Martine Urrutigoïty

Biocatalysis in Organic Solvents with a Polymer-Bound Horseradish Peroxidase

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.

Application of the reaction of dithioesters with ε-amino groups in lysine to the chemical modification of proteins

Abstract The reaction of lysine with dithioesters was applied to horseradish peroxidase donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) using car☐ymethyl dithiotridecanoate: three to four lysine residues were modified. The modified enzyme was soluble and active in diethyl ether. Papain (EC 3.4.22.2) was modified with car☐ymethyl dithiobenzoate: two lysine residues were modified. The modified enzyme was soluble and active in dimethylsulfoxide. From these results it is concluded that dithioesters are efficient reagents for the modification of peripheral lysine residues of proteins. Aromatic dithioesters, less reactive but more selective, should be recommended for thiol-dependent enzymes such as papain.

Use of pyrocarbonates for chemical modification of histidine residues of horseradish peroxidase

Abstract In an attempt to alter the catalytic properties of horseradish peroxidase (HRP, EC 1.11.1.7), various electrophiles were employed to modify histidine residues in this enzyme. Pyrocarbonates were found to be particularly effective, and their chromatic effect was exploited to determine the number of modified histidine residues directly by uv spectroscopy. We also developed a method for assay of histidines using diethyl pyrocarbonate, which could be extended to determination of these residues in other proteins. We showed that the catalytic activity of HRP was not affected by modification of histine residues, especially His 170, by small-sized substituents not containing reactive groups. On the other hand, electron-rich substituents, especially those with a heteroatom such as sulfur, disrupt the heme structure without producing the catalytic properties of cytochrome P450.

Effect of linking allyl and aromatic chains to histidine 170 in horseradish peroxidase.

Histidine residues in horseradish peroxidase (HRP) were modified chemically with diethyl pyrocarbonate, 4,omega-dibromoacetophenone or diallylpyrocarbonate. Histidines were chosen as His-170, the fifth ligand of the heme iron atom, forms part of the active site of this enzyme. Good yields of hemoprotein were obtained in all cases. Analysis by HPLC of peptides obtained after tryptic digestion showed that His-170 of HRP was in fact modified. The specific activity remained satisfactory after chemical modification of the histidine residues, and so the active site of HRP can thus be altered without a dramatic loss of hemoprotein or peroxidase activity. This may open routes to the preparation of novel biocatalysts.

Dichlorido[(S,RS)‐1‐diphenylphosphino‐2‐(ethylsulfanylmethyl)ferrocene]palladium(II)

The reaction of enantiomerically pure planar chiral ferrocene phosphine thioether with bis(acetonitrile)dichloridopalladium yields the title square-planar mononuclear palladium complex as an enantiomerically pure single diastereoisomer, [PdFe(C5H5)(C20H20PS)Cl2]. The planar chirality of the ligand is retained in the complex and fully controls the central chirality on the S atom. The absolute configuration, viz. S for the planar chirality and R for the S atom, is unequivocally determined by refinement of the Flack parameter.

Chemical Modification of Aspartic Acid and Arginine Residues in Horseradish Peroxidase Role of ASP 43 and ARG 38 in the Catalytic Cycle

In an attempt to alter the catalytic properties of horseradish peroxidase (HRP, EC 1.11.1.7), aspartic, glutamic and arginine residues were modified using ethanedithiol and diacetyl. Modification of Asp and Glu led to a marked increase in Vmax along with denaturation of the protein. The thiol groups introduced were thought to be responsible, despite being situated on the periphery of the molecule as shown by the modification of the apoenzyme. The role of Arg 38 in the activation of hydrogen peroxide was indicated by the modifications of both enzyme and apoenzyme. An amino acid residue close to Arg 38 was thought to take over its function after blocking the group.