Jerome Souppe

Chemical modification of horseradish peroxidase with ethanal-methoxypolyethylene glycol: Solubility in organic solvents, activity, and properties

Abstract The oxidation of polyethylene glycol monomethyl ethers (MW 350, 1990, and 5000) by the Moffatt-Swern method to the corresponding aldehyde is described. These aldehydes are used to modify horseradish peroxidase (HRP) by a reductive amination. The modification of two to three e-NH2 groups of the enzyme was observed. The isoelectric point of the native HRP (pI 8.8) was shifted to pI 5.5 on modification. The modified enzymes have an activity close to that of the native enzyme. Only the enzyme modified with the aldehyde MW 5000 (HRP 5000) was soluble and active in organic solvents like toluene, dioxane, and methylene chloride. In toluene, HRP 5000 was more sensitive to hydrogen peroxide inhibition than in buffer. At room temperature, it is more stable in toluene than in buffer.

Assay of phenylethanolamine N-methyltransferase activity using high-performance liquid chromatography with ultraviolet absorbance detection

A simple and rapid method for measuring phenylethanolamine N-methyltransferase (PNMT) activity by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. This assay requires a partially purified PNMT preparation derived from bovine adrenals, with noradrenaline and S-adenosyl-L-methionine (SAM) as co-substrates. After incubation, the reaction is stopped by addition of acid and the reaction mixture is analysed directly by HPLC. The enzymatically formed S-adenosyl-L-homocysteine (SAH) is detected at 258 nm and determined. Under optimum conditions, the stability of SAH allowed automation of the HPLC detection. This assay was validated by the determination of the kinetic properties of PNMT. Km values for noradrenaline and SAM defined in this assay (16 and 5.7 microM, respectively) are consistent with previously published values. This assay is simple enough to be used for large series of measurements of PNMT activity testing new methyl acceptors, potential inhibitors or PNMT activity in adrenal medulla.

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.

Crude extracts of yeasts in biocatalysed organic reactions: the example of Pichia pastoris

Abstract This paper deals with enzymatic activities present in dialyzed crude extracts of methanol-grown Pichia pastoris : oxidation of NADH by O 2 , degradation of NADH, secondary alcohol dehydrogenase and formate dehydrogenase activities. The use of crude extracts of the yeast as a biocatalyst is discussed with respect to the different enzyme activities in order to choose the best experimental conditions to carry out bioconversions involving NADH or NAD + regenerations.

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.