no intermediate enzyme species, such as a semiquinoid flavin, was detected. The rate for anaerobic reduction of the noncovalently bound FAD and the covalently bound FAD by sarcosine were 31 and 6.7 s-1, respectively. The latter value was smaller than the value of respective Vmax/e0 obtained by the overall reaction kinetics (Vmax/e0: the maximum velocity per enzyme concentration). Both rate constants for oxidation of the two FADs by molecular oxygen were 100 s-1. A reaction scheme of sarcosine oxidase is proposed to account for the data obtained; 70% of the enzyme functions via a fully reduced enzyme, and 30% of the enzyme goes along a side-path, without forming the fully reduced enzyme. In addition, it is suggested that the reactivity of noncovalently bound FAD with sarcosine is affected by the oxidation-reduction state of the covalently bound FAD, in contrast to the reactivity of the covalently bound FAD with molecular oxygen, which is independent of the oxidation-reduction state of the noncovalently bound FAD.

Key residue responsible for catalytic activities in the antibodies elicited against N-methyl mesoporphyrin

Abstract Five catalytic and nine non-catalytic antibodies for insertion of a metal ion into porphyrin were generated by immunization with N-methyl mesoporphyrin (N-MMP) as hapten, which was designed to mimic the distortion of porphyrin toward a transition-state geometry in the reaction. In order to determine the features responsible for the catalytic activity, we characterized the properties of the catalytic and non-catalytic antibodies. The catalytic antibodies did not have higher affinity to N-MMP than the non-catalytic ones. All the antibodies, except one non-catalytic antibody, combined with ferric N-methyl mesoporphyrin (N-MMP-Fe) to form the respective antibody·N-MMP-Fe complex. The binding affinity of cyanide to ferric iron in the complexes agreed with that of free N-MMP-Fe, indicating that the protruding side of N-MMP-Fe in the complexes is exposed to solvents. All the complexes of the catalytic antibodies had a peroxidase-like activity, whereas those of the non-catalytic ones did not. This suggests that the metalation activity associates with the peroxidase-like one, so that there is a common residue acting as catalyst for both reactions. The amino acid sequence alignment shows that the catalytic antibodies contain a homologous heavy chain sequence in the third complementarity-determining region. Based on the results, the possibility that Asp(H96) in the region is the key residue responsible for the metalation and peroxidase-like activities is discussed.

Chemical modification of a catalytic antibody that accelerates the hydrolysis of carbonate esters.

Catalytic antibody, 4A1, catalyzes the hydrolysis of p-nitrophenyl alkyl carbonate. To determine the amino acid residues related to the catalytic activity of the antibody, we studied the effect of Tyr-, Trp-, and Lys-selective reagents on the catalytic activity and determined the amino acid sequences around the modified amino acid residues. We found that the Tyr-selective reagent is the most effective one and the modification of one Tyr residue results in the complete loss of the catalytic activity. The modified Tyr residue is identified to be Tyr-32 in the CDR-1 of the L chain.

A catalytic antibody that accelerates the hydrolysis of carbonate esters. Prediction of the binding-site structure of the substrate.

Monoclonal antibodies catalyzing the hydrolysis of p-nitrophenyl alkyl carbonate were obtained using p-nitrophenyl phosphonate as hapten. One of the antibodies, 4A1, has a relatively high activity for the substrate having a bulky group. To determine the amino acid residues related to the binding of the bulky group, we determined the amino acid sequences of VL and VH regions of 4A1 by the cycle sequencing method, built the three-dimensional structure of the V regions, labeled 4A1 with [14C]phenyl glyoxal in the presence and absence of I-1 or I-13, and analyzed the labeled incubation mixture with SDS–PAGE. From these results, the possibility that Arg-H28 of the heavy chain is involved in binding the bulky group of the substrate is discussed.

Kinetics of Formation of Antibody-Ferric Porphyrin Complex with Peroxidase Activity

The antibody 2B4 combines with ferric mesoporphyrin to form an antibody-ferric mesoporphyrin complex which has a peroxidase activity. Formation of the complex was investigated by measuring the absorption in the Soret region after mixing the antibody and ferric mesoporphyrin. A rapid increase and a gradual decrease in the absorption were observed, and the respective first-order rate constants were obtained. From the dependence of values of the rate constants on the concentration of ferric mesoporphyrin, the complex formation was explained by a plausible mechanism, in which the antibody associated with ferric mesoporphyrin to form the first complex followed by a conformational change to the second complex. The first complex had almost the same peroxidase activity as that of the second complex. Our results suggests that the antibody acquires the peroxidase activity as soon as ferric mesoporphyrin is incorporated into its binding site, and that there will be no protein ligand to the iron center of ferric mesoporphyrin in the complex.