Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme.
Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-) ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0) values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower (0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of another affinity site specific to the polysaccharides.
Yoshinaga
K
K
The United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-4, Kagoshima, Japan.
Fujisue
M
M
Abe
J
J
Hanashiro
I
I
Takeda
Y
Y
Muroya
K
K
Hizukuri
S
S
D000643:Ammonium Chloride / Q000494:pharmacology; D001974:Bromosuccinimide / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D007700:Kinetics; D011133:Polysaccharide-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000461:Rhodophyta / Q000201:enzymology*
D000643:Ammonium Chloride; D011133:Polysaccharide-Lyases; C080474:alpha-1,4-glucan lyase; C023911:pectate lyase; D001974:Bromosuccinimide; D005022:Ethyldimethylaminopropyl Carbodiimide; D002118:Calcium