Collect. Czech. Chem. Commun.
1993, 58, 681-692
https://doi.org/10.1135/cccc19930681
Mode of Action and Partial Purification of the Active Centre of exo-Poly-α-D-galacturonosidase from Selenomonas ruminantium
Kveta Heinrichováa, Július Heinrichb, Mária Dzúrováa and Alexander Zioleckic
a Institute of Chemistry, Slovak Academy of Sciences, 842 38 Bratislava, Slovak Republic
b Institute of Pharmacy, Komensky University, 842 32 Bratislava, Slovak Republic
c Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 03 110 Jablonna, Poland
Abstract
In the presented paper are summarized results of the study of the mode of action, dimensions and arrangement of the active centre of the exo-poly-α-D-galacturonosidase, (poly(1,4-α-D-galactosiduronate) digalacturonohydrolase, E.C. 3.2.1.82) produced by the bacteria Selenomonas ruminantium. With this aim we determined experimentally values of Michaelis constants and limiting rates for the catalytic hydrolysis of linear oligo(D-galactosiduronates) of the degree of polymeration in the range of 3 to 8, at pH 7.0 and the temperature 30 °C. We calculated molecular activities k0 and parameters k0/Km from these values. From the dependence of logk0 and logk0/Km on the degree of polymerization five subsites of the active centre were determined, with the catalytic site being situated between the second and third one. Kinetic data were used for the calculation of the affinities of the fourth and fifth subsites A4 and A5 in accordance with the theory of Hiromi. Product analysis of non-labelled oligo(D-galactosiduronates) and compounds labelled with [1-3H] at the reducing end anabled to ascertain approximately the value for the first subsite A1 of the active centre and to study the mode of action of the exo-poly-α-D-galacturonosidase from Selenomonas ruminantium.
