Collection of Czechoslovak Chemical Communications - digital archive 

Abstract

The dimension and arrangement of the active center in carrot poly(1→4-α-D-galacturonide)galacturonohydrolase (E.C.3.2.1.67) is studied. Molecular activities k₀ and parameters k₀/K_m were calculated from the experimentally determined Michaelis constants K_m and maximum rates of catalytic hydrolysis of linear oligo(D-galactosiduronates) (polymerization degree n = 2 to 7) at pH 5.0 and 30 °C. From the dependence of log k₀ and log k₀/K_m on n we derived that the active center consists of six subsites, the catalytic site being situated between the first and second subsite. In accord with the theory of Hiromi [Hiromi K.: Biochem. Biophys. Res. Commun. 40, 1 (1970)], the kinetic data were used for calculation of affinities (A_i) of the third (A₃) to the sixth (A₆) subsite. Two possible models were studied for the action of carrot poly(galacturonate)hydrolase which catalyzes the gradual terminal hydrolytic cleavage of oligo(D-galactosiduronates) from the non-reducing end of the molecule. The distribution of products in monomolecular hydrolysis of penta(D-galactosiduronate) under optimal conditions (pH and temperature) indicates a multi-chain enzymatic attack with predominant single collision. The kinetic results of the enzyme degradation are in good accord with the above-mentioned assumption.