Partial Characterization of the Active Site of Carrot Poly(galacturonate) Hydrolase and Degradation of Oligo(D-galactosiduronates)

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/Km were calculated from the experimentally determined Michaelis constants Km 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/Km 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 (Ai) 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.