Relationship of exo-beta-D-galactofuranosidase kinetic parameters to the number of phosphodiesters in Penicillium fellutanum peptidophosphogalactomannan: enzyme purification and kinetics of glycopeptide and galactofuran chain hydrolysis.
Extracellular Penicillium fellutanum exo-beta-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-beta-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 x 10(3) M(-1) s(-1), that for 1-O-methyl-beta-D-galactofuranoside is 1.1 x 10(4) M(-1) s(-1), that for pP(2)GM(ii) is 1.7 x 10 (4) M(-1) s(-1), and those for 5-O-beta-D-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 x 10(5) and 4.1 x 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-beta-D-galactofuranosidase.
Tuekam
B A
BA
Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611-0700, USA.
Park
Y I
YI
Unkefer
C J
CJ
Gander
J E
JE
D002240:Carbohydrate Sequence; D005690:Galactose / Q000031:analogs & derivatives; D006020:Glycopeptides / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry*; D006026:Glycoside Hydrolases; D006868:Hydrolysis; D007700:Kinetics; D008351:Mannans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010755:Organophosphates / Q000737:chemistry*; D010407:Penicillium / Q000201:enzymology* / Q000254:growth & development; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D006020:Glycopeptides; D006025:Glycosaminoglycans; D008351:Mannans; D009844:Oligosaccharides; D010755:Organophosphates; C012990:galactomannan; D006026:Glycoside Hydrolases; C023112:exo-beta-D-galactofuranosidase; D001616:beta-Galactosidase; D005690:Galactose