ABSTRACT
Extracellular Penicillium fellutanumexo-β-d-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP2GMii and pP25GMii(containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-O-methyl-β-d-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP2GMii or pP25GMii. Thek
cat/Km
value for pP25GMii is 1.7 × 103M−1 s−1, that for 1-O-methyl-β-d-galactofuranoside is 1.1 × 104 M−1 s−1, that for pP2GMii is 1.7 × 10 4M−1 s−1, and those for 5-O-β-d-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 × 105 and 4.1 × 105 M−1s−1, respectively. Variability in thek
cat/Km
values is due primarily to differences in Km
values; thek
−1/k
1 ratio likely provides the most influence on Km. k
cat increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pPxGMii added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml−1h−1. No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pPxGMii and of related polymers is proportional to thek
cat/Km
value of each polymer. The kinetic data show that thek
cat/Km
value increases as the number of phosphodiesters of pPxGMiidecreases, also resulting in an increase in the activity of exo-β-d-galactofuranosidase.
Kinetics of ATP and inorganic phosphate release during hydrolysis of ATP by rabbit skeletal actomyosin subfragment 1. Oxygen exchange between water and ATP or phosphate.
