Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity.
The extracellular poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis T1, which hydrolyzes both hydrophobic poly(3-hydroxybutyrate) and water-soluble oligomers of D(-)-3-hydroxybutyrate, lost its hydrolyzing activity toward the hydrophobic substrate on mile trypsin treatment, but retained its activity toward water-soluble oligomers. The molecular mass of the trypsin-treated enzyme was 44 kDa, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, which was 6 kDa smaller than that of the native enzyme (50 kDa). The trypsin-treated enzyme seemed to be less hydrophobic than the native one, because it was rather weakly adsorbed to a hydrophobic butyl-Toyopearl column compared with the native enzyme, and showed no ability to bind to poly(3-hydroxybutyrate), to which the native enzyme tightly bound. These results suggest that, in addition to a catalytic site, the enzyme has a hydrophobic site, which is not essential for the hydrolysis of water-soluble oligomers, but is necessary for the hydrolysis of hydrophobic substrates, and this hydrophobic site is removed from the enzyme by the action of trypsin.
Fukui
T
T
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Narikawa
T
T
Miwa
K
K
Shirakura
Y
Y
Saito
T
T
Tomita
K
K
D000421:Alcaligenes / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
D010446:Peptide Fragments; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase; D014357:Trypsin