MODE OF ACTION AND PARTIAL-PURIFICATION OF THE ACTIVE-CENTER OF EXO-POLY-ALPHA-D-GALACTURONOSIDASE FROM SELENOMONAS-RUMINATIUM

In the presented paper are summarized results of the study of the mode of action, dimensions and arrangement of the active centre of the CXO -poly-alpha-D-galacturonosidase, (poly(1,4-alpha-D-galactosiduronate) digalacturonohydrolase, E.C. 3.2.1.82) produced by the bacteria Seleno monas ruminantium. With this aim we determined experimentally values o f 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-degrees-C. We c alculated molecular activities k0 and parameters k0/K(m) from these va lues. From the dependence of log ko and log k0/K(m) on the degree of p olymerization rive subsites of the active centre were determined. with the catalytic site being situated between the second and third one. K inetic data were used for the calculation of the affinities of the fou rth and fifth subsites A4 and A5 in accordance with the theory of Hiro mi. Product analysis of non-labelled oligo(D-galactosiduronates) and c ompounds labelled with [1-H-3] al the reducing end enabled to ascertai n approximately the value for the first subsite Al of the active centr e and lo study the mode of action of the exo-poly-alpha-D-galacturonos idase from Selenomonas ruminantium.