Relationship of Exo-β- d -Galactofuranosidase Kinetic Parameters to the Number of Phosphodiesters in Penicillium fellutanum Peptidophosphogalactomannan: Enzyme Purification and Kinetics of Glycopeptide and Galactofuran Chain Hydrolysis

Abstract

ABSTRACT Extracellular Penicillium fellutanum exo-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 pP 2 GM ii and pP 25 GM ii (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 pP 2 GM ii or pP 25 GM ii . The k cat / K m value for pP 25 GM ii is 1.7 × 10 3 M −1 s −1 , that for 1- O -methyl-β- d -galactofuranoside is 1.1 × 10 4 M −1 s −1 , that for pP 2 GM ii is 1.7 × 10 4 M −1 s −1 , and those for 5- O -β- d -galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 × 10 5 and 4.1 × 10 5 M −1 s −1 , respectively. Variability in the k cat / K m values is due primarily to differences in K m values; the k −1 / k 1 ratio likely provides the most influence on K m . 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 pP x GM ii 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 −1 h −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 pP x GM ii and of related polymers is proportional to the k cat / K m value of each polymer. The kinetic data show that the k cat / K m value increases as the number of phosphodiesters of pP x GM ii decreases, also resulting in an increase in the activity of exo-β- d -galactofuranosidase.