Collection of Czechoslovak Chemical Communications - digital archive 

Abstract

Alcohol dehydrogenase (E.C.1.1.1.1) from germinating pea seedlings was modified by treatment with diethyl pyrocarbonate. The inactivation rate is proportional to the molar concentration of the modifying agent; the inactivation was almost complete in fifty minutes at a diethyl pyrocarbonate concentration of 5 . 10^-6 mol/l. The histidine content calculated from the absorbance difference at 240 nm was 3.43 residues per molecule of native and 4.75 residues per molecule of demetalized enzyme. A correlation of the absorbance difference at 240 nm with a 100% loss of enzymatic activity shows that 1.22 histidine residue is essential for the activity of alcohol dehydrogenase. The dependence of the inactivation rate constant on the pH of the medium indicates that the treatment of pea alcohol dehydrogenase with diethyl pyrocarbonate results in the modification of one group only with a pK of 7.1, well corresponding to the imidazole group of histidine. The enzyme is partially protected against inactivation by NADH at a concentration close to the Michaelis constant for NADH. The treatment of the ethoxyformylated enzyme with hydroxylamine followed by dialysis restored the activity of pea alcohol dehydrogenase by 88%.